Riesbeck K, Chen D, Kemball-Cook G, McVey JH, George AJ, Tuddenham EG, Dorling A, Lechler RI (1998) Expression of hirudin fusion proteins in mammalian cells: a strategy for prevention of intravascular thrombosis., Circulation 98 (24) pp. 2744-2752
BACKGROUND: Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. METHODS AND RESULTS: An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti-hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P-selectin fusion proteins accumulated in adrenocorticotropic hormone-containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane. CONCLUSIONS: Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.
Chen D, Ma L, Tham EL, Maresh S, Lechler RI, Mcvey JH, Dorling A (2013) Fibrocytes mediate intimal hyperplasia post-vascular injury and are regulated by two tissue factor-dependent mechanisms, Journal of Thrombosis and Haemostasis 11 (5) pp. 963-974
Background: CD34+ ±-smooth muscle actin (SMA)+ cells mediate intimal hyperplasia (IH) after mechanical endoluminal injury. We previously found that IH is tissue factor (TF) dependent. The precise phenotype of the CD34+ cells mediating IH is unknown and the mechanisms of TF are also unknown. Objective: To define the phenotype of cells mediating IH and compare the effects of inhibiting TF on different subsets of CD34+ cells. Methods: Endoluminal injury was induced in C57BL/6 and two strains of mice expressing a human tissue factor pathway inhibitor (hTFPI) fusion protein on different subsets of CD34+ cells. Confocal microscopy, immunocytofluorescence and real-time PCR were used to determine phenotype. Results: Neointimal cells in C57BL/6 mice were defined as a subset of fibrocytes (CD34+CD45+collagen-1+) expressing SMA, CD31, TIE-2, CXCR4 and CXCL12. Similar cells circulated post-injury and were also found in mice expressing hTFPI on CD34+CD31+ cells, though in these mice, hTFPI inhibited CD31+ fibrocyte hyperplasia, so no IH developed. Mice with hTFPI on all CD34+ ±-SMA+ cells repaired arteries back to a pre-injured state. No CD31+ fibrocytes were found in these mice unless an anti-hTFPI antibody was administered. Similar findings in protease activated receptor (PAR)-1-deficient mice suggested hTFPI prevented thrombin signaling through PAR-1. In vitro, thrombin increased the number of CD31+ fibrocytes. Conclusions: Inhibition of TF on CD31+ fibrocytes inhibits IH whereas inhibition on all CD34+ ±-SMA+ cells (or PAR-1 deficiency) inhibits the appearance of CD31+ fibrocytes and promotes repair. These data enhance our understanding of IH and suggest novel ways to promote regenerative repair. © 2013 International Society on Thrombosis and Haemostasis.
Bachli EB, Nathwani AC, Pech C, Tuddenham EGD, McVey JH (1998) Characterisation of nuclear factors binding to the 5 ' untranslated region of the human tissue factor gene., BLOOD 92 (10) pp. 92B-92B W B SAUNDERS CO
David D, Santos AI, Jonson D, Tuddenham EGD, McVey JH, Kemball-Cook G (1999) Stable expression and characterization of CRM- (C329S) and a CRM+ (G1948D) factor VIII mutants using the B-domain deleted FVIIISQ variant, THROMBOSIS AND HAEMOSTASIS pp. 3-3 F K SCHATTAUER VERLAG GMBH
Morichika S, Shima M, Tanaka I, Nakai B, Suzuki H, Nogami K, Hato T, McVey J, Tuddenham EGD, Saenko E, Scandella D, Yoshioka A (1996) A case of factor VIII C2 antigen positive hemophilia a with inhibitor recognizing the 44kDa Al domain: Identification of a large deletion from exon 4 TO 7., BLOOD 88 (10) pp. 2616-2616 W B SAUNDERS CO
Palmer DB, McVey JH, Purohit R, Picard J, Dyson PJ (1998) Characterization of a recent retroposon insertion on mouse chromosome 2 and localization of the cognate parental gene to chromosome 11., Mamm Genome 9 (2) pp. 103-106
The genomic sequence on mouse Chromosome (Chr) 2 corresponding to a previously identified novel cDNA has been characterized. The genomic organization of this locus, adjacent to the beta 2 microglobulin gene, has the properties of a processed gene or retroposon including the presence of a short flanking direct repeat, a polyadenylation signal/poly A tract, and the absence of introns. Analysis of inbred and wild-derived Mus DNAs reveals the retroposon to be a feature only of M. m. domesticus subspecies, suggesting that the insertion event is relatively recent. This notion is supported by the presence of an open reading frame and the lack of sequence divergence in the flanking direct repeats. The complex chromatin configuration characteristic of this region in mouse and human is not, therefore, related to this cDNA. The cognate parental gene encoding the cDNA was mapped to Chr 11. A further, more ancient retroposon present in many Mus species localizes to Chr 17.
Daxin C, Riesbeck K, McVey J, Kembell-Cook G, Tuddenham EGD, Lechler RI, Dorling A (2000) Expression of novel anticoagulant fusion proteins inhibits factor Xa- and thrombin-induced activation of porcine vascular endothelial cells., TRANSPLANTATION 69 (8) pp. S383-S383 LIPPINCOTT WILLIAMS & WILKINS
Gomez K, Laffan MA, Kemball-Cook G, Pasi J, Layton M, Singer JD, Tuddenham EG, McVey JH (2004) Two novel mutations in severe factor VII deficiency., Br J Haematol 126 (1) pp. 105-110
We have characterized the molecular defect in two families with severe factor VII (FVII) deficiency. In family I, the proband was found to be homozygous for a novel 18 bp deletion in exon 8 (g.10896-10913del) resulting in the in-frame deletion of six amino acids in the serine protease domain. Molecular modelling suggests the deletion is likely to disrupt folding of the FVII molecule. The reduced FVII antigen (21 U/dl) and negligible activity (0.4 U/dl) in the patient's plasma indicated that the deletion affected both the secretion/stability and function of the mutant protein. In family II, the proband was found to be a compound heterozygote for a novel missense mutation (g.7884G>A; FVII G117R) in exon 5 encoding the EGF2 domain of FVII and a nonsense mutation (g.8960C>T; FVII R152X) in exon 6. Extensive sequence comparison in a wide evolutionary context suggested that the Gly117 residue is critical for structure of FVII. The grossly reduced FVII antigen (1.1 U/dl) and activity (0.4 U/dl) plasma values indicate the mutation primarily affected the folding/secretion or stability of the protein.
TAKAMIYA O, KEMBALLCOOK G, MARTIN DMA, COOPER DN, VONFELTEN A, MEILI E, HANN I, PRANGNELL DR, LUMLEY H, TUDDENHAM EGD, MCVEY JH (1993) DETECTION OF MISSENSE MUTATIONS BY SINGLE-STRAND CONFORMATIONAL POLYMORPHISM (SSCP) ANALYSIS IN 5 DYSFUNCTIONAL VARIANTS OF COAGULATION FACTOR-VII, HUMAN MOLECULAR GENETICS 2 (9) pp. 1355-1359 OXFORD UNIV PRESS UNITED KINGDOM
Mansvelt EP, Laffan M, McVey JH, Tuddenham EG (1998) Analysis of the F8 gene in individuals with high plasma factor VIII: C levels and associated venous thrombosis., Thromb Haemost 80 (4) pp. 561-565
High FVIII:C levels have previously been shown to be an independent risk factor for thrombosis with 4.8 times higher potential risk of thrombosis in individuals with FVIII:C levels greater than 1.5 u/ml. Recently, we found that raised FVIII:C levels are largely attributable to elevated FVIII:Ag levels. The determinants of FVIII:Ag levels are unclear and might be partly genetic. The promoter of the F8 gene has recently been characterised we therefore investigated the promoter and the 3' terminus of the F8 gene for possible polymorphisms associated with raised FVIII:Ag levels in 62 selected individuals with a thrombotic tendency. We confirm previous reports that raised FVIII:C levels are largely attributable to an elevation in FVIII:Ag and this is also associated with elevation of vWF; non-O blood group: relatively short APTT and relatively low APC ratio. We screened 1140 bp of the proximal promoter including the protein binding sites identified by DNase I footprint analysis by SSCP, however no polymorphisms were identified. Direct DNA sequence analysis of the region -542 to +165 failed to identify any sequence polymorphisms. The recently described polymorphism in the polyadenylation cleavage site in the prothrombin gene associated with increased prothrombin activity prompted us to screen the region surrounding the 3' terminus of the F8 gene for polymorphisms but we found none.
Baker AH, Mcvey JH, Waddington SN, Di Paolo NC, Shayakhmetov DM (2007) The influence of blood on In Vivo adenovirus bio-distribution and transduction, Molecular Therapy 15 (8) pp. 1410-1416
Intravascular delivery of adenovirus (Ad) vectors is being developed for liver-directed gene therapy for targeting disseminated disease in cancer therapeutics and for targeting non-hepatic tissues and organs through vector engineering strategies. The utility of Ad vectors is not limited to serotype 5 (Ad5), and many alternate human serotypes and non-human serotypes of Ad are currently being investigated. Critical to intravascular delivery of Ad is the interaction of the virus with host blood cells and plasma proteins, because immediate contact is observed following injection. Although incompletely understood, recent studies suggest that these interactions are critical in dictating the particle bio-distribution and resulting transduction properties of Ad in vivo. For example, plasma proteins-in particular, vitamin K-dependent coagulation zymogens-are able to directly bind to Ad, and "bridge" the virus to receptors in the liver. Unraveling and characterizing these mechanisms will be of fundamental importance both for understanding basic Ad biology in vivo and for refinement and optimization of Ad vectors for human gene therapy.
Dorling A, Chen D, Riesbeck K, McVey J, Kemball-Cook G, Tuddenham EG, Lechler RI (2000) Regulated endothelial cell expression of novel anticoagulants: a strategy for the prevention and therapy of intravascular thrombosis., Transplant Proc 32 (5)
MCVEY JH, IMANAKA Y, NISHIMURA T, OBRIEN DP, BOLTONMAGGS PHB, TUDDENHAM EGD (1995) IDENTIFICATION OF A NOVEL MECHANISM OF HUMAN GENETIC-DISEASE - A MISSENSE MUTATION CAUSING FXI DEFICIENCY THROUGH A CHANGE IN MESSENGER-RNA STABILITY, THROMBOSIS AND HAEMOSTASIS 73 (6) pp. 1442-1442 F K SCHATTAUER VERLAG GMBH
McVey JH (1999) Tissue Factor pathway,BEST PRACTICE & RESEARCH CLINICAL HAEMATOLOGY 12 (3) pp. 361-372
Blood coagulation is initiated in response to vessel damage in order to preserve the integrity of the mammalian vascular system. The coagulation cascade can also be initiated by mediators of the inflammatory response, and fibrin deposition has been noted in a variety of pathological states. The cascade of coagulation zymogen activations which leads to clot formation is initiated by exposure of flowing blood to Tissue Factor (TF), the cellular receptor and cofactor for Factor VII (FVII). FVII binds to the receptor in a I:I stoichiometric complex and is rapidly activated. FVIIa undergoes an active site transition upon binding TF in the presence of calcium which enhances the fundamental properties of the enzyme. This results in rapid autocatalytic activation of FVII to FVIIa, thereby amplifying the response by generating more TF-FVIIa complexes. The TF-FVIIa activates both FIX and FX. Further FXa generation by the FIXa-FVIIIa-Ca2+-phospholipid complex is required to sustain the coagulation mechanism, since the TF-FVIIa complex is rapidly inactivated by Tissue Factor pathway inhibitor (TFPI). TFPI circulates in plasma, is associated with vascular cell surface and is released from platelets following stimulation by thrombin. TFPI requires the formation of an active TF-FVIIa complex and FXa generation before inhibition can occur. TFPI prevents further participation of TF in the coagulation process by forming a stable quaternary complex, TF-FVIIa-FXa-TFPI.
Bachli E, Pech CM, Johnson KM, Tuddenham EGD, McVey JH (2000) Coagulation factors Xa and IIa but not VIIa induce a proinflammatory response in dermal fibroblasts., BLOOD 96 (11) pp. 636A-+ AMER SOC HEMATOLOGY
Chen D, Carpenter A, Abrahams J, Chambers RC, Lechler RI, McVey JH, Dorling A (2008) Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1-dependent leukocyte recruitment in vivo, JOURNAL OF EXPERIMENTAL MEDICINE 205 (8) pp. 1739-1746 ROCKEFELLER UNIV PRESS
Salooja N, McVey J, Carmeliet P, Tuddenham EGD (1997) Construction of a mouse model of factor IX deficiency., BLOOD 90 (10) pp. 4647-4647 W B SAUNDERS CO
Riesbeck K, Dorling A, Kemball-Cook G, McVey JH, Jones M, Tuddenham EG, Lechler RI (1997) Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface., Thromb Haemost 78 (6) pp. 1488-1494
Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.
Boswell EJ, Tuddenham EGD, McVey JH (1997) Expression and characterisation of site-directed variants of the EGF-1 domain of human factor VII, THROMBOSIS AND HAEMOSTASIS pp. PS41-PS41 F K SCHATTAUER VERLAG GMBH
Chen D, Weber M, McVey JH, Kemball-Cook G, Tuddenham EG, Lechler RI, Dorling A (2004) Complete inhibition of acute humoral rejection using regulated expression of membrane-tethered anticoagulants on xenograft endothelium., Am J Transplant 4 (12) pp. 1958-1963
Xenotransplantation promises an unlimited supply of organs for clinical transplantation. However, an aggressive humoral immune response continues to limit the survival of pig organs after transplantation into primates. Because intravascular thrombosis and systemic coagulopathy are prominent features of acute humoral xenograft rejection, we hypothesized that expression of anticoagulants on xenogeneic vascular endothelium might inhibit the process. Hearts from novel transgenic mice, expressing membrane-tethered fusion proteins based on human tissue factor pathway inhibitor and hirudin, respectively, were transplanted into rats. In contrast to control non-transgenic mouse hearts, which were all rejected within 3 days, 100% of the organs from both strains of transgenic mice were completely resistant to humoral rejection and survived for more than 100 days when T-cell-mediated rejection was inhibited by administration of ciclosporin A. These results demonstrate the critical role of coagulation in the pathophysiology of acute humoral rejection and the potential for inhibiting rejection by targeting the expression of anticoagulants to graft endothelial cells. This genetic strategy could be applied in a clinically relevant species such as the pig.
Pugh RE, McVey JH, Tuddenham EG, Hancock JF (1995) Six point mutations that cause factor XI deficiency., Blood 85 (6) pp. 1509-1516
We have identified six novel types of mutation that cause factor XI deficiency, an inherited bleeding disorder. Two are point mutations that interfere with the normal splicing of exons in the mRNA and four are point mutations that result in amino acid substitutions. One of these amino acid substitutions (Asp 16-->His) is near the amino terminal end of the protein. The other three amino acid substitutions (Leu 302-->Pro, Thr 304-->Ile, and Glu 323-->Lys) are in the fourth apple domain, a region that mediates dimerization of identical subunits of factor XI. All four amino acid substitutions cause a reduction in the amount of factor XI secreted from cells grown in vitro.
Duffy MR, Bradshaw AC, Parker AL, McVey JH, Baker AH (2011) A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes, Journal of Virology 85 (20) pp. 10914-10919
Hepatocyte transduction following intravenous administration of adenovirus 5 (Ad5) is mediated by interaction between coagulation factor X (FX) and the hexon. The FX serine protease (SP) domain tethers the Ad5/FX complex to hepatocytes through binding heparan sulfate proteoglycans (HSPGs). Here, we identify the critical HSPG-interacting residues of FX. We generated an FX mutant by modifying seven residues in the SP domain. Surface plasmon resonance demonstrated that mutations did not affect binding to Ad5. FX-mediated, HSPG-associated cell binding and transduction were abolished. A cluster of basic amino acids in the SP domain therefore mediates surface interaction of the Ad/FX complex. © 2011, American Society for Microbiology.
Schwaab R, Ludwig M, Kochhan L, Oldenburg J, McVey JH, Egli H, Brackmann HH, Olek K (1991) Detection and characterisation of two missense mutations at a cleavage site in the factor VIII light chain., Thromb Res 61 (3) pp. 225-234
Haemophilia A is an X-linked bleeding disorder caused by a deficiency of factor VIII. As an essential cofactor in the intrinsic clotting cascade, factor VIII is activated and subsequently inactivated by proteolytic cleavages involving factor IIa (thrombin), factor Xa and activated protein C (APC). Investigation of the thrombin cleavage sites at amino acids 372 and 1689 of the factor VIII protein by oligonucleotide screening, DNA amplification and direct sequencing, enabled us to identify two missense mutations in 441 unrelated haemophiliacs. A C-to-T transition, which leads to the substitution of cysteine for arginine at position 1689, was found in a severely affected patient and a previously undescribed G-to-A substitution, causing replacement of arginine1689 with histidine, was found in a patient with mild disease.
Bold RL, McVey JH, Tuddenham EGD, Kemball-Cook G (1999) Recombinant expression and characterization of site-directed variants of the human factor VIIa active site catalytic triad, THROMBOSIS AND HAEMOSTASIS pp. 98-99 F K SCHATTAUER VERLAG GMBH
Alshinawi CR, Boswell EJ, Felice AE, Tuddenham EGD, McVey JH (1997) Characterization of two naturally occurring variants of human factor VII, THROMBOSIS AND HAEMOSTASIS pp. P1689-P1689 F K SCHATTAUER VERLAG GMBH
Mumford AD, Chen D, Dorling A, Kemball-Cook G, McVey JH (2005) Generation of a polyclonal rabbit anti-mouse tissue factor antibody by nucleic acid immunisation., Thromb Haemost 93 (1) pp. 160-164
Tissue factor (TF) the cellular receptor and cofactor for factor VII, initiates coagulation and has also been implicated in several coagulation-independent functions, including inflammation, angiogenesis and tumour metastasis. Investigations of TF expression in mouse models of these processes has been limited by the availability of antibodies that specifically recognise mouse TF. We have generated a rabbit polyclonal antibody to mTF by DNA immunisation. This has yielded an antiserum that recognises native mTF in immunohistochemical and flow cytometric analyses. Furthermore, the antiserum is inhibitory in coagulation assays. This antiserum will be a valuable investigative tool in the analysis of mTF expression.
Bachli EB, Tuddenham EGD, McVey JH (1998) Tissue factor expression in a transformed endothelial cell line., BLOOD 92 (10) pp. 92B-92B W B SAUNDERS CO
Kemball-Cook G, Johnson DJ, Takamiya O, Banner DW, McVey JH, Tuddenham EG (1998) Coagulation factor VII Gln100 --> Arg. Amino acid substitution at the epidermal growth factor 2-protease domain interface results in severely reduced tissue factor binding and procoagulant function., J Biol Chem 273 (14) pp. 8516-8521
We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln100 --> Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X. We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant. In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.
Kritz AB, Nicol CG, Dishart KL, Nelson R, Holbeck S, Von Seggern DJ, Work LM, McVey JH, Nicklin SA, Baker AH (2007) Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop.,Mol Ther 15 (4) pp. 741-749
Adenoviral vectors are commonly used for liver-directed gene therapy following systemic administration owing to their strong propensity for hepatocyte transduction. However, many disease applications would benefit from the delivery of adenoviruses to alternate tissues via this route. Research has thus focused on stripping the virus of native hepatic tropism in conjunction with modifying virus capsid proteins to incorporate novel tropism. Recently, the KO1S* adenovirus serotype 5 fiber mutant, devoid of both coxsackie and adenovirus receptor binding in the fiber knob domain and mutated at the putative heparan sulphate proteoglycan binding site in the fiber shaft, was shown to possess strikingly poor hepatic tropism in mice, rats, and non-human primates. Thus, it is an ideal candidate for retargeting strategies. We therefore assessed the ability of peptide-modified KO1S* fibers to retarget adenovirus. Peptide insertions were well tolerated and virions produced to high titers. However, expected retargeting at the level of transduction was not observed, despite cell-binding studies showing enhanced vector targeting at the cell surface. Cy3 labeling studies showed retarded trafficking of S*-containing fibers. Taken together, our data demonstrates that KO1S* mutant fibers are ineffective for cell retargeting strategies.
McVey JH, Lal K, Imanaka Y, Kemball-Cook G, Bolton-Maggs PHB, Tuddenham EGD (2005) Characterization of blood coagulation factor XIT4751, Thrombosis and Haemostasis 93 (6) pp. 1082-1088
PCR-SSCP and DNA sequence analysis of a factor XI (FXI) deficient patient (FXI:C 39 U/dL; FXI:Ag 27 U/dL) identified a C to T transition in exon 12 of the FXI gene (F11 c. 1521C>T) that predicts the substitution of Thr475 by Ile (FXI T4751) within the serine protease domain of FXI. This mutation destroys a consensus sequence for N-linked glycosylation, N473-Y-T475, known to be utilized in vivo. The FXIT4751 variant was generated by site-directed mutagenesis, together with other variants that could help explain the phenotype, and recombinant FXI variants were expressed in Chinese hamster ovary cells. FXI:Ag expression was analysed by Western blot analysis, ELISA and immunocytochemical staining. Wild-type FXI:Ag was secreted at high levels, however the mutant (FXIT4751) was secreted very poorly. Substitution of Thr475 by Ala, Pro, Lys or Arg (all of which abolish the glycosylation consensus sequence) also severely reduced the level of secreted FXI:Ag suggesting that glycosylation at Asn473 is required for folding or secretion. Concordant with this hypothesis the conservative substitution of Thr475 by Ser (which preserves the glycosylation consensus sequence) had no effect on FXI secretion. Thr/Ser475 is highly conserved in serine protease domains but the glycosylation site (Asn473) is not. Surprisingly, substitution of Asn473 by Ala (which removes the N-linked glycosylation site) had no effect on the levels of FXI:Ag secreted. In conclusion, although the FXI-T4751 mutation destroys an N-linked glycosylation consensus sequence, the cause of failure to secrete FXI is not the loss of a glycosylation site but rather a direct effect of the substitution of this highly conserved residue. © 2005 Schattauer GmbH, Stuttgart.
Mumford AD, McVey JH (2004) Tissue Factor in the myocardium: Evidence of roles in haemostasis and inflammation, Disease Markers 20 (6) pp. 353-358
The interaction between cell-surface tissue factor (TF) and the plasma coagulation factor VII (FVII) initiates the coagulation network that leads to the generation of thrombin and the formation of a fibrin clot. Thrombin also activates cellular protease activated receptors (PARs) through which it activates components of the inflammatory pathway. TF is expressed constitutively by cardiomyocytes and evidence from mice transgenic for a human TF mini-gene that express very low levels of human TF suggests that the TF-FVII interaction is critical for haemostasis within the heart. Pathological contact between TF and FVII may occur in the heart during ischaemia-reperfusion (I-R) injury and this may lead to activation of coagulation and thrombin generation. Evidence from animal models now suggests that thrombin is an important mediator of inflammation in I-R injury. The coagulation pathway therefore represents a novel therapeutic target for intervention in the prevention of I-R injury. © 2004 - IOS Press and the authors. All rights reserved.
Parker AL, McVey JH, Doctor JH, Lopez-Franco O, Waddington SN, Havenga MJE, Nicklin SA, Baker AH (2007) Influence of coagulation factor zymogens on the infectivity of adenoviruses pseudotyped with fibers from subgroup D, Journal of Virology 81 (7) pp. 3627-3631
Recent evidence supports a role for vitamin K-dependent coagulation zymogens in adenovirus serotype 5 (Ad5, subgroup C) infection of hepatocytes. Here, we assessed the effect of virus-zymogen interaction on cellular transduction using a panel of fiber (f)-pseudotyped viruses derived from subgroup D (f47, f33, f24, f45, f17, f30). Each virus directly bound factor X (FX) as determined by surface plasmon resonance, resulting in enhanced cell surface binding. Infection of HepG2 cells was promoted by FX but not by FVII or FIX, while transduction of CHO cells was blocked in heparan sulfate proteoglycan-deficient cells. This suggests a broad role for FX in adenovirus infectivity. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Kochhan L, Lalloz MR, Oldenburg J, McVey JH, Olek K, Brackmann HH, Tuddenham EG, Schwaab R (1994) Haemophilia A diagnosis by automated fluorescent DNA detection of ten factor VIII intron 13 dinucleotide repeat alleles., Blood Coagul Fibrinolysis 5 (4) pp. 497-501
Haemophilia A is a recessive X linked bleeding disorder caused by deficiency or functional abnormality of coagulation factor VIII. This disease usually has no visible phenotype in female carriers; hence, great efforts are made to offer all haemophilia A families accurate carrier diagnosis. Significant progress in this direction was made with the identification of the intron 13 variable number tandem repeat (VNTR), which is hitherto the most informative single marker within the factor VIII gene. The authors have established intron 13 VNTR detection in their laboratory by adapting its analysis to an automated sequencer using different primers of which one is fluorescent dye labelled. With this method, which is more rapid and convenient than that originally described, 67 haemophilia A families of German origin were screened and two new alleles (alleles 17 and 25) were identified. The informativeness of the VNTR in these families based on the patients maternal X chromosomes (134) is about 67%.
Albrecht C, McVey JH, Elliott JI, Sardini A, Kasza I, Mumford AD, Naoumova RP, Tuddenham EGD, Szabo K, Higgins CF (2005) A novel missense mutation in ABCA1 results in altered protein trafficking and reduced phosphatidylserine translocation in a patient with Scott syndrome, BLOOD 106 (2) pp. 542-549 AMER SOC HEMATOLOGY
Steen M, Norstrom EA, Tholander AL, Bolton-Maggs PHB, Mumford A, McVey JH, Tuddenham EGD, Dahlback B (2004) Functional characterization of factor V-Ile359Thr: a novel mutation associated with thrombosis, BLOOD 103 (9) pp. 3381-3387 AMER SOC HEMATOLOGY
Ma J, Duffy MR, Deng L, Dakin RS, Uil T, Custers J, Kelly SM, McVey JH, Nicklin SA, Baker AH (2015) Manipulating adenovirus hexon hypervariable loops dictates immune neutralisation and coagulation factor X-dependent cell interaction in vitro and in vivo.,PLoS Pathog 11 (2)
Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX "coating" also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.
Alba R, Bradshaw AC, Parker AL, Bhella D, Waddington SN, Nicklin SA, Van Rooijen N, Custers J, Goudsmit J, Barouch DH, McVey JH, Baker AH (2009) Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: Effect of mutagenesis on FX interactions and gene transfer, Blood 114 (5) pp. 965-971
Recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus (Ad) serotype 5-mediated liver transduction in vivo. FX binds to the adenovirus hexon hypervariable regions (HVRs). Here, we perform a systematic analysis of FX binding to Ad5 HVRs 5 and 7, identifying domains and amino acids critical for this interaction. We constructed a model of the Ad5-FX interaction using crystallographic and cryo-electron microscopic data to identify contact points. Exchanging Ad5 HVR5 or HVR7 from Ad5 to Ad26 (which does not bind FX) diminished FX binding as analyzed by surface plasmon resonance, gene delivery in vitro, and liver transduction in vivo. Exchanging Ad5-HVR5 for Ad26-HVR5 produced deficient virus maturation. Importantly, defined mutagenesis of just 2 amino acids in Ad5-HVR5 circumvented this and was sufficient to block liver gene transfer. In addition, mutation of 4 amino acids in Ad5-HVR7 or a single mutation at position 451 also blocked FX-mediated effects in vitro and in vivo. We therefore define the regions and amino acids on the Ad5 hexon that bind with high affinity to FX thereby better defining adenovirus infectivity pathways. These vectors may be useful for gene therapy applications where evasion of liver transduction is a prerequisite. © 2009 by The American Society of Hematology.
Elliott JI, McVey JH, Higgins CF (2005) The P2X7 receptor is a candidate product of murine and human lupus susceptibility loci: a hypothesis and comparison of murine allelic products., Arthritis Res Ther 7 (3) pp. R468-R475
Systemic lupus erythematosus and its murine equivalent, modelled in the New Zealand Black and New Zealand White (NZB x NZW)F1 hybrid strain, are polygenic inflammatory diseases, probably reflecting an autoimmune response to debris from cells undergoing programmed cell death. Several human and murine loci contributing to disease have been defined. The present study asks whether the proinflammatory purinergic receptor P2X7, an initiator of a form of programmed cell death known as aponecrosis, is a candidate product of murine and human lupus susceptibility loci. One such locus in (NZB x NZW)F1 mice is lbw3, which is situated at the distal end of NZW chromosome 5. We first assess whether NZB mice and NZW mice carry distinct alleles of the P2RX7 gene as expressed by common laboratory strains, which differ in sensitivity to ATP stimulation. We then compare the responses of NZB lymphocytes, NZW lymphocytes and (NZB x NZW)F1 lymphocytes to P2X7 stimulation. NZB and NZW parental strains express the distinct P2X7-L and P2X7-P alleles of P2RX7, respectively, while lymphocytes from these and (NZB x NZW)F1 mice differ markedly in their responses to P2X7 receptor stimulation. NZB mice and NZW mice express functionally distinct alleles of the proinflammatory receptor, P2X7. We show that current mapping suggests that murine and human P2RX7 receptor genes lie within lupus susceptibility loci lbw3 and SLEB4, and we argue that these encode a product with the functional characteristics consistent with a role in lupus. Furthermore, we argue that aponecrosis as induced by P2X7 is a cell death mechanism with characteristics that potentially have particular relevance to disease pathogenesis.
MCVEY JH, TAKAMIYA O, TAMAGNINI G, VALENTE V, FIDALGO T, LAYTON M, TUDDENHAM EGD (1995) EXCLUSION OF THE 1ST EGF-1 DOMAIN OF FACTOR-VII BY A SPLICE-SITE MUTATION CAUSES LETHAL FACTOR-VII DEFICIENCY, THROMBOSIS AND HAEMOSTASIS 73 (6) pp. 1439-1439 F K SCHATTAUER VERLAG GMBH
Chen D, Abrahams JM, Smith LM, Mcvey JH, Lechler RI, Dorling A (2008) Regenerative repair after endoluminal injury in mice with specific antagonism of protease activated receptors on CD34(+) vascular progenitors, BLOOD 111 (8) pp. 4155-4164 AMER SOC HEMATOLOGY
MCVEY JH (1994) TISSUE FACTOR PATHWAY,BAILLIERES CLINICAL HAEMATOLOGY 7 (3) pp. 469-484
Blood coagulation is initiated in response to vessel damage in order to preserve the integrity of the mammalian vascular system. The coagulation cascade can also be initiated by mediators of the inflammatory response, and fibrin deposition has been noted in a variety of pathological states. The cascade of coagulation zymogen activations which leads to clot formation is initiated by exposure of flowing blood to tissue factor (TF), the cellular receptor and cofactor for factor VII (FVII). FVII binds to the receptor in a 1:1 stoichiometric complex and is rapidly activated. FVIIa undergoes an active site transition upon binding TF in the presence of calcium which enhances the fundamental properties of the enzyme. This results in rapid autocatalytic activation of FVII to VIIa thereby amplifying the response by generating more TF-VIIa complexes. The TF-VIIa activates both FIX and FX. Further FXa generation by the IXa-VIIIa-Ca2+-phospholipid complex is required to sustain the coagulation mechanism, since the TF-VIIa complex is rapidly inactivated. Structure and function studies have identified a number of regions on both TF and FVII involved in this interaction. It is clear, however, that the molecular structures of TF, FVII and the TF-VII complex will have to be solved before we fully understand this complex interaction. The activity of the TF-VIIa complex is controlled by two inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin III (AT-III). TFPI circulates in plasma, is associated with vascular cell surface and is released from platelets following stimulation by thrombin. TFPI requires the formation of an active TF-VIIa complex and FXa generation before inhibition can occur. Similarly, AT-III which is unable to inhibit circulating FVIIa requires the formation of the TF-VIIa complex. TFPI prevents further participation of TF in the coagulation process by forming a stable quaternary complex, TF-VIIa-Xa-TFPI. In contrast, the AT-III-VIIa complex is thought to dissociate from TF allowing it to interact with additional FVII-VIIa. TFPI has been considered the primary regulator of TF-VIIa activity during haemostasis. Whether AT-III in the presence of glycosaminoglycans on cell surfaces expressing TF can function as an auxiliary second physiological regulator is not known. © 1994 Baillière Tindall. All rights reserved.
Parker AL, McVey JH, Waddington SN, Buckley SMK, Francischetti IMB, Monteiro RQ, Nicklin SA, Baker AH (2008) An exosite within the human FX serine protease domain mediates cell transduction of AD5: FX complexes, HUMAN GENE THERAPY 19 (4) pp. 407-407 MARY ANN LIEBERT INC
Chen D, Riesbeck K, McVey JH, Kemball-Cook G, Tuddenham EGD, Lechler RI, Dorling A (1999) Regulated endothelial cell expression of novel molecules with anticoagulant properties: A strategy for the prevention and therapy of vascular rejection, THROMBOSIS AND HAEMOSTASIS pp. 12-12 F K SCHATTAUER VERLAG GMBH
Palmer DB, McVey JH, Robinson PJ, Dyson PJ (1992) The chromatin structure of the mouse beta-2-microglobulin locus., Differentiation 51 (3) pp. 201-207
Within the promoter regions of both major histocompatibility complex (MHC) class I genes and the beta 2-microglobulin (beta 2m) gene, there are a number of common regulatory elements suggesting co-ordinate control. However, there is also evidence to suggest that beta 2m and class I are differentially regulated, indicating that these genes may have distinct regulatory elements. We sought to explore this question by analysing DNase I hypersensitive (DH) sites flanking the beta 2m gene. Five DH sites have been found within the vicinity of the beta 2m gene. One of these sites (DH1) located within the promoter region, correlates with the transcriptional activity of beta 2m since it is weak in embryonal (beta 2m negative) cell lines. The remaining DH sites (2-5) are located downstream of the beta 2m gene. The most proximal downstream site, (DH2) located 5.5 kb from the last exon, was observed only in embryonal cell lines, indicating possible involvement in the downregulation of beta 2m. Furthermore, this site is markedly diminished in differentiated F9 cells. Possible roles for the remaining sites are discussed, in particular relationship to a second transcriptional unit identified in the vicinity. In addition, a similar analysis reveals a cluster of DH sites located downstream from the last exon of the human beta 2m gene.
McVey JH (2003) Your bleeding heart: lessons from low tissue factor expression in mice., Trends Pharmacol Sci 24 (6) pp. 269-272
Tissue factor (TF) is the cellular receptor and cofactor for blood coagulation factor VII (FVII). Exposure of flowing blood to cells that express TF leads to the initiation of blood coagulation. A recent study of mice expressing low levels of TF has demonstrated the importance of TF and FVII in maintaining adequate haemostasis within the heart. In addition, the study indicates that the heart is subject to a succession of minor bleeds most probably as a result of repetitive minor mechanical injury to the blood vessels.
David D, Santos IMA, Johnson K, Tuddenham EGD, McVey JH (2003) Analysis of the consequences of premature termination codons within factor VIII coding sequences, JOURNAL OF THROMBOSIS AND HAEMOSTASIS 1 (1) pp. 139-146 BLACKWELL PUBL LTD
Zhang B, Cunningham MA, Nichols WC, Bernat JA, Seligsohn U, Pipe SW, McVey JH, Schulte-Overberg U, de Bosch NB, Ruiz-Saez A, White GC, Tuddenham EGD, Kaufman RJ, Ginsburg D (2003) Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex, NATURE GENETICS 34 (2) pp. 220-225 NATURE PUBLISHING GROUP
EDBROOKE MR, MCVEY JH, PARKER D, CRAIG RK (1984) EUTOPIC AND ECTOPIC EXPRESSION OF THE HUMAN CALCITONIN GENE, METABOLIC BONE DISEASE & RELATED RESEARCH 5 (4) pp. 207-207 PERGAMON-ELSEVIER SCIENCE LTD
Hampshire DJ, Cairo A, Dolan G, Giansily-Blaizot M, Gomez K, Goodeve AC, Kemball-Cook G, Ludlam CA, Mcvey JH, Oldenburg J, Perkins SJ, Peyvandi F, Rallapalli PM (2015) Eahad-DB: a combined coagulation factor variant databases resource for the clinical and scientificcommunities, JOURNAL OF THROMBOSIS AND HAEMOSTASIS 13 pp. 997-997 WILEY-BLACKWELL
Pattinson JK, Millar DS, McVey JH, Grundy CB, Wieland K, Mibashan RS, Martinowitz U, Tan-Un K, Vidaud M, Goossens M, Sampietro M, Mannucci PM, Krawczak M, Reiss J, Zoll B, Whitmore D, Bowcock S, Wensley R, Ajani A, Mitchell V, Rizza C, Maia R, Winter P, Mayne EE, Schwartz M, Green PJ, Kakkar VV, Tuddenham EGD, Cooper DN (1990) The molecular genetic analysis of hemophilia A: A directed search strategy for the detection of point mutations in the human factor VIII gene, Blood 76 (11) pp. 2242-2248
A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA TGA). 372 (CGC TGC). 372 (CGC CAC). and 1689 (CGC TGC). These are functionally important cleavage sites for either activated protein C or Thrombin. Further novel C T transitions were identified in the remaining arginine codons screened (-5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking. © 1990 by The American Society of Hematology.
Cytokines such as interferon-gamma (IFNgamma) released by resident uterine immune cells are thought to influence the expression of differentiated function in the human endometrium. Decidualization of the stromal cell compartment is confined to the superficial endometrial layer in the nonpregnant uterus. To explore the molecular mechanism underlying the spatial expression of the decidual phenotype, the effect of IFNgamma on the induction of two well characterized markers of endometrial stromal (ES) cell differentiation, PRL and tissue factor (TF), has been investigated. IFNgamma antagonizes cAMP-mediated PRL protein and messenger RNA expression in primary ES cell cultures through inhibition of decidual PRL promoter activity. In parallel, IFNgamma stimulates Stat-1 (signal transducer and activator of transcription-1) expression, phosphorylation, and translocation to the nucleus. Exogenously expressed Stat-1 potently represses decidual PRL promoter activation, indicating the potential for the inhibitory effects of IFNgamma to be mediated by Stat-1. We demonstrate that although the coactivator CREB-binding protein/p300 is essential for decidual PRL transcription, this coactivator does not appear to be the target for IFNgamma-mediated repression. By contrast, IFNgamma has little effect on cAMP-mediated TF expression, but induces TF in ES cells not exposed to a decidualizing stimulus. This suggested that in vivo TF expression may not be restricted to decidualizing cells of the superficial layer and was confirmed by immunohistochemical analysis demonstrating intense TF staining in the basal stromal compartment during the regeneration phase of the cycle. The differential sensitivity of decidualization-associated genes to IFNgamma illustrates its potential role as a selective biological response modifier that influences regional function within the endometrium.
Shrivastava S, McVey JH, Dorling A (2007) The interface between coagulation and immunity., Am J Transplant 7 (3) pp. 499-506
Coagulation proteases are involved in generating fibrin after vascular injury (hemostasis) but they also have multiple other effects, many of which are mediated independently of fibrin generation, via interactions with specific cell membrane-expressed "protease activated receptors". In inflammation, this family of proteins has a complex influence, the facets of which are still incompletely understood, though a common feature in different models appears to be amplification of innate signals that are initially generated by pathogenic elements or, in the context of transplantation, ischemia or anti-graft antibodies, for instance. There is increasing evidence that these proteases may also have specific effects on cells involved in adaptive immunity and on cells that mediate chronic inflammation and fibrosis. Understanding whether these effects are relevant in the responses generated against transplanted organs is important, as it could lead ultimately to the development of novel ways to promote long-term graft survival.
Chen DX, Riesbeck K, Kemball-Cook G, McVey JH, Tuddenham EGD, Lechler RI, Dorling A (1999) Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins, TRANSPLANTATION 67 (3) pp. 467-474 LIPPINCOTT WILLIAMS & WILKINS
Kemball-Cook G, Tuddenham EGD, McVey JH (2007) Normal Haemostasis,In: Postgraduate Haematology: Fifth Edition pp. 783-807
Lenaerts L, Baker AH, McVey JH, Waddington S, Verbeken E, Naesens L (2007) Effect of coagulation factors on liver targeting of mouse adenovirus type 1, HUMAN GENE THERAPY 18 (10) pp. 1032-1032 MARY ANN LIEBERT INC
Alba R, Bradshaw AC, Coughlan L, Denby L, McDonald RA, Waddington SN, Buckley SMK, Greig JA, Parker AL, Miller AM, Wang H, Lieber A, Van Rooijen N, McVey JH, Nicklin SA, Baker AH (2010) Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors, Blood 116 (15) pp. 2656-2664
A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophagedepleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c+, ER-TR7+, and MAdCAM-1+ cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46+ mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo. © 2010 by The American Society of Hematology.
McVey JH (1994) Staden Plus., Methods Mol Biol 25 pp. 171-179
Pipe SW, Miao HZ, Kucab PF, McVey JH, Kaufman RJ (2005) The secretion efficiency of factor VIII can be regulated by the size and oligosaccharide content of the B domain., BLOOD 106 (11) pp. 203A-203A AMER SOC HEMATOLOGY
Lenaerts L, McVey JH, Baker AH, Denby L, Nicklin S, Verbeken E, Naesens L (2009) Mouse adenovirus type 1 and human adenovirus type 5 differ in endothelial cell tropism and liver targeting, Journal of Gene Medicine 11 (2) pp. 119-127
Background: For adenovirus vectors derived from human serotype 5 (Ad5), the efficiency and safety after intravascular delivery is hindered by their sequestration in nontarget tissues, predominantly the liver. The latter is largely dictated by adenovirus binding to blood coagulation zymogens. In addition, several target cells, such as endothelial and smooth muscle cells, are difficult to transduce by Ad5 due to the low expression of the primary coxsackie-adenovirus receptor (CAR). Therefore, alternative adenovirus serotypes are being explored. Methods: In the present study, we assessed the tropism of mouse adenovirus type 1 (MAV-1), a nonhuman adenovirus for which cellular attachment is CAR-independent. Results: The typical replication of MAV-1 in endothelial cells as observed in vivo was not reflected in elevated attachment to primary and continuous endothelial cells in cell culture. Remarkably, MAV-1 displayed a higher affinity for primary human smooth muscle cells than recombinant Ad5 (rAd5). Attachment of MAV-1 to human and mouse cells of hepatocyte origin was not altered by physiological concentrations of human coagulation factor XI (FXI) or the vitamin K-dependent FIX, FX and FVII. By contrast, attachment of Ad5-derived vectors was enhanced at least eight-fold by FX. Using surface plasmon resonance, MAV-1 was shown to directly associate with human FX and murine FX and FIX but, opposite to rAd5, this interaction did not lead to enhanced cellular attachment. In intravenously injected severe combined immunodeficiency mice, distribution of MAV-1 to the liver was markedly lower than that observed with rAd5. Conclusions: Our data on the tropism of MAV-1 suggest that this virus may find utility in the field of gene therapy. Copyright © 2008 John Wiley & Sons, Ltd.
Morichika S, Shima M, Kamisue S, Tanaka I, Imanaka Y, Suzuki H, Shibata H, Pemberton S, Gale K, McVey J, Tuddenham EGD, Yoshioka A (1997) Factor VIII gene analysis in Japanese CRM-positive and CRM-reduced haemophilia A patients by single-strand conformation polymorphism, British Journal of Haematology 98 (4) pp. 901-906
Haemophilia A is the most common X-linked blood coagulation disorder; it is caused by deficiency of factor VIII activity (FVIII:C). Half of the affected patients do not have detectable levels of FVIII protein in their plasma, whereas about 5% have normal levels of the FVIII antigen (FVIII:Ag) (> 50 u/dl), and are called cross-reacting material (CRM) positive (CRM+ or A+). About 45% of patients have reduced levels of the FVIII:Ag (1-50 u/dl), classified as CRM reduced (CRM(R) or A(R)). We screened the FVIII gene of 13 Japanese patients (five CRM+ and eight CRM(R)) by single-strand conformation polymorphism, and identified 11 different mutations in 13 patients by analysing all 26 exons (Trp255Cys, Tyr473Cys, Gly479Arg, Arg531His, Thr667Arg, Arg1689Cys, Arg1941Gln, Arg2150His, Arg2159Cys, Thr2245Ala and Gly2285Val). Seven mutations were identified in the A domains (four in the A2 domain). All the mutations are point mutations resulting in missense codons. Four mutations (Trp255Cys, Thr667Arg, Thr2245Ala and Gly2285Val) have not been described previously.
Chen D, Shrivastava S, Ma L, Tham EL, Abrahams J, Coe JD, Scott D, Lechler RI, McVey JH, Dorling A (2012) Inhibition of thrombin receptor signaling on ±-smooth muscle actin + CD34 + progenitors leads to repair after murine immune vascular injury, Arteriosclerosis, Thrombosis, and Vascular Biology 32 (1) pp. 42-49
Objective-: The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on ±-smooth muscle actin (±-SMA) + cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). Methods and Results-: BALB/c aortas (H-2 d) transplanted into ±-TFPI- transgenic (Tg) mice (H-2 d) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2 d alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34 + (but not CD34 -) cells inhibited IH in WT recipients, indicating the phenotype of ±-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34 + cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45 + myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed ±-SMA and were recruited to the neointima. In contrast, the ±-SMA + human TFPI + CD34 + cells recruited in Tg recipients were from a CD45 - lineage. WT CD34 + cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. Conclusion-: Specific inhibition of thrombin generation or PAR-1 signaling on ±-SMA + CD34 + cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage. © 2011 American Heart Association, Inc.
McVey JH, Tsai M-C, Chen K-D, Wang C-C (2016) Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling, CELL DEATH DISCOVERY
We previously demonstrated PAR2 starts upstreamed with tissue factor (TF) and factor VII (FVII), inhibited autophagy via mTOR signaling in HCC. However, the mechanism underlying for merging functions of PAR2 with the coagulation system in HCC progression remained unclear. The present study aimed to investigate the role of TF, FVII and PAR2 in tumor progression of HCC. The expressions of TF, FVII and PAR2 from HCC specimens were evaluated by immunohistochemical stains and western blotting. We found that the expression of FVII, but not TF and PAR2, directly related to the vascular invasion and the clinical staging. Importantly, a lower level of FVII expression was significantly associated with the longer disease-free survival. The addition of FVII but not TF induced the expression of PAR2 and phosphorylation of ERK1/2, whereas knockdown of FVII decreased PAR2 expression and ERK1/2 phosphorylation in HCC cell lines. Furthermore, levels of phosphor-TSC2 (Ser664) were increased after treatment with FVII and PAR2 agonist whereas these were significantly abolished in the presence of a potent and specific MEK/ERK inhibitor U0126. Moreover, mTOR knockdown highly reduced Hep3B migration, which could be reverted by FVII but not TF and PAR2. These results indicated that FVII/PAR2 signaling through MEK/ERK and TSC2 axis for mTOR activation has potent effects on the migration of HCC cells. In addition, FVII/PAR2 signaling elicits an mTOR-independent signaling, which promotes hepatoma cell migration in consistent with the clinical observations. Our study indicates that levels of FVII, but not TF, are associated with tumor migration and invasiveness in HCC, and provides clues that evaluation of FVII expression in HCC may be useful as a prognostic indicator in patients with HCC and may form an alternative target for further therapy
McVey JH, Boswell EJ, Takamiya O, Tamagnini G, Valente V, Fidalgo T, Layton M, Tuddenham EG (1998) Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency., Blood 92 (3) pp. 920-926
We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5' splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor-like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the triangle upEGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.
Salooja N, McVey J, Tuddenham EGD (1998) Towards gene therapy for haemophilia B: Two sensitive ELISAS for mouse FIX antigen., BRITISH JOURNAL OF HAEMATOLOGY 101 pp. 83-83 BLACKWELL SCIENCE LTD
Bolton-Maggs PHB, Hay CRM, Shanks D, Mitchell MJ, McVey JH (2007) The importance of tissue factor source in the management of factor VII deficiency, Thrombosis and Haemostasis 97 (1) pp. 151-152
Nathwani AC, Gale KM, Pemberton KD, Crossman DC, Tuddenham EG, McVey JH (1994) Efficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter., Br J Haematol 88 (1) pp. 122-128
Tissue factor (TF) is a cellular receptor and cofactor for factor VII/VIIa which initiates the blood coagulation cascade. We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the human TF gene in human umbilical vein endothelial cells (HUVEC). Using a chloramphenicol acetyltransferase (CAT) reporter gene, we attempted to transfect primary cultured HUVEC (passage 3-4) with calcium phosphate coprecipitation, DEAE Dextran, lipopolyamine-coated DNA or electroporation. Electroporation in HEPES-buffered saline of 1 x 10(7) cells at 200V and 250 microF was found to be optimal. Using these conditions, varying lengths of TF 5'-flanking sequences coupled to the CAT reporter gene were tested in transient expression studies. CAT expression corrected for variation in transfection efficiency and cell viability revealed that the sequences between -111 and +14 base pairs are essential for minimal transcriptional activity. This region contains consensus sequences for a TATA box and three Sp1 binding sites. A domain from -382 to -111bp, which contains two AP-1 consensus elements, promoted high levels of gene expression. This transcriptional activity was repressed by 50% with constructs containing sequences between -550 and -382 bp. A further 2-fold drop in transcription activity was attributed to the region between -948 and -550 bp. These results suggest that the basal transcription of the human TF gene in HUVEC is mediated through at least two negative regulatory elements upstream of the proximal promoter domain. The proximal promoter region which contains two AP-1 sites is essential for efficient transcription.
CRAIG RK, EDBROOKE MR, RILEY JH, MCVEY JH, PARKER D (1985) DIFFERENTIAL EXPRESSION OF THE HUMAN CALCITONIN - CGRP GENE IN MEDULLARY-THYROID CARCINOMA AND LUNG-CARCINOMA CELL-LINES, RECENT RESULTS IN CANCER RESEARCH 99 pp. 71-78 RESEARCH INST TECHNICAL CHEMISTRY OF HUNGARIAN ACAD SCIENCES
Bachli EB, Pech CM, Johnson KM, Johnson DJ, Tuddenham EG, McVey JH (2003) Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts., J Thromb Haemost 1 (9) pp. 1935-1944
UNLABELLED: Coagulation factors (F)VIIa, FXa and thrombin are implicated in cellular responses in vascular, mesenchymal and inflammatory cells. Fibroblasts are the most abundant cells in connective tissue, and damage to blood vessels places coagulation factors in contact with these and other cell types. OBJECTIVES: To investigate cellular responses of primary dermal fibroblasts to FVIIa, FXa and thrombin by following changes in expression of candidate proteins: monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF), and to determine the expression of receptors implicated in signaling by these coagulation factors. METHODS: Steady-state mRNA levels were quantified by RNase protection assay, and protein secretion by ELISA. PAR gene expression was assessed by ribonuclease protection assay and conventional and quantitative reverse-transcription-polymerase chain reaction. RESULTS: FVIIa did not induce the candidate genes. In contrast, FXa and thrombin induced MCP-1 mRNA and protein secretion strongly, IL-8 moderately, and IL-6 weakly. Neither FXa nor thrombin induced VEGF mRNA or protein secretion, although FXa induced VEGF protein secretion in lung fibroblasts. Comparison of the presence of candidate receptors in the two fibroblast subtypes demonstrated higher levels of PAR-1 and PAR-3 in lung fibroblasts relative to their dermal counterparts and the additional expression of PAR-2. CONCLUSIONS: FXa and thrombin induce expression of MCP-1, IL-8 and IL-6, and distribution and expression of PARs on dermal fibroblasts is reduced relative to their lung counterparts. Tissue origin may influence the cellular response of fibroblasts to coagulation proteases.
Denby L, Work LM, Von Seggern DJ, Wu E, Mcvey JH, Nicklin SA, Baker AH (2007) Development of renal-targeted vectors through combined in vivo phage display and capsid engineering of adenoviral fibers from serotype 19p, MOLECULAR THERAPY 15 (9) pp. 1647-1654 NATURE PUBLISHING GROUP
Waddington SN, McVey JH, Bhella D, Parker AL, Barker K, Atoda H, Pink R, Buckley SMK, Greig JA, Denby L, Custers J, Morita T, Francischetti IMB, Monteiro RQ, Barouch DH, van Rooijen N, Napoli C, Havenga MJE, Nicklin SA, Baker AH (2008) Adenovirus Serotype 5 Hexon Mediates Liver Gene Transfer, Cell 132 (3) pp. 397-409
Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. © 2008 Elsevier Inc. All rights reserved.
Ventura C, Santos AIM, Tavares A, Gago T, Lavinha J, McVey JH, David D (2000) Molecular genetic analysis of factor XI deficiency: Identification of five novel gene alterations and the origin of type II mutation in Portuguese families, THROMBOSIS AND HAEMOSTASIS 84 (5) pp. 833-840 F K SCHATTAUER VERLAG GMBH
McIntosh J, Lenting PJ, Rosales C, Lee D, Rabbanian S, Raj D, Patel N, Tuddenham EGD, Christophe OD, McVey JH, Waddington S, Nienhuis AW, Gray JT, Fagone P, Mingozzi F, Zhou S-Z, High KA, Cancio M, Ng CYC, Zhou J, Morton CL, Davidoff AM, Nathwani AC (2013) Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant, BLOOD 121 (17) pp. 3335-3344 AMER SOC HEMATOLOGY
Gomez K, McVey JH (2006) Tissue factor initiated blood coagulation., Front Biosci 11 pp. 1349-1359
Tissue factor (TF) is the cellular receptor and cofactor for blood coagulation factor (F) VII. Exposure of flowing blood to cells that express TF leads to the initiation of blood coagulation. Blood coagulation is tightly regulated to generate a local fibrin clot at the site of vascular injury without compromising blood flow in the vasculature. This chapter describes the initiation and propagation of the response and how it is ultimately down-regulated to prevent widespread inappropriate blood coagulation.
Greig JA, Buckley SMK, Waddington SN, Parker AL, Bhella D, Pink R, Rahim AA, Morita T, Nicklin SA, McVey JH, Baker AH (2009) Erratum: "Influence of coagulation factor X on in vitro and in vivo gene delivery by adenovirus (Ad) 5, Ad35, and chimeric Ad5/Ad35 vectors" (Molecular Theraphy (2009) 10.1038/mt.2009.152), Molecular Therapy 17 (10)
The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins.
McVEY JH (2012) Factor seven-activating protease: does it do what it says on the tin?, JOURNAL OF THROMBOSIS AND HAEMOSTASIS 10 (5) pp. 857-858 WILEY-BLACKWELL
McVey JH (2009) FVIIa gene delivery: Potential treatment for hemophilia?, Blood 113 (16) pp. 3649-3650
By using an AAV vector that contains a liver-specific promoter, Margaritis and colleagues demonstrate that FVIIa gene delivery leads to long-term expression of FVIIa in treated hemophilic dogs. This results in a partial correction of hemostatic parameters and an absence of spontaneous bleeding episodes.
Parker AL, Waddington SN, Buckley SMK, Custers J, Havenga MJE, Van Rooijen N, Goudsmit J, McVey JH, Nicklin SA, Baker AH (2009) Effect of neutralizing sera on factor X-mediated adenovirus serotype 5 gene transfer, Journal of Virology 83 (1) pp. 479-483
The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated cellular binding and transduction of Ad5-based vectors in vitro. Neutralizing activity correlated well with the ability to inhibit Ad5-mediated liver transduction, suggesting that prescreening patient sera in this manner accurately predicts the efficacy of Ad5-based gene therapies. Neutralization in vitro can be partially bypassed by pseudotyping with Ad45 fiber protein, indicating that a proportion of neutralizing antibodies are directed against the Ad5 fiber. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
CROSSMAN DC, CARR DP, MCVEY JH, PEARSON JD, TUDDENHAM EGD (1989) TISSUE FACTOR PRODUCTION BY HUMAN ENDOTHELIUM - MEASUREMENT OF MESSENGER-RNA AND PROTEIN-LEVELS IN RESPONSE TO LIPOPOLYSACCHARIDE OR PHORBOL ESTER, BRITISH JOURNAL OF HAEMATOLOGY 73 (3) pp. 438-438 BLACKWELL SCIENCE LTD
Greig JA, Buckley SMK, Waddington SN, Parker AL, Bhella D, Pink R, Morita T, Custers J, Goudsmit J, Nicklin SA, McVey JH, Baker AH (2009) Influence of Factor X on In Vitro and In Vivo Gene Delivery by Ad5 and Ad35 Vectors, MOLECULAR THERAPY 17 pp. S325-S325 NATURE PUBLISHING GROUP
Holland PW, Harper SJ, McVey JH, Hogan BL (1987) In vivo expression of mRNA for the Ca++-binding protein SPARC (osteonectin) revealed by in situ hybridization., J Cell Biol 105 (1) pp. 473-482
In situ hybridization is used to survey the tissue-specific and developmental expression of the cloned mouse gene Sparc, coding for a protein homologous to the bovine Ca++-binding protein, osteonectin. High levels of SPARC RNA are found in osteoblasts and odontoblasts. In addition, high grain counts are associated with a variety of other cell types in the embryo and newborn mouse, including parietal endoderm, deciduum, whisker follicles (connective tissue sheath), peripheral nerve trunk, skin (dermis), and stomach (submucosa). Spatially restricted but high levels of SPARC mRNA are also seen in the adult adrenal glands, testis, and ovary. This pattern of differential gene expression demands a reassessment of the function originally proposed for osteonectin, and predicts a much wider role for the protein in a variety of biological processes.
McVey JH, Boswell E, Mumford AD, Kemball-Cook G, Tuddenham EG (2001) Factor VII deficiency and the FVII mutation database., Hum Mutat 17 (1) pp. 3-17
Factor VII (FVII) is a zymogen for a vitamin K-dependent serine protease essential for the initiation of blood coagulation. It is synthesized primarily in the liver and circulates in plasma at a concentration of approximately 0.5 microg/ml (10 nmol/L). The FVII gene (F7) is located on chromosome 13 (13q34), consists of 9 exons, and spans approximately 12kb. It encodes a mature protein of 406 amino acids, which has an N-terminal domain (Gla) post-translationally modified by gamma-carboxylation of glutamic acid residues, two domains with homology to epidermal growth factor (EGF1 and 2), and a C-terminal serine protease domain. The single chain zymogen is activated by proteolytic cleavage at Arg152-Ile153. There are 238 individuals described in the world literature with mutations in their F7 genes (FVII mutation database; europium.csc. mrc.ac.uk). Complete absence of FVII activity in plasma is usually incompatible with life, and individuals die shortly after birth due to severe hemorrhage. The majority of individuals with mutations in their F7 gene(s), however, are either asymptomatic or the clinical phenotype is unknown. In general, a severe bleeding phenotype is only observed in individuals homozygous for a mutation in their F7 genes with FVII activities (FVII:C) below 2% of normal, however, a considerable proportion of individuals with a mild-moderate bleeding phenotype have similar FVII:C by in vitro assay. The failure of in vitro tests to differentiate between these groups may be due to lack of sensitivity in the assays to the very low amounts of FVII:C, which are sufficient to initiate coagulation in vivo. A number of polymorphisms have been identified in the F7 gene and some have been shown to influence plasma FVII antigen levels.
Griffith ME, Turner AN, McVey J, Pusey CD (1996) Production of recombinant proteinase 3 recognised by human autoantibodies., JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 7 (9) pp. A2271-A2271 WILLIAMS & WILKINS
IMANAKA Y, MCVEY JH, NISHIMURA T, BOLTONMAGGS P, LLOYD J, TUDDENHAM EGD (1993) IDENTIFICATION AND CHARACTERIZATION OF MUTATIONS IN THE FACTOR-XI GENE OF NON-JEWISH FXI DEFICIENT PATIENTS, THROMBOSIS AND HAEMOSTASIS 69 (6) pp. 752-752 F K SCHATTAUER VERLAG GMBH
Binny C, McIntosh J, Della Peruta M, Kymalainen H, Tuddenham EGD, Buckley SMK, Waddington SN, McVey JH, Spence Y, Morton CL, Thrasher AJ, Gray JT, Castellino FJ, Tarantal AF, Davidoff AM, Nathwani AC (2012) AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage, Blood 119 (4) pp. 957-966
We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII 1% by 7 weeks. Readministration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth. © 2012 by The American Society of Hematology.
Nathwani AC, Pemberton KD, Bachli EB, Tuddenham EGD, McVey JH (1997) Expression of the human tissue factor gene in endothelial cells is downregulated by sequences in the 5' untranslated region., BLOOD 90 (10) pp. 1295-1295 W B SAUNDERS CO
McVey JH, Lal K, Imanaka Y, Nishimura T, Kemball-Cook G, Bolton-Maggs PHB, Tuddenham EGD (1999) Identification and characterization of the molecular defect in blood coagulation factor Xi T475I, THROMBOSIS AND HAEMOSTASIS pp. 285-285 F K SCHATTAUER VERLAG GMBH
Shibata M, Shima M, Morichika S, McVey J, Tuddenham EGD, Tanaka I, Suzuki H, Nogami K, Minamoto Y, Hato T, Saenko EL, Scandella D, Yoshioka A (2000) An alloantibody recognizing the FVIII A1 domain in a patient with CRM reduced haemophilia a due to deletion of a large portion of the A1 domain DNA sequence, THROMBOSIS AND HAEMOSTASIS 84 (3) pp. 442-448 SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN
Goodeve AC, Reitsma PH, Mcvey JH (2011) Nomenclature of genetic variants in hemostasis, Journal of Thrombosis and Haemostasis 9 (4) pp. 852-855
Takamiya O, Abe S, Yoshioka A, Nakajima K, McVey JH, Tuddenham EG (1995) Factor VIIShinjo: a dysfunctional factor VII variant homozygous for the substitution Gln for Arg at position 79., Haemostasis 25 (3) pp. 89-97
We report a factor VII (FVII) variant, FVIIShinjo, characterized by normal FVII antigen levels and variable procoagulant activity using tissue thromboplastin from different sources. Normal FVII activity is obtained using human placenta thromboplastin but low activity using rabbit or bovine brain thromboplastin. Exons 2-8 and the intron-exon junctions of the FVII genes of the propositus were amplified by PCR from DNA extracted from peripheral white blood cells, and screened by single-strand conformational polymorphism (SSCP) analysis. DNA fragments showing aberrant mobility were cloned and sequenced. We detected a single-point mutation, a homozygous G to A transition at nucleotide position 6,055 in exon 4, which results in the substitution of Arg 79 by Gln in the first EGF-like domain. This mutation results in a loss of a site for the restriction endonuclease MspI. The Msp I digestion pattern of the PCR-amplified exon 3+4 fragments from each member of the family was determined. The Msp I haplotypes were consistent with this G to A transition being associated with reduced FVII activity as detected using thromboplastins from various species. We conclude that the Arg 79 to Gln substitution in the first EGF-like domain of FVII identified in the propositus is responsible for the inherited FVII abnormality in this Japanese family. We postulate that one of the sites of interaction between FVII and tissue thromboplastin includes Arg 79 in the first EGF-like domain of factor VII.
PEMBERTON KD, TUDDENHAM EGD, MCVEY JH (1995) TISSUE LOCALIZATION OF MURINE TISSUE FACTOR (TF) ANTIGEN BY IMMUNOSTAINING WITH A POLYCLONAL CHICKEN ANTI-HUMAN TF ANTIBODY, THROMBOSIS AND HAEMOSTASIS 73 (6) pp. 1180-1180 F K SCHATTAUER VERLAG GMBH
PUGH RE, MCVEY JH, TUDDENHAM EGD, HANCOCK JF (1995) 6 POINT MUTATIONS THAT CAUSE FACTOR-XI DEFICIENCY, BLOOD 85 (6) pp. 1509-1516 W B SAUNDERS CO
Hampshire DJ, Gomez K, Goodeve AC, Kemball-Cook G, Ludlam CA, McVey JH, Oldenburg J, Perkins SJ, Peyvandi F, Rallapalli PM (2014) The EAHAD coagulation factor variant databases, JOURNAL OF THROMBOSIS AND HAEMOSTASIS 12 pp. 105-106 WILEY-BLACKWELL
Maroney SA, Ferrel JP, Pan S, White TA, Simari RD, Mcvey JH, Mast AE (2009) Temporal expression of alternatively spliced forms of tissue factor pathway inhibitor in mice, Journal of Thrombosis and Haemostasis 7 (7) pp. 1106-1113
Background: Mouse tissue factor pathway inhibitor (TFPI) is produced in three alternatively spliced isoforms that differ in domain structure and mechanism for cell surface binding. Tissue expression of TFPI isoforms in mice was characterized as an initial step for identification of their physiological functions. Methods and Results: Sequence homology demonstrates that TFPI± existed over 430Ma while TFPI² and TFPI³ evolved more recently. In situ hybridization studies of heart and lung did not reveal any cells exclusively expressing a single isoform. Although our previous studies have demonstrated that TFPI± mRNA is more prevalent than TFPI² or TFPI³ mRNA in mouse tissues, western blot studies demonstrated that TFPI² is the primary protein isoform produced in adult tissues, while TFPI± is expressed during embryonic development and in placenta. Consistent with TFPI² as the primary isoform produced within adult vascular beds, the TFPI isoform in mouse plasma migrates like TFPI² in SDS-PAGE and mice have a much smaller heparin-releasable pool of plasma TFPI± than humans. Conclusions: The data demonstrate that alternatively spliced isoforms of TFPI are temporally expressed in mouse tissues at the level of protein production. TFPI± and TFPI² are produced in embryonic tissues and in placenta while adult tissues produce almost exclusively TFPI². © 2009 International Society on Thrombosis and Haemostasis.
David D, Saenko EL, Santos IMA, Johnson DJD, Tuddenham EGD, McVey JH, Kemball-Cook G (2001) Stable recombinant expression and characterization of the two haemophilic factor VIII variants C329s (CRM-) and G1948D (CRMr), British Journal of Haematology 113 (3) pp. 604-615
In haemophilia A, the functional defect at the molecular level of most factor VIII (FVIII) missense mutations remains unknown. Site-directed mutagenesis of B domain-deleted FVIII cDNA (FVIIISQ) was used to introduce two mutations associated with severe cross-reacting material (CRM)-negative (FVIII-C329S) or mild/moderate CRM-reduced (FVIII-G1948D) haemophilia A. Wild-type (FVIIISQ-WT) and variant FVIIISQ proteins were successfully expressed after stable transfection in Chinese hamster ovary (CHO) cells, and partially characterized at the intracellular, molecular and functional levels. Reverse transcription polymerase chain reaction analysis confirmed that both transcription and mRNA processing appeared normal in CHO cells transfected with both the wild-type and two variant constructs. In contrast to FVIIISQ-WT, immunofluorescence analysis of both CRM- and CRMr variants showed intracellular FVIII accumulation within the rough endoplasmic reticulum, suggesting secretion defects in transfected CHO cells. Immunoblot analysis of the FVIIISQ variant proteins that were secreted showed that they were expressed as mixed populations of uncleared 170 kDa polypeptides, processed 90 kDa heavy chains and 80 kDa light chains, similar to FVIIISQ-WT. Phenotypic analysis of the B domain-deleted FVIIISQ variants expressed in CHO cells correlated well with the patients' reduced FVIII activity and, in addition, surface plasmon resonance studies demonstrated that both missense mutations were associated with increased rates of A2 domain dissociation following thrombin activation. We conclude that the mutations found are responsible for the haemophilia A phenotype, through intracellular retention and decreased stability of the active cofactor FVIIIa.
Shrivastava S, Ma L, Tham E-L, McVey JH, Chen D, Dorling A (2013) Protease-activated receptor-2 signalling by tissue factor on dendritic cells suppresses antigen-specific CD4(+) T-cell priming, IMMUNOLOGY 139 (2) pp. 219-226 WILEY-BLACKWELL
Gomez K, McVey JH, Tuddenham E (2005) Inhibition of coagulation by macromolecular complexes., Haematologica 90 (11) pp. 1570-1576
The role of vertebrate blood coagulation is to rapidly prevent the loss of body fluids following vascular injury without compromising blood flow through either the uninjured or damaged vessels. To achieve this the coagulation network is initiated and regulated by a complex network of interactions that are under the control of both positive and negative feedback loops that result in controlled fibrin deposition and platelet activation only at the site of injury. Anticoagulant molecules play key roles in preventing inappropriate initiation of coagulation as well as down-regulating thrombin generation at the site of injury. Tissue factor pathway inhibitor (TFPI) inhibits the initiation complex, antithrombin (AT) inhibits the active serine proteases directly, whereas the activated protein C pathway inhibits coagulation by inactivating the cofactors V and VIII. In this review the structure and function of these anticoagulant molecules and their inhibitory complexes is discussed.
Mumford AD, McVey JH, Morse CV, Gomez K, Steen M, Norstrom EA, Tuddenham EGD, Dahlback B, Bolton-Maggs PHB (2003) Factor V I359T: A novel mutation associated with thrombosis and resistance to activated protein C, British Journal of Haematology 123 (3) pp. 496-501
We report a kindred in which two siblings suffered spontaneous venous thromboses in the second decade of life. Further investigation showed reduced coagulation factor V (FV) activity and activated protein C resistance (APCR) ratio but no other thrombophilic abnormalities. The reduction in APCR ratio persisted in a modified APCR assay in which FV activity was normalized between test and control plasmas. Analysis of the FV gene showed that the thrombotic individuals had a complex genotype that included two novel point mutations c.529G>T and c.1250T>C resulting in FV E119X and FV I359T substitutions inherited on different alleles. Individuals in the kindred with FV E119X or FV I359T substitutions alone were asymptomatic. We suggest that the FV I359T substitution confers prothrombotic risk and APCR, but that this is only clinically manifest when co-inherited with the FV E119X allele. The FV I359T substitution creates a new consensus sequence for N-linked glycosylation within the FV heavy chain and we speculate that this abnormal glycosylation may disrupt activated protein C-mediated proteolysis of the variant FV and FVa.
Lalloz MR, McVey JH, Pattinson JK, Tuddenham EG (1991) Haemophilia A diagnosis by analysis of a hypervariable dinucleotide repeat within the factor VIII gene., Lancet 338 (8761) pp. 207-211
The diagnosis of haemophilia A and the identification of carriers has greatly improved with knowledge of the structure of the gene for factor VIII. This has permitted the defect to be tracked in families by the study of restriction fragment length polymorphisms (RFLPs), irrespective of the nature of the molecular defect. However, this approach is time-consuming and the information yielded falls away as more polymorphisms are added. Within the factor VIII gene lies another source of polymorphism, a dinucleotide repeat sequence of varying length known as (CA)n. Conventional mapping localised this (CA)n repeat to intron 13. The polymerase chain reaction, used to examine (CA)n variability in genomic DNA from 25 males and 67 females, revealed eight allelic bands between n = 16 and n = 24. 91% of females were heterozygous for this repeat, and family studies showed X-linked mendelian inheritance with allelic frequencies ranging from 1% to 45%. The intron 13 (CA)n repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. The analysis requires DNA from other family members, and relatives of sporadic cases of haemophilia A are only amenable to exclusion. Nonetheless, this intron 13 (CA)n repeat provides the most highly informative marker so far available for factor VIII gene tracking studies in haemophilia A kindreds and a result can be available within a day.
McVey JH, Lal K, Imanaka Y, Kemball-Cook G, Bolton-Maggs PH, Tuddenham EG (2005) Characterisation of blood coagulation factor XI T475I., Thromb Haemost 93 (6) pp. 1082-1088
PCR-SSCP and DNA sequence analysis of a factor XI (FXI) deficient patient (FXI:C 39 U/dL; FXI:Ag 27 U/dL) identified a C to T transition in exon 12 of the FXI gene (F11 c.1521C>T) that predicts the substitution of Thr475 by Ile (FXI T475I) within the serine protease domain of FXI. This mutation destroys a consensus sequence for N-linked glycosylation, N473-Y-T475, known to be utilized in vivo. The FXIT475I variant was generated by site-directed mutagenesis, together with other variants that could help explain the phenotype, and recombinant FXI variants were expressed in Chinese hamster ovary cells. FXI:Ag expression was analysed by Western blot analysis, ELISA and immunocytochemical staining. Wild-type FXI:Ag was secreted at high levels, however the mutant (FXI T475I) was secreted very poorly. Substitution of Thr475 by Ala, Pro, Lys or Arg (all of which abolish the glycosylation consensus sequence) also severely reduced the level of secreted FXI:Ag suggesting that glycosylation at Asn473 is required for folding or secretion. Concordant with this hypothesis the conservative substitution of Thr475 by Ser (which preserves the glycosylation consensus sequence) had no effect on FXI secretion. Thr/Ser475 is highly conserved in serine protease domains but the glycosylation site (Asn473) is not. Surprisingly, substitution of Asn473 by Ala (which removes the N-linked glycosylation site) had no effect on the levels of FXI:Ag secreted. In conclusion, although the FXI-T475I mutation destroys an N-linked glycosylation consensus sequence, the cause of failure to secrete FXI is not the loss of a glycosylation site but rather a direct effect of the substitution of this highly conserved residue.
Connolly DJ, Cotterill LA, Hederer RA, Thorpe CJ, Travers PJ, McVey JH, Dyson J, Robinson PJ (1993) A cDNA clone encoding the mouse Qa-1a histocompatibility antigen and proposed structure of the putative peptide binding site., J Immunol 151 (11) pp. 6089-6098
We have isolated a cDNA clone which encodes the Qa-1a histocompatibility Ag from a library prepared from Con A-activated B10.BR mouse spleen cells. The clone encodes a protein of 322 amino acids with three potential N-glycosylation sites. The coding sequence shows strongest similarity with that of the T23d gene of DBA/2 mice which encodes the Qa-1b molecule. Molecular modeling of the putative peptide-combining site indicates most of the differences between Qa-1a and Qa-1b are located peripheral to the binding cleft, with only two amino acid substitutions, at positions 9 and 24, which might affect peptide binding. Many features of the Qa-1 binding cleft are also conserved in the rat RTBM.1 and in human HLA-E molecules. This suggests that all of these molecules may associate with structurally similar peptides.
Greig JA, Buckley SMK, Waddington SN, Parker AL, Bhella D, Pink R, Rahim AA, Morita T, Nicklin SA, McVey JH, Baker AH (2009) Influence of coagulation factor X on in vitro and in vivo gene delivery by adenovirus (Ad) 5, Ad35, and chimeric Ad5/Ad35 vectors, Molecular Therapy 17 (10) pp. 1683-1691
The binding of coagulation factor X (FX) to the hexon of adenovirus (Ad) 5 is pivotal for hepatocyte transduction. However, vectors based on Ad35, a subspecies B Ad, are in development for cancer gene therapy, as Ad35 utilizes CD46 (which is upregulated in many cancers) for transduction. We investigated whether interaction of Ad35 with FX influenced vector tropism using Ad5, Ad35, and Ad5/Ad35 chimeras: Ad5/ fiber(f)35, Ad5/penton(p)35/f35, and Ad35/f5. Surface plasmon resonance (SPR) revealed that Ad35 and Ad35/f5 bound FX with approximately tenfold lower affinities than Ad5 hexon-containing viruses, and electron cryomicroscopy (cryo-EM) demonstrated a direct Ad35 hexon:FX interaction. The presence of physiological levels of FX significantly inhibited transduction of vectors containing Ad35 fibers (Ad5/f35, Ad5/ p35/f35, and Ad35) in CD46-positive cells. Vectors were intravenously administered to CD46 transgenic mice in the presence and absence of FX-binding protein (X-bp), resulting in reduced liver accumulation for all vectors. Moreover, Ad5/f35 and Ad5/p35/f35 efficiently accumulated in the lung, whereas Ad5 demonstrated poor lung targeting. Additionally, X-bp significantly reduced lung genome accumulation for Ad5/f35 and Ad5/ p35/f35, whereas Ad35 was significantly enhanced. In summary, vectors based on the full Ad35 serotype will be useful vectors for selective gene transfer via CD46 due to a weaker FX interaction compared to Ad5.
Neerman-Arbez M, Johnson KM, Morris MA, McVey JH, Peyvandi F, Nichols WC, Ginsburg D, Rossier C, Antonarakis SE, Tuddenham EGD (1999) Molecular analysis of the ERGIC-53 gene in 35 families with combined factor V-factor VIII deficiency, Blood 93 (7) pp. 2253-2260
Combined factor V-factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder in which the levels of both coagulation factors V and VIII are diminished. The F5F8D locus was previously mapped to a 1-cM interval on chromosome 18q21. Mutations in a candidate gene in this region, ERGIC-53, were recently found to be associated with the coagulation defect in nine Jewish families. We performed single-strand conformation and sequence analysis of the ERGIC-53 gene in 35 F5F8D families of different ethnic origins. We identified 13 distinct mutations accounting for 52 of 70 mutant alleles. These were 3 splice site mutations, 6 insertions and deletions resulting in translational frameshifts, 3 nonsense codons, and elimination of the translation initiation codon. These mutations are predicted to result in synthesis of either a truncated protein product or no protein at all. This study revealed that F5F8D shows extensive allelic heterogeneity and all ERGIC-53 mutations resulting in F5F8D are 'null.' Approximately 26% of the mutations have not been identified, suggesting that lesions in regulatory elements or severe abnormalities within the introns may be responsible for the disease in these individuals. In two such families, ERGIC-53 protein was detectable at normal levels in patients' lymphocytes, raising the further possibility of defects at other genetic loci.
McVey JH (2010) O tissue factor, where art thou?, Blood 116 (5) pp. 676-677
In this issue of Blood, Pawlinski and colleagues identify myeloid cells and an unidentified nonhematopoietic cell(s) as the source of TF responsible for intravascular coagulation in a mouse model of endotoxemia, excluding a role for EC, VSMC, and platelet cell TF expression.1.
OBRIEN DP, GALE K, ANDERSON JS, MCVEY JH, MEADE T, MILLER G, TUDDENHAM EGD (1991) FVII-304 GLN - A DYSFUNCTIONAL FVII MOLECULE WITH REDUCED AFFINITY FOR TISSUE FACTOR, THROMBOSIS AND HAEMOSTASIS 65 (6) pp. 769-769 F K SCHATTAUER VERLAG GMBH
Johnson KM, McVey JH (1999) Molecular Analysis in Factor XI Deficiency., 31 pp. 211-221
Factor XI (FXI) is the zymogen precursor of an active serine protease that participates in the contact phase of coagulation. Synthesized in the liver, it circulates in the plasma in a noncovalent complex with high molecular weight kininogen (1) at a normal concentration of 5 ¼g/mL. (For clinical purposes, the normal range is defined as 50-150 U/dL) (2). FXI circulates as a homodimeric glycoprotein with a mass of 160 kDa.
Chen D, Riesbeck K, McVey JH, Kemball-Cook G, Tuddenham EG, Lechler RI, Dorling A (1999) Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins., Transplantation 68 (6) pp. 832-839
BACKGROUND: Thrombotic vascular occlusion resulting in infarction occurs during hyperacute rejection of allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. A similar process is also found in disorders of diverse etiology including atherosclerosis, vasculitis, and disseminated intravascular coagulation. METHODS: We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue factor pathway inhibitor and the leech anticoagulant hirudin and demonstrated their functional efficacy in vitro. These constructs have now been modified by the addition of a P-selectin sequence to the cytoplasmic tail to localize them in Weibel-Palade bodies. They have been transfected into Weibel-Palade body-positive endothelial cells isolated from the inferior vena cava of normal pigs. RESULTS: In resting endothelial cells, fusion protein expression colocalized with P-selectin and was confined to Weibel-Palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activation with phorbol ester the anticoagulant proteins were rapidly relocated to the cell surface where they specifically inhibited the clotting of human plasma. CONCLUSIONS: Novel anticoagulant molecules may prove useful therapeutic agents for gene therapy in thrombotic disease and postangioplasty or for transgenic expression in animals whose organs may be used for clinical xenotransplantation. Expression in vascular endothelial cells may be regulated by inclusion of P-selectin cytoplasmic sequence, to restrict cell surface expression to activated endothelium.
Elliott JI, Mumford AD, Albrecht C, Collins PW, Giddings JC, Higgins CF, Tuddenham EGD, McVey JH (2004) Characterisation of lymphocyte responses to Ca2+ in Scott syndrome, Thrombosis and Haemostasis 91 (2) pp. 412-415
Imanaka Y, Lal K, Nishimura T, Bolton-Maggs PH, Tuddenham EG, McVey JH (1995) Identification of two novel mutations in non-Jewish factor XI deficiency., Br J Haematol 90 (4) pp. 916-920
We have studied two heterozygous unrelated CRM- non-Jewish FXI-deficient patients. Neither of the patients carries a previously described mutation. Their FXI genes were screened by SSCP analysis following PCR amplification of each exon and the flanking intronic sequences. DNA fragments showing aberrant mobility were cloned and sequenced. The following mutations were identified: in case 1, a T to G transition in exon 12 results in the substitution of Phe-442 by Val (FXI-F442V); in case 2 a C to A transition in exon 5 results in the substitution of Cys-128 by a nonsense codon (FXI-C128X). The missense mutation results in a substitution within the protease domain of FXI. Molecular modelling locates this residue in a structurally conserved region of the protease domain and the amino acid substitution may therefore interfere with either chain folding and subsequent secretion or the stability of the protein in plasma. We conclude that the mutations which we have identified are responsible for the inherited abnormality in these patients.
O'Brien DP, Kemball-Cook G, Hutchinson AM, Martin DM, Johnson DJ, Byfield PG, Takamiya O, Tuddenham EG, McVey JH (1994) Surface plasmon resonance studies of the interaction between factor VII and tissue factor. Demonstration of defective tissue factor binding in a variant FVII molecule (FVII-R79Q)., Biochemistry 33 (47) pp. 14162-14169
The blood coagulation cascade is initiated when vessel injury allows factor VII (FVII) to form a complex with tissue factor (TF). Complete deficiency of FVII causes a lethal bleeding diathesis, but individuals with moderately reduced FVII levels are often asymptomatic. Some of these individuals have circulating partially functional FVII, as a result of point missense mutations in critical parts of the molecule. One such mutation has been reported at position 79 in the first epidermal growth factor-like (EGF) domain of FVII, where an arginine residue has been replaced by glutamine. There is controversy as to whether or not this mutation reduces the affinity of the FVII/TF interaction compared to wild-type FVII. To address this problem, we have expressed recombinant FVII-R79Q and subjected it to detailed biochemical analysis. One-stage FVII:C assays show the variant FVII to have reduced activity with respect to the wild type. Rates of autoactivation and activation by FXa to the two-chain molecule were identical for wild-type and variant FVII. The Vmax for FX activation was lower for the mutant as measured using an amidolytic assay for FX activity. In contrast, the Km for FX was lower for the variant than the wild-type molecule. Peptidyl substrate hydrolysis was virtually identical for both variant and normal FVIIa in the presence and absence of TF. The variant has reduced affinity for TF as measured by surface plasmon resonance. FVII-R79Q has an association rate constant (kassoc) one-fifth of that of normal FVII, but a similar kdiss, resulting in a decrease in the affinity of the enzyme for its cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)
Ward NJ, Buckley SMK, Waddington SN, VandenDriessche T, Chuah MKL, Nathwani AC, McIntosh J, Tuddenham EGD, Kinnon C, Thrasher AJ, McVey JH (2011) Codon optimization of human factor VIII cDNAs leads to high-level expression, Blood 117 (3) pp. 798-807
Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A. © 2011 by The American Society of Hematology.
Graham A, Papalopulu N, Lorimer J, McVey JH, Tuddenham EG, Krumlauf R (1988) Characterization of a murine homeo box gene, Hox-2.6, related to the Drosophila Deformed gene., Genes Dev 2 (11) pp. 1424-1438
The Hox-2 locus on chromosome 11 represents one of the major clusters of homeo-box-containing genes in the mouse. We have identified two new members (Hox-2.6 and Hox-2.7), which form part of this cluster of seven linked genes, and it appears that the Hox-2 locus is related by duplication and divergence to at least one other mouse homeo box cluster, Hox-1. The Hox-2.6 gene encodes a predicted protein of 250 amino acids, which displays extensive similarity in multiple regions to certain mouse, human, Xenopus, and zebra fish homeo domain proteins. The Drosophila Deformed (Dfd) gene also shares these same regions of similarity, and based on this sequence conservation, we suggest that Hox-2.6 forms part of a vertebrate 'Dfd-like' family. Hox-2.6 is expressed in fetal and adult tissues and is modulated during the differentiation of F9 teratocarcinoma stem cells. In situ hybridization analysis of mouse embryos shows that the Hox-2.6 is expressed in ectodermal derivatives: spinal cord, hindbrain, dorsal root ganglia, and the Xth cranial ganglia. In the central nervous system, expression is observed in the most posterior parts of the spinal cord, with the anterior limit residing in a region of the hindbrain and no expression in the mid- or forebrain. In mesodermal structures, Hox-2.6 is expressed in the kidney, the mesenchyme of the stomach and lung, and the longitudinal muscle layer of the gut. Expression has not been observed in derivatives of embryonic endoderm. The patterns of Hox-2.6 expression in both mesoderm and ectoderm are spatially restricted and may reflect a role for the gene in the response to or establishment of positional cues in the embryo.
Greig JA, McVey JH, Waddington SN, Parker AL, Buckley SMK, Havenga MJE, Nicklin SA, Baker AH (2008) Factor X enhances binding and transduction of human cancer cell lines by adenovirus (Ad) serotype 5 vectors but not by Ad35, HUMAN GENE THERAPY 19 (4) pp. 398-398 MARY ANN LIEBERT INC
Chen D, Xia M, Hayford C, Tham ELL, Semik V, Hurst S, Chen Y, Tam HH, Pan J, Wang Y, Tan X, Lan HY, Shen H, Kakkar VV, Xu Q, McVey JH, Dorling A (2015) Expression of human tissue factor pathway inhibitor on vascular smooth muscle cells inhibits secretion of macrophage migration inhibitory factor and attenuates atherosclerosis in ApoE-/- mice.,Circulation 131 (15) pp. 1350-1360
BACKGROUND: Tissue factor (TF) and coagulation proteases are involved in promoting atherosclerosis, but the molecular and cellular bases for their involvement are unknown. METHODS AND RESULTS: We generated a new strain (ApX4) of apolipoprotein E-deficient mice expressing a membrane-tethered human tissue factor pathway inhibitor fusion protein on smooth muscle actin-positive cells, including vascular smooth muscle cells (SMCs). ApX4 mice developed little atherosclerosis on either a normal chow or high-fat diet. Lipid levels were similar to those in parental ApoE(-/-) mice, and there was no detectable difference in systemic (circulating) tissue factor pathway inhibitor levels or activity. The small lipid-rich lesions that developed had markedly reduced leukocyte infiltrates, and in contrast to ApoE(-/-) mice, SMCs did not express macrophage migratory inhibitory factor (MIF), including at sites distant from atheromatous lesions. Low levels of circulating MIF in ApX4 mice normalized to levels seen in ApoE(-/-) mice after injection of an inhibitory anti-human tissue factor pathway inhibitor antibody, which also led to MIF expression by tissue factor-positive medial SMCs. MIF production by SMCs in ApoE(-/-) mice in vitro and in vivo was shown to be dependent on tissue factor and protease-activated receptor signaling, which were inhibited in ApX4 mice. CONCLUSIONS: Our data indicate that tissue factor plays a hitherto unreported role in the generation of MIF by SMCs in atherosclerosis-prone ApoE(-/-) mice, inhibition of which significantly prevents the development of atherosclerosis, through inhibition of leukocyte recruitment. These data significantly enhance our understanding of the pathophysiology of this important pathology and suggest new potential translational strategies to prevent atheroma formation.
Owen F, Poulter M, Shah T, Collinge J, Lofthouse R, Baker H, Ridley R, McVey J, Crow TJ (1990) An in-frame insertion in the prion protein gene in familial Creutzfeldt-Jakob disease., Brain Res Mol Brain Res 7 (3) pp. 273-276
In a pedigree with Creutzfeldt-Jakob disease we identified a 144-bp insertion in the open reading frame of the prion protein (PrP) gene. The insertion is in-frame and codes for 6 extra uninterrupted octapeptide repeats in addition to the 5 that are normally present in the N-terminal region of the protein. The possibility that this mutation may prove relevant to elucidating the mechanism of horizontal transmission of the spongiform encephalopathies is discussed.
Chen DX, Giannopoulos K, Shiels PG, Webster Z, McVey JH, Kemball-Cook G, Tuddenham E, Moore M, Lechler R, Dorling A (2004) Inhibition of intravascular thrombosis in murine endotoxemia by targeted expression of hirudin and tissue factor pathway inhibitor analogs to activated endothelium, BLOOD 104 (5) pp. 1344-1349 AMER SOC HEMATOLOGY
David D, Schwaab R, Santos AIM, McVey J, Tuddenham EGD (1998) Impact of premature stop codons on factor VIII and recombinant re-FVIIISQ pre-mRNA processing, correlation with inhibitor formation, EUROPEAN JOURNAL OF HUMAN GENETICS 6 pp. 125-125 STOCKTON PRESS
Hamid C, McVey J, Riddell A, Gomez K, Slatter D, Chowdary P (2015) Prothrombin complex concentrate can increase thrombin generation in the presence of low platelet number, JOURNAL OF THROMBOSIS AND HAEMOSTASIS 13 pp. 880-880 WILEY-BLACKWELL
Pattinson JK, McVey JH, Boon M, Ajani A, Tuddenham EG (1990) CRM+ haemophilia A due to a missense mutation (372----Cys) at the internal heavy chain thrombin cleavage site., Br J Haematol 75 (1) pp. 73-77
We have used the polymerase chain reaction (PCR) and differential oligonucleotide melting to screen for mutations in selected CpG dinucleotides in the factor VIII genes of haemophilia A patients. By this means we have identified and confirmed by sequencing a novel point mutation in codon 372 (CGC) of the factor VIII gene of a moderately severe CRM+ haemophiliac. The first C of this codon has been substituted by T resulting in the non-conservative substitution of cysteine for arginine at an essential thrombin cleavage site in factor VIII. Analysis of three intragenic restriction fragment length polymorphisms was uninformative in the patient's family. However, DNA analysis for the specific mutation shows one sister and the patient's mother to be carriers, and the other sister to be normal. This DNA analysis confirmed the results of phenotype analysis by factor VIII coagulant to von Willebrand factor antigen ratios for the females at risk. The two carrier females had low factor VIII coagulant activity and excess VIII antigen as predicted but the non-carrier sister also had anomalously high VIII antigen in her plasma. When feasible, mutation specific DNA analysis is able to resolve the difficulties posed by variable phenotype data and unknown level of mutation in sporadic haemophilia A.
McVey JH, Michaelides K, Hansen LP, Ferguson-Smith M, Tilghman S, Krumlauf R, Tuddenham EG (1993) A G-->A substitution in an HNF I binding site in the human alpha-fetoprotein gene is associated with hereditary persistence of alpha-fetoprotein (HPAFP)., Hum Mol Genet 2 (4) pp. 379-384
A family displaying hereditary persistence of alpha-fetoprotein (HPAFP) in adult life was detected in an antenatal screening programme for spina bifida. RFLP linkage analysis shows that the trait is linked with the albumin-AFP locus. The molecular mechanism responsible for the post-natal repression of the AFP gene is unknown. We wished to determine the molecular mechanism underlying HPAFP in this family. Sequence analysis of the 5'-flanking sequences of their gene revealed a GA substitution at position -119 associated with the trait. This substitution occurs in a potential HNF I binding site, and increases the similarity of the sequence to a consensus HNF I recognition site. In a competitive gel retardation assay the mutant sequence binds HNF I alpha more tightly than the wild type sequence. Furthermore, 5'-flanking sequences of the human AFP gene containing the G-->A substitution direct a higher level of CAT expression in transfected human hepatoma cells than the wild type sequences. We conclude that the G-->A substitution at position -119 of the AFP gene is the mutation causing HPAFP in this family. These results highlight the importance of this HNF I binding site in the developmental regulation of the AFP gene.
Millar D, Kemball-Cook G, McVey J, Tuddenham E, Mumford A, Attock G, Reverter J, Lanir N, Parapia L, Reynaud J, Meili E, Von Felton A, Martinowitz U, Prangnell D, Krawczak M, Cooper D (2000) Molecular analysis of the genotype-phenotype relationship in factor VII deficiency, Human Genetics 107 (4) pp. 327-342
Factor VII (FVII) deficiency is a rare haemorrhagic condition, normally inherited as an autosomal recessive trait, in which clinical presentation is highly variable and correlates poorly with laboratory phenotype. The FVII (F7) gene was sequenced in 48 unrelated individuals with FVII deficiency, yielding a total of 23 novel lesions including 15 missense mutations, 2 micro-deletions, 5 splice junction mutations and a single base-pair substitution in the 5' untranslated region. Family studies were performed in order to distinguish the contributions of individual mutant F7 alleles to the clinical and laboratory phenotypes. Specific missense mutations were evaluated by molecular modelling in the context of the FVIIa-tissue factor crystal structure. Single base-pair substitutions in splice sites and the 5' untranslated region were studied by in vitro splicing assay and luciferase reporter gene assay, respectively. All probands were also typed for four previously reported F7 polymorphisms. In the majority of cases of FVII deficiency studied here, consideration of both mutational and polymorphism data permitted the derivation of plausible explanations for the FVII activity and antigen levels measured in the laboratory. Inter-familial variation in FVII activity and the antigen levels of heterozygous relatives of probands was found to be significantly higher than intra-familial variation, consistent with the view that the nature of the F7 gene lesion(s) segregating in a given family is a prime determinant of laboratory phenotype. Although no relationship could be discerned between laboratory phenotype and polymorphism genotype, the frequencies of the A2 and M2 polymorphic alleles were significantly higher in the FVII-deficient individuals tested than in controls. This suggests that the presence of these alleles may have served to increase the likelihood of pathological F7 gene lesions coming to clinical attention.
Rahim AA, Wong AM, Ahmadi S, Hoefer K, Buckley SMK, Hughes DA, Nathwani AN, Baker AH, McVey JH, Cooper JD, Waddington SN (2012) In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression, Gene Therapy 19 (9) pp. 936-946
The efficient delivery of genetic material to the developing fetal brain represents a powerful research tool and a means to supply therapy in a number of neonatal lethal neurological disorders. In this study, we have delivered vectors based upon adenovirus serotype 5 (Ad5) and adeno-associated virus (AAV) pseudotypes 2/5, 2/8 and 2/9 expressing green fluorescent protein to the E16 fetal mouse brain. One month post injection, widespread caudal to rostral transduction of neural cells was observed. In discrete areas of the brain these vectors produced differential transduction patterns. AAV2/8 and 2/9 produced the most extensive gene delivery and had similar transduction profiles. All AAV pseudotypes preferentially transduced neurons whereas Ad5 transduced both neurons and glial cells. None of the vectors elicited any significant microglia-mediated immune response when compared with control uninjected mice. Whole-body imaging and immunohistological evaluation of brains 9 months post injection revealed long-term expression using these non-integrating vectors. These data will be useful in targeting genetic material to discrete or widespread areas of the fetal brain with the purpose of devising therapies for early neonatal lethal neurodegenerative disease and for studying brain development. © 2012 Macmillan Publishers Limited. All rights reserved.
Melchionna T, Karegli J, Farrar CA, McVey JH, Dorling A, Sacks SH, Smith RAG (2010) Integrating cytotopic strategy in transplantation: (1) Design of locally-acting coagulation inhibitors for use with anti-complement therapeutics, MOLECULAR IMMUNOLOGY 47 (13) pp. 2203-2203 PERGAMON-ELSEVIER SCIENCE LTD
Davidson CJ, Tuddenham EG, McVey JH (2003) 450 million years of hemostasis., J Thromb Haemost 1 (7) pp. 1487-1494
In mammalian blood coagulation, five proteases (factor VII [FVII]; factor IX [FIX]; factor X [FX]; protein C [PC] and prothrombin [PT]) act with five cofactors (tissue factor [TF]; factor V [FV]; factor VIII [FVIII]; thrombomodulin and protein S) to control the generation of fibrin. Biochemical evidence, molecular cloning data and comparative sequence analysis support the existence of all components of this network in all jawed vertebrates, and strongly suggest that it evolved before the divergence of teleosts over 430 million years ago. Phylogenetic analysis of the amino acid sequences of the Gla-EGF1-EGF2-SP domain serine proteases (FVII, FIX, FX, PC) and the A domain-containing cofactors (FV and FVIII) strongly supports the evolution of the blood coagulation network through two rounds of gene duplication, and supports the hypothesis that vertebrate evolution benefited from two global genome duplications. The jawless vertebrates (hagfish and lamprey) that diverged over 450 million years ago have a blood coagulation network involving TF, PT and fibrinogen. Preliminary evidence indicates that they may have a smaller complement of Gla-EGF1-EGF2-SP domain proteins, suggesting the existence of a 'primitive' coagulation system in jawless vertebrates.
David D, Santos AIM, Johnson K, Tuddenham EGD, McVey JH (1999) mRNA processing, translation and protein secretion from factor VIII genes containing premature stop codons: No correlation with inhibitor development, THROMBOSIS AND HAEMOSTASIS pp. 238-239 F K SCHATTAUER VERLAG GMBH
Takacs K, Du Roure C, Nabarro S, Dillon N, McVey JH, Webster Z, MacNeil A, Bartok L, Higgins C, Gray D, Merkenschlager M, Fisher AG (2004) The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 (46) pp. 16298-16303 NATL ACAD SCIENCES
Di Matteo M, Samara-Kuko E, Ward NJ, Waddingon SN, McVey JH, Chuah MKL, Vanden Driessche T (2015) Erratum: Hyperactive PiggyBac Transposons for Sustained and Robust Liver-targeted Gene Therapy (Molecular Therapy (2014) 22 (1614-1624) DOI:10.1038/mt.2014.131), Molecular Therapy 23 (10)
Smith RAG, Karegli J, Melchionna T, Farrar CA, McVey JH, Dorling A, Sacks SH (2009) Towards an integrated cytotopic strategy for graft modification in transplantation-Complement and coagulation, MOLECULAR IMMUNOLOGY 46 (14) pp. 2833-2833 PERGAMON-ELSEVIER SCIENCE LTD
Griffith ME, Turner AN, McVey J, Pusey CD (1997) Expression of intracellular and secreted recombinant proteinase 3 recognized by ANCA, KIDNEY INTERNATIONAL 52 (1) pp. 277-277 BLACKWELL SCIENCE INC
Lalloz MR, Schwaab R, McVey JH, Michaelides K, Tuddenham EG (1994) Haemophilia A diagnosis by simultaneous analysis of two variable dinucleotide tandem repeats within the factor VIII gene., Br J Haematol 86 (4) pp. 804-809
Haemophilia A is a bleeding disorder caused by defects in the gene coding for the co-factor, factor VIII (FVIII). The few available intragenic restriction fragment length polymorphisms (RFLPs) currently used in carrier detection and prenatal diagnosis of haemophilia A are informative in only about 65% of cases. We earlier reported a multi-allelic dinucleotide tandem repeat, (CA)n, specific to intron 13, which remains the single most informative marker within the FVIII gene. We here report a second informative dinucleotide repeat of the form (GT)n (AG)n, located to intron 22 of the FVIII gene. The polymerase chain reaction (PCR) method was used to examine the variability of the repeat in 60 individuals (75 X-chromosomes) and revealed four alleles. The calculated heterozygosity rate is 45%, and family studies showed X-linked mendelian inheritance. The intron 22 dinucleotide repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. We now show that by simultaneous amplification of the intron 13 and 22 repeats using PCR all alleles for both markers are detectable on a single polyacrylamide gel. The information thus obtained from a single multiplexed analysis is greater than from multiple RFLP analyses. Hence, rapid haplotype determination by simultaneous amplification and detection of two intragenic dinucleotide repeats should supersede less informative RFLP analysis.
McVey JH, Gomez K, Tuddenham EGD (2010) Normal Haemostasis,In: Green AR, Hoffbrand AV, Catovsky D, Tuddenham EGD (eds.), Postgraduate Haematology (39) 39 pp. 746-771
Tuddenham EG, McVey JH (1998) The genetic basis of inhibitor development in haemophilia A., Haemophilia 4 (4) pp. 543-545
Inhibitor development is now the main complication of replacement therapy in haemophilia A. Given that most severely affected patients make no detectable factor VIII, it is perhaps surprising that only approximately 30% actually mount an immune response to factor VIII as a foreign antigen. Those that do mostly have major factor VIII gene lesions. The association of HLA genotype with inhibitors in patients with identical mutations is weak. Environmental factors may be more important than genetic in antibody response to factor VIII.
White K, Buening H, McVey J, Murphy G, Work L, Hallek M, Nicklin S, Baker A (2007) Development and characterisation of viral vectors targeted to atherosclerotic plaques, HEART 93 pp. A99-A99 B M J PUBLISHING GROUP
White K, Büning H, Kritz A, Janicki H, McVey J, Perabo L, Murphy G, Odenthal M, Work LM, Hallek M, Nicklin SA, Baker AH (2008) Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions, Gene Therapy 15 (6) pp. 443-451
Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.
Crossman DC, Carr DP, Tuddenham EG, Pearson JD, McVey JH (1990) The regulation of tissue factor mRNA in human endothelial cells in response to endotoxin or phorbol ester., J Biol Chem 265 (17) pp. 9782-9787
Tissue factor (TF) is the membrane-bound glycoprotein whose cofactor activity with factor VIIa causes activation of the extrinsic pathway of coagulation. The transition of endothelium to a procoagulant state by agents such as bacterial lipopolysaccharide (LPS) is the result of TF expression by these cells. The mechanism of TF induction in human umbilical vein endothelial cells (HUVEC) was investigated in response to LPS and phorbol 12-myristate 13-O-acetate (PMA). Northern blot analysis of total RNA from HUVEC showed a rapid rise in TF mRNA levels which was maximal at 2 h and had fallen to low levels by 6 h following both LPS (10 micrograms/ml) and PMA (10 ng/ml) stimulation. Nuclear-run on experiments showed at most a 2-fold increase in transcription of the TF gene following LPS stimulation but a 10-fold increase following PMA stimulation. In addition 24-h pre-incubation with PMA desensitized HUVEC to further PMA exposure, but caused no alteration in the response to LPS. Cycloheximide (10 micrograms/ml) alone caused induction of TF mRNA. Treatment of cells previously exposed to LPS for 1 or 4 h with actinomycin D indicated a 12-fold difference in the TF mRNA half-life. Therefore the rapid accumulation of TF mRNA in HUVEC stimulated by LPS is largely a result of an increase in mRNA stability rather than an increased rate of transcription of the gene.
Kritz AB, Nicol CG, Dishart KL, Nelson R, Holbeck S, Von Seggern DJ, Work LM, McVey JH, Nicklin SA, Baker AH (2007) Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop,MOLECULAR THERAPY 15 (4) pp. 741-749
NATURE PUBLISHING GROUP
Waddington SN, Parker AL, Havenga M, Nicklin SA, Buckley SMK, McVey JH, Baker AH (2007) Targeting of adenovirus serotype 5 (Ad5) and 5/47 pseudotyped vectors in vivo: Fundamental involvement of coagulation factors and redundancy of CAR binding by Ad5, Journal of Virology 81 (17) pp. 9568-9571
Vitamin K-dependent coagulation factors can promote adenoviral cell transduction in vitro. In vivo, warfarin pretreatment ablates liver targeting of an adenovirus serotype 5 (Ad5) vector deleted of CAR binding capability. Here, we assess in vivo transduction and biodistribution of Ad5 vectors with nonmodified fibers (Ad5) and a serotype 47 fiber-pseudotyped Ad5 (Ad5/47; subgroup D) virus following intravascular injection. Warfarin reduced liver transduction by both viruses. However, no impact on early liver virus accumulation was observed, suggesting no effect on Kupffer cell interactions. Hence, coagulation factors play a pivotal role in selectively mediating liver hepatocyte transduction of Ad5 and Ad5/47 vectors. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Kemball-Cook G, Garner I, Imanaka Y, Nishimura T, O'Brien DP, Tuddenham EG, McVey JH (1994) High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector., Gene 139 (2) pp. 275-279
Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.
OBJECTIVES.: Intravascular thrombosis remains a major barrier to successful pig-to-primate xenotransplantation. However, the precise factors initiating thrombosis are unknown. In this study, we investigated the contribution of recipient platelets and monocytes. METHODS.: Primary pig aortic endothelial cells (PAECs) were incubated with combinations of fresh or heat-inactivated human plasma, platelets, or monocytes, after which they were separated and analyzed individually by flow cytometry for tissue factor (TF) expression and for their ability to clot recalcified normal or factor-VII-deficient plasma. RESULTS.: Procoagulant porcine TF was induced in PAECs only by fresh human plasma, and not by heat-inactivated plasma, platelets, or monocytes. In contrast, procoagulant human TF was induced on platelets and monocytes after incubation with PAEC, irrespective of whether the plasma was present or not. In addition, human platelets caused the shedding of procoagulant TF-expressing aggregates from PAEC. CONCLUSIONS.: This work defines a cell-based in vitro assay system to address complex interactions among PAECs, human platelets, and monocytes. The induction of procoagulant TF on PAECs by fresh human plasma was most likely dependent on xenoreactive natural antibody and complement present in fresh human plasma. In contrast, the shedding of procoagulant platelet-PAEC aggregates, induced by human platelets, and the induction of procoagulant TF on human platelets and monocytes by PAEC, occurred independently of these factors. These results suggest that different mechanisms may contribute to the initiation of thrombosis after xenotransplantation, some of which may not be influenced by the further manipulation of the immune response against pig xenografts.
Michaelides K, McVey JH, Lal K, Tuddenham EGD (1999) The cloning and characterisation of the mouse platelet membrane glycoprotein IX gene, THROMBOSIS AND HAEMOSTASIS pp. 47-48 F K SCHATTAUER VERLAG GMBH
Houston P, Dickson MC, Ludbrook V, White B, Schwachtgen JL, McVey JH, Mackman N, Reese JM, Gorman DG, Campbell C, Braddock M (1999) Fluid shear stress induction of the tissue factor promoter in vitro and in vivo is mediated by Egr-1, Arteriosclerosis, Thrombosis, and Vascular Biology 19 (2) pp. 281-289
Hemodynamic forces such as fluid shear stress have been shown to modulate the activity of an expanding family of genes involved in vessel wall homeostasis and the pathogenesis of vascular disease. We have investigated the effect of shear stress on tissue factor (TF) gene expression in human endothelial cells (ECs) and in a rat arterial model of occlusion. As measured by reverse transcriptase polymerase chain reaction, exposure of ECs to 1.5 N/m2 shear stress resulted in a time-dependent induction of endogenous TF transcripts of over 5-fold. Transient transfection of TF promoter mutants into cultured ECs suggests the involvement of the transcription factor Egr-1 in mediating the response of the TF promoter to shear stress. To address the importance of flow induction of Egr-1 in vivo, we have established a flow- restricted rat arterial model and determined the level of expressed Egr-1 and TF at the site of restricted flow using immunohistochemistry. We report an increase in the level of Egr-1 and TF protein in ECs expressed at the site of restricted flow. Elevated expression of Egr-1 and TF is restricted to a highly localized area, as evidenced by the fact that no significant increase in level can be detected at arterial sites distal to the site of occlusion. These findings suggest a direct role for Egr-1 in flow-mediated induction of TF and further substantiate the importance of shear stress as a modulator of vascular endothelial gene function in vivo.
Waddington SN, Mcvey JH, Bhella D, Parker AL, Barker K, Atoda K, Pink R, Buckley SMK, Greig JA, Denby L, Custers J, Morita T, Francischetti IMB, Monteiro RQ, Barouch DH, van Rooijen N, Napoli C, Havenga M, Nicklin SA, Baker AH (2008) A critical role for the adenovirus serotype 5 hexon in liver gene transfer, HUMAN GENE THERAPY 19 (4) pp. 409-409 MARY ANN LIEBERT INC
Mumford AD, Chen D, Dorling A, Kemball-Cook G, McVey JH (2005) Generation of a polyclonal rabbit anti-mouse tissue factor antibody by nucleic acid immunisation, Thrombosis and Haemostasis 93 (1) pp. 160-164
Tissue factor (TF) the cellular receptor and cofactor for factor VII, initiates coagulation and has also been implicated in several coagulation-independent functions, including inflammation, angiogenesis and tumour metastasis. Investigations of TF expression in mouse models of these processes has been limited by the availability of antibodies that specifically recognise mouse TF. We have generated a rabbit polyclonal antibody to mTF by DNA immunisation. This has yielded an antiserum that recognises native mTF in immunohistochemical and flow cytometric analyses. Furthermore, the antiserum is inhibitory in coagulation assays. This antiserum will be a valuable investigative tool in the analysis of mTF expression. © 2005 Schattauer GmbH, Stuttgart.
Chen D, McVey JH, Dorling A (2013) Enhanced effect of inhibition of thrombin on endothelium in murine endotoxaemia: Specific inhibition of thrombocytopenia, Thrombosis Research 132 (6) pp. 750-756
Introduction In systemic endotoxaemia, bacterial lipopolysaccharide causes the rapid expression of tissue factor (TF) and disseminated intravascular coagulation and in animal models, anticoagulants limit pathology and promote survival. Recent studies have emphasised the importance of TF expressed by mononuclear cells for initiating thrombin generation during endotoxaemia and suggested that endothelial cell TF is of little relevance. However, the precise importance of endothelium for intravascular thrombin generation has not been established. In this study, we compared the effect of equivalent levels of hirudin tethered to either endothelium or platelets and monocytes. Materials and Methods CD31-Hir-Tg mice express a vesicle-targeted, membrane-tethered hirudin fusion protein on endothelium, platelets and monocytes. Bone marrow chimeras between these mice and C57BL/6 were generated The level of intravascular hirudin expressed during endotoxaemia was quantified by inhibition studies using an anti-hirudin antibody and reference to the circulating thrombin anti-thrombin complexes generated in control mice given soluble hirudin. Results and Conclusions Antibody inhibition studies indicated that individual chimeras expressed similar levels of hirudin fusion protein on endothelium alone as on platelets and leukocytes combined and accordingly, the levels of thrombin anti-thrombin complexes and fibrinogen in each chimera were similar, indicating equivalent inhibition of thrombin generation. However, mice with hirudin on endothelium alone developed significantly less thrombocytopenia. These results suggest a hitherto unrecognized role of endothelium in thrombin-dependent platelet sequestration during endotoxaemia. The data have implications for the development of therapeutic strategies based on targeted anticoagulation to limit disseminated intravascular coagulation. © 2013 Elsevier Ltd.
Davidson CJ, Hirt RP, Lal K, Snell P, Elgar G, Tuddenham EG, McVey JH (2003) Molecular evolution of the vertebrate blood coagulation network., Thromb Haemost 89 (3) pp. 420-428
In mammalian blood coagulation 5 proteases, factor VII (FVII), factor IX (FIX), factor X (FX), protein C (PC) and prothrombin act with two cofactors factor V and factor VIII to control the generation of fibrin. Biochemical evidence and molecular cloning data have previously indicated that blood coagulation involving tissue factor, prothrombin and fibrinogen is present in all vertebrates. Using degenerate RT-PCR we have isolated and characterized novel cDNAs with sequence identity to the blood coagulation serine proteases and cofactors from chicken and the puffer fish (Fugu rubripes). Sequence alignments, phylogenetic and comparative sequence analysis all support the existence of the Gla-EGF1-EGF2-SP domain serine proteases FVII, FIX, FX, PC and the A1-A2-B-A3-C1-C2 domain protein cofactors FV and FVIII in these species. These results strongly suggest that the blood coagulation network is present in all jawed vertebrates and evolved before the divergence of tetrapods and teleosts over 430 million years ago; and that vertebrate blood coagulation may have benefited from two rounds of gene or whole genome duplication. Sequences identified in Fugu coding for additional FVII-like, FIX-like and PC-like sequences support the possibility of further tandem and large-scale duplications in teleosts. Comparative sequence analyses of amino acid residues in the active site region suggest these additional sequences have evolved new and as yet unknown functions.
Wang H, Liu Y, Li Z, Tuve S, Stone D, Kalyushniy O, Shayakhmetov D, Verlinde CLM, Stehle T, McVey J, Baker A, Peng KW, Roffler S, Lieber A (2008) In vitro and in vivo properties of adenovirus vectors with increased affinity to CD46, Journal of Virology 82 (21) pp. 10567-10579
Gene transfer vectors containing adenovirus (Ad) serotype 35 (Ad35) fibers have shown promise for cancer and stem cell gene therapy. In this study, we attempted to improve the in vitro and in vivo infection properties of these vectors by increasing their affinity to the Ad35 fiber receptor CD46. We constructed Ad vectors containing either the wild-type Ad35 fiber knob (Ad5/35) or Ad35 knob mutants with 4-fold- and 60-fold-higher affinity to CD46 (Ad5/35+ and Ad5/35++, respectively). In in vitro studies with cell lines, the higher affinities of Ad5/35+ and Ad5/35++ to CD46 did not translate into correspondingly higher transduction efficiencies, regardless of the CD46 receptor density present on cells. However, in vivo, in a mouse model with preestablished CD46high liver metastases, intravenous injection of Ad5/35++ resulted in more-efficient tumor cell transduction. We conclude that Ad5/35 vectors with increased affinity to CD46 have an advantage in competing with non-CD46-mediated sequestration of vector particles after intravenous injection. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Nomura S, Hashmi S, McVey JH, Ham J, Parker M, Hogan BL (1989) Evidence for positive and negative regulatory elements in the 5'-flanking sequence of the mouse sparc (osteonectin) gene., J Biol Chem 264 (21) pp. 12201-12207
We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the murine Sparc (osteonectin) gene in parietal endoderm cells and in F9 embryonal carcinoma cells induced to differentiate into parietal endoderm with retinoic acid and cyclic AMP. Varying lengths of flanking sequences extending up to 3.0 kilobase pairs 5' of the transcription initiation site were linked to the bacterial chloramphenicol transacetylase gene in the Bluescript M13- vector. The constructs were tested in transient assays, using a beta-galactosidase plasmid as a transfection control. Sequences between 78 and 169 base pairs upstream of the cap site are the minimum required for cell-type specific promoter activity; this region is dominated by two oligopurine/oligopyrimidine stretches or "GAGA" boxes and is highly conserved between the mouse and bovine genes. Addition of the sequence between -169 and -449, which includes part or all of a third GAGA box, results in increased parietal endoderm specific transcription, up to a maximum of 6.3-fold higher than in undifferentiated F9 cells. Further addition of sequences between -449 and -638 markedly reduces promoter activity in both cell types but parietal endoderm-specific activity is restored in constructs containing 2.2 and 3.0 kilobase pairs of flanking DNA. In addition, we have identified sequences related to the consensus sequence for steroid response elements, one of which is able to confer progesterone-enhanced transcription when tested with a heterologous promoter in steroid responsive cells. These results suggest that negative and positive elements normally interact to regulate the temporal and tissue-specific patterns of Sparc gene transcription seen in vivo.
Krumlauf R, Holland PW, McVey JH, Hogan BL (1987) Developmental and spatial patterns of expression of the mouse homeobox gene, Hox 2.1., Development 99 (4) pp. 603-617
The Hox 2.1 gene forms part of a cluster of homeobox-containing genes on mouse chromosome 11. Analysis of Hox 2.1 cDNAs isolated from an 8 1/2-day p.c. mouse embryo library predicts that the gene encodes a 269 amino acid protein (Mr, 29,432). This deduced protein contains a homeobox 15 amino acids from the carboxy terminus and is very rich in serine and proline. A second partially conserved region present in several other genes containing homeoboxes, the hexapeptide Ile-Phe-Pro-Trp-Met-Arg, is located 12 amino acids upstream of the homeodomain and is encoded by a separate exon. Analysis of Hox 2.1 gene expression reveals a complex and tissue-specific series of RNA transcripts in a broad range of fetal tissues (lung, spinal cord, kidney, gut, spleen, liver and visceral yolk sac). Comparison of the temporal patterns of gene expression during development and in the adult suggests that Hox 2.1 is regulated independently in different tissues. Evidence is also presented that transcripts from other loci have extensive homology to the Hox 2.1 gene in sequences outside of the homeobox. In situ hybridization shows that Hox 2.1 transcripts are regionally localized in the spinal cord in an apparent anterior-posterior gradient extending from the hind brain. The distribution of RNA also displays a cell-type specificity in the lung, where mesodermal cells surrounding the branching epithelial cell layer accumulate high levels of Hox 2.1 transcripts.
McVey JH, Nomura S, Kelly P, Mason IJ, Hogan BL (1988) Characterization of the mouse SPARC/osteonectin gene. Intron/exon organization and an unusual promoter region., J Biol Chem 263 (23) pp. 11111-11116
Two overlapping cosmids have been isolated containing the entire murine gene for SPARC (osteonectin), a Ca2+-binding, phosphorylated glycoprotein associated with extracellular matrix synthesis and remodeling. The gene contains 10 exons and covers 26.5 kilobase pairs of DNA. Exon analysis shows that the two N-terminal glutamic acid-rich sequences which are predicted to undergo conformational change upon binding of calcium, as well as the C-terminal EF-hand Ca2+-binding domain are each encoded by a single exon. Comparative analysis of the exon sequence does not support the idea that the SPARC gene has evolved by shuffling of exons from other Ca2+-binding proteins. The 5' flanking region of the SPARC gene, which promotes transcription when placed in front of the bacterial chloramphenicol acetyltransferase gene, contains neither "TATA" nor "CAAT" box sequences. However, unlike most other genes lacking these motifs, mapping of the 5' end of the SPARC gene by RNase protection and primer extension analysis reveals only a single major and one minor transcription start site. The upstream region to -120 includes six repeats of the sequence GGAGG, two repeats of the sequence 5' GGAGG A/C GGAGGG 3', and a potential transcription factor AP-2 binding site.
Shestopal SA, Kurasawa JH, Karnaukhova E, McVey J, Lee TK, Sarafanov AG (2015) Expression, purification and characterization of codon optimized coagulation factor VIII, JOURNAL OF THROMBOSIS AND HAEMOSTASIS 13 pp. 822-823 WILEY-BLACKWELL
Brown PJ, Gill AC, Nugent PG, McVey JH, Tomley FM (2001) Domains of invasion organelle proteins from apicomplexan parasites are homologous with the Apple domains of blood coagulation factor XI and plasma pre-kallikrein and are members of the PAN module superfamily, FEBS LETTERS 497 (1) pp. 31-38 ELSEVIER SCIENCE BV
EDBROOKE MR, PARKER D, MCVEY JH, RILEY JH, SORENSON GD, PETTENGILL OS, CRAIG RK (1985) EXPRESSION OF THE HUMAN CALCITONIN CGRP GENE IN LUNG AND THYROID-CARCINOMA, EMBO JOURNAL 4 (3) pp. 715-724 OXFORD UNIV PRESS
Chen DX, Riesbeck K, McVey JH, Kemball-Cook G, Tuddenham EGD, Lechler RI, Dorling A (2001) Human thrombin and FXa mediate porcine endothelial cell activation; modulation by expression of TFPI-CD4 and hirudin-CD4 fusion proteins, XENOTRANSPLANTATION 8 (4) pp. 258-265 MUNKSGAARD INT PUBL LTD
Chen D, Weber M, Shiels PG, Dong R, Webster Z, Mcvey JH, Kemball-Cook G, Tuddenham EGD, Lechler RI, Dorling A (2006) Postinjury vascular intimal hyperplasia in mice is completely inhibited by CD34+ bone marrow-derived progenitor cells expressing membrane-tethered anticoagulant fusion proteins, Journal of Thrombosis and Haemostasis 4 (10) pp. 2191-2198
Background: Coagulation proteins promote neointimal hyperplasia and vascular remodelling after vessel injury, but the precise mechanisms by which they act in vivo remain undetermined. Objectives: This study, using an injury model in which the neointima is derived from bone marrow (BM)-derived cells, compared inhibition of tissue factor or thrombin on either BM-derived or existing vascular smooth muscle cells. Methods: Two transgenic (Tg) mouse strains expressing membrane-tethered tissue factor pathway inhibitor (TFPI) or hirudin (Hir) fusion proteins driven by an ± smooth muscle actin (SMA) promoter were generated (±-TFPI-Tg and ±-Hir-Tg) and the phenotype after wire-induced endovascular injury was compared with that in wild-type (WT) controls. Results: WT mice developed progressive neointimal expansion, whereas injury in either Tg was followed by repair back to a preinjured state. This was also seen when WT mice were reconstituted with BM from Tg mice but not when Tgs were reconstituted with WT BM, in which injury was followed by slowly progressive neointimal expansion. Injection of CD34+ cells from Tg mice into injured WT mice resulted in the accumulation of fusion protein-expressing cells from day 3 onwards and an absence of neointimal hyperplasia in those areas. Conclusions: Neointimal development after wire-induced endovascular injury in mice was completely inhibited when BM-derived cells infiltrating the damaged artery expressed membrane tethered anticoagulant fusion proteins under an ±-SMA promoter. These findings enhance our understanding of the pathological role that coagulation proteins play in vascular inflammation. © 2006 International Society on Thrombosis and Haemostasis.
Mumford AD, Laffan M, O'Donnell J, McVey JH, Johnson DJ, Manning RA, Kemball-Cook G (2002) A Tyr346-->Cys substitution in the interdomain acidic region a1 of factor VIII in an individual with factor VIII:C assay discrepancy., Br J Haematol 118 (2) pp. 589-594
The interdomain acidic region a1 is a unique structural feature of coagulation factor VIII (FVIII) and may mediate the proteolytic activation of FVIII and the inactivation of FVIIIa. We report an individual with a Tyr346-->Cys substitution within region a1, who presented with a one-stage FVIII activity (FVIII:C) of 0.34 iu/ml (normal range 0.5-2.0) but normal two-stage FVIII:C and FVIII antigen values. In a factor Xa (FXa)-generation assay for FVIII in which the activation time with thrombin was varied, the variant plasma showed normal FVIII:C at both short and long activation times. However, at intermediate activation times the FXa generation of the variant plasma was less than that of normal pooled plasma. In a modified one-stage FVIII:C assay in which partially purified FVIII was activated with thrombin at low concentrations, the variant FVIII showed less activation than wild-type FVIII, although this defect corrected with increasing concentrations of thrombin. When partially purified variant FVIII was activated with a large molar excess of thrombin, the subsequent rate of decay of FVIII:C was greater for variant FVIII. The complex defects in activation and inactivation displayed by FVIII Tyr346-->Cys support the hypothesis that the a1 sequence is a key regulator of FVIII activity.
PEMBERTON KD, MARSHALL H, KRUMLAUF R, TUDDENHAM EGD, MCVEY JH (1995) MULTIPLE ENHANCERS MAY REGULATE TISSUE FACTOR (TF) EXPRESSION IN TRANSGENIC MICE, THROMBOSIS AND HAEMOSTASIS 73 (6) pp. 1180-1180 F K SCHATTAUER VERLAG GMBH
Parker AL, Nicol CG, Waddington SN, Buckley SM, Shayakhmetov DM, Ni SH, Lieber A, McVey JH, Denby L, Nicklin SA, Baker AH (2006) Hepatic Tropism of Adenoviral Type 5 Vectors Can Be Mediated by Multiple Coagulation Factors, MOLECULAR THERAPY 13 pp. S143-S143 NATURE PUBLISHING GROUP
Factor VII (FVII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation. A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVII activity when tested in a one-stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVII antigen. Variant FVII purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of normal FVII. Also, the variant FVII was unable to compete with wild-type FVII for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinant human TF to the variant FVII compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus' gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the FVII molecule is important for its function.
Paleolog EM, Crossman DC, McVey JH, Pearson JD (1990) Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells., Blood 75 (3) pp. 688-695
We examined the effect of cytokines on basal and agonist-stimulated release of von Willebrand factor (vWf) by human endothelial cells. Treatment of endothelial cells for up to 48 hours with human recombinant or purified interleukin 1 (IL-1) or human recombinant tumor necrosis factor-alpha (TNF-alpha) did not significantly affect constitutive secretion of vWf or intracellular levels of vWf, although basal prostacyclin (PGI2) production was markedly enhanced. In contrast, both IL-1 and TNF-alpha modulated vWf release in response to thrombin or phorbol ester. Pretreatment of endothelial cells for 2 hours with either cytokine enhanced by up to threefold the stimulatory effect of a subsequent 60-minute exposure to thrombin. Addition of cycloheximide (5 micrograms/mL) during the preincubation abolished this enhancement. Moreover, if the cytokine pretreatment time was extended to 24 hours, agonist-stimulated vWf release was significantly suppressed. Cytokine treatment for 2 or 24 hours had no detectable effect on levels of vWf messenger RNA. The effects of cytokines were not the result of contamination with bacterial lipopolysaccharide and were not attributable to endothelial cell injury. These results show that cytokines have little or no direct effect on vWf release from endothelial cells but can significantly modulate its acute release in response to other stimuli in a complex time- and dose-dependent manner.
Ludlam CA, Cairo A, Dolan G, Gomez K, Goodeve AC, Hampshire DJ, Kemball-Cook G, Mcvey JH, Oldenburg J, Perkins SJ, Peyvandi F, Rallapalli PM (2015) The European Association for Haemophilia and Allied Disorders Coagulation Factor Variant Databases, HAEMOPHILIA 21 pp. 37-37 WILEY-BLACKWELL
Parker AL, Waddington SN, Nicol CG, Shayakhmetov DM, Buckley SM, Denby L, Kemball-Cook G, Ni S, Lieber A, McVey JH, Nicklin SA, Baker AH (2006) Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes, BLOOD 108 (8) pp. 2554-2561 AMER SOC HEMATOLOGY
Shestopal S, Hao J, Karnaukhova E, Liang Y, Ovanesov M, Lin M, Kurasawa J, Lee T, McVey J, Sarafanov A (2017) Expression and characterization of a codonoptimized blood coagulation factor VIII,Journal of Thrombosis and Haemostasis 15 (4) pp. 709-720
Background: Production of recombinant factor VIII (FVIII) is challenging due to its low expression. It was previously shown that codon-optimization of a B domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well-recognized that synonymous mutations may affect the protein structure and function. Objectives: To compare biochemical properties of a BDD-FVIII expressed from codon-optimized (CO) and the wild-type (WT) cDNAs. Methods: Each variant of the BDD-FVIII was expressed in several independent CHO cell lines, generated using a lentiviral platform. The proteins were purified by two-step affinity chromatography and analyzed in parallel by: PAGE-Western blot (including thrombin cleavage), mass-spectrometry, circular dichroism, surface plasmon resonance, chromogenic, clotting and thrombin generation assays. Results and Conclusion: The average yield of the CO was 7-fold higher than WT, while both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low-density lipoprotein receptor-related protein 1. The CO preparations had averagely 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure due to considerably higher protein concentrations during the production. We concluded that the codon-optimization of the BDD-FVIII resulted in significant increase of its expression and did not affect the structure-function properties.
Most of diabetic cardiovascular complications are attributed to endothelial dysfunction and impaired angiogenesis. Endoplasmic Reticulum (ER) and oxidative stresses were shown to play a pivotal role in the development of endothelial dysfunction in diabetes. Hemeoxygenase-1 (HO-1) was shown to protect against oxidative stress in diabetes; however, its role in alleviating ER stress-induced endothelial dysfunction remains not fully elucidated. We aim here to test the protective role of HO-1 against high glucose-mediated ER stress and endothelial dysfunction and understand the underlying mechanisms with special emphasis on oxidative stress, inflammation and cell death. Human Umbilical Vein Endothelial Cells (HUVECs) were grown in either physiological or intermittent high concentrations of glucose for 5 days in the presence or absence of Cobalt (III) Protoporphyrin IX chloride (CoPP, HO-1 inducer) or 4-Phenyl Butyric Acid (PBA, ER stress inhibitor). Using an integrated cellular and molecular approach, we then assessed ER stress and inflammatory responses, in addition to apoptosis and angiogenic capacity in these cells. Our results show that HO-1 induction prevented high glucose-mediated increase of mRNA and protein expression of key ER stress markers. Cells incubated with high glucose exhibited high levels of oxidative stress, activation of major inflammatory and apoptotic responses [nuclear factor (NF)-ºB and c-Jun N-terminal kinase (JNK)] and increased rate of apoptosis; however, cells pre-treated with CoPP or PBA were fully protected. In addition, high glucose enhanced caspases 3 and 7 cleavage and activity and augmented cleaved poly ADP ribose polymerase (PARP) expression whereas HO-1 induction prevented these effects. Finally, HO-1 induction and ER stress inhibition prevented high glucose-induced reduction in NO release and impaired the angiogenic capacity of HUVECs, and enhanced vascular endothelial growth factor (VEGF)-A expression. Altogether, we show here the critical role of ER stress-mediated cell death in diabetes-induced endothelial dysfunction and impaired angiogenesis and underscore the role of HO-1 induction as a key therapeutic modulator for ER stress response in ischemic disorders and diabetes. Our results also highlight the complex interplay between ER stress response and oxidative stress.
Dissection of pleiotropic effects of missense mutations, rarely investigated in inherited diseases, is fundamental to understanding genotype-phenotype relationships. Missense mutations might impair mRNA processing in addition to protein properties.
As a model for hemophilia A we investigated the highly prevalent F8 c.6046c>t/p.R2016W (exon 19) mutation. In expression studies exploiting lentiviral vectors, we demonstrated that the amino acid change impairs both factor VIII (FVIII) secretion (antigen 11.0±0.4% of wild-type) and activity (6.0±2.9%). Investigations in patients? ectopic F8 mRNA and with minigenes showed that the corresponding nucleotide change also decreases correct splicing to 70±5%, which is predicted to lower further FVIII activity (4.2±2%), consistently with patients? levels (a (p.G2013R) reduced exon inclusion to 41±3% and the c.6053a>g (p.E2018G) to 28±2%, similarly to a variant affecting the 5? splice site (c.6113a>g, p.N2038S, 26±2%), which displayed normal protein features upon recombinant expression. The p.G2013R remarkably reduced both antigen (7.0±0.9%) and activity (8.4±0.8%), while the p.E2018G produced a dysfunctional molecule (antigen, 69.0±18.1%; activity, 19.4±2.3%).
In conclusion, differentially altered mRNA and protein patterns produce a gradient of residual activity, and clarify genotype-phenotype relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Altogether we provide a clear example of interplay between mRNA and protein mechanisms of disease that certainly operate in shaping many other inherited disorders.
Karegli J, Melchionna T, Farrar C, Greenlaw R, Smolarek D, Horsfield C, Charif R, McVey JH, Dorling A, Sacks S, Smith R (2017) Thrombalexins: Cell-Localized Inhibition of Thrombin and its Effects in a Model of HighRisk Renal Transplantation,American Journal of Transplantation 17 (1) pp. 272-280
Allograft transplantation into sensitised recipients with anti-donor antibodies results in accelerated antibody-mediated rejection (AMR), complement activation and graft thrombosis. We have developed a membrane-localizing technology of wide applicability that enables therapeutic agents including anticoagulants to bind to cell surfaces and protect the donor endothelium. We describe here how this technology has been applied to thrombin inhibitors to generate a novel class of drugs termed thrombalexins (TLN). Using a rat model of hyperacute rejection we have investigated the potential of one such inhibitor (TLN-1) to prevent acute antibody-mediated thrombosis in the donor organ. TLN-1 alone was able to reduce intragraft thrombosis and significantly delay rejection. The results confirm a pivotal role for thrombin in AMR in vivo. This approach targets donor organs rather than the recipient and is intended to be directly translatable to clinical use.
Purpose of review: The role of tissue factor (TF) in the initiation of blood coagulation network leading to the generation of a fibrin clot has been well defined over the past 50 years. Although much is known about this sequence of events and its regulation many important questions remain unresolved. More recently, a complex role for TF in cellular processes independent of fibrin generation has emerged. This review summarizes some of the advances in this field. Recent findings: TF is the cellular receptor and cofactor for factor VII/VIIa however controversy still surrounds expression of TF within the vasculature, the role of circulating microvesicle pools of TF and mechanisms of ?encryption? of TF activity. However, there have been significant advances in the role of TF initiated cell signalling. Lastly, an alternatively spliced TF transcript has been identified and some insights into its role in cancer cell metastasis/proliferation have been elucidated. Summary: Understanding of TF structure-function has increased substantially however multiple controversies still surround some aspects of its regulation. TF has emerged as a pivotal player in orchestrating not only fibrin generation but in wound repair. Derangement of these repair processes contributes significantly to the pathophysiology of a number of disease processes.
Most diabetic cardiovascular complications are mediated by endothelial dysfunction and impaired angiogenesis. Endoplasmic Reticulum [ER] and oxidative stresses were shown to play a pivotal role in the development of endothelial dysfunction in diabetes. The cytoprotective effects of Hemeoxygenase-1 [HO-1] were extensively studied; however, its role in alleviating ER stress-induced endothelial dysfunction remains elusive. We aim here to test the role of HO-1 against high glucose-mediated ER stress response and endothelial dysfunction and understand the underlying mechanisms with special emphasis on oxidative stress, inflammation and cell death.
Primary Human Umbilical Vein Endothelial cells [HUVECs] were harbored in culture medium containing high glucose (33 mM) for 5 days with 8 hrs intermittent recovery periods to mimic the diabetic milieu. Using a wide array of molecular biology techniques, we were able to show that this chronic and intermittent exposure of HUVECs to high glucose significantly increased mRNA and protein expression of key ER stress markers namely, binding immunoglobulin protein [BiP], activation transcription factor-4 [ATF-4], CCAAT-enhancer-binding protein homologous protein [CHOP], and phosphorylated eukaryotic initiation factor2± [p-eIF-2±]. In addition, there was a significant elevation in ROS associated with significant increased phosphorylation of p47phox regulatory subunit of NADPH oxidase [NOX]. Moreover, inflammatory and apoptotic responses were also elicited, featured mainly by significant increase in phosphorylation/activation of IºB kinase [IKK] and c-Jun, and upregulation of IL-6, whereas apoptosis was featured by showing significantly increased caspase3/7 activity and cell death. Vascular endothelial growth factor-A [VEGF-A] expression, Nitric Oxide [NO] production, and tube formation capacity were also significantly inhibited by high glucose. Pre-treatment by HO-1 inducer Cobalt protoporphyrin IX [CoPP] significantly abolished all the observed effects with high glucose.
Altogether, to the best of our knowledge, we present for the first time the role of HO-1 induction as a potential antagonist to ER stress response against high glucose mediated endothelial dysfunction and impaired angiogenesis.
Fibrocytes are myeloid lineage cells implicated in wound healing, repair and fibrosis. We previously showed that fibrocytes are mobilized into the circulation after vascular injury, including the immune-mediated injury that occurs after allogeneic transplantation. A common response to inflammatory vascular injury is intimal hyperplasia (IH), which, alongside vascular remodeling, results in progressive loss of blood flow, downstream ischaemia and end-organ fibrosis. This forms the pathological basis of transplant arteriosclerosis and other diseases including post-angioplasty re-stenosis. In investigating whether fibrocytes contribute to IH, we previously showed that subpopulations expressing smooth muscle actin and CD31 are recruited to the site of injury and accumulate in the neointima. Expression of tissue factor (TF) by these ?CD31+ myofibrocytes? is needed for progressive neointimal expansion, such that TF inhibition limits the neointima to a single layer of cells by day 28 post-injury. The aim of this study was to determine pathophysiological mediators downstream of TF that contribute to myofibrocyte-orchestrated IH. We first show that myofibrocytes make up a significant component of the neointima 28 days following injury. Using a previously defined adoptive transfer model, we then show that CD31+ myofibrocytes get recruited early to the site of injury; this model allows manipulations of the adoptively transferred cells to study how IH develops. Having confirmed that inhibition of TF on adoptively transferred cells prevents IH, we then show that TF, primarily through the generation of thrombin, induces secretion of angiopoietin-2 by myofibrocytes and this directly stimulates proliferation, inhibits apoptosis and induces CXCL-12 production by neointimal cells, including non-fibrocytes, all of which promote progressive IH in vivo. Prior incubation to inhibit angiopoietin-2 secretion by or block TIE-2 signaling on adoptively transferred fibrocytes inhibits IH. These novel data indicate that angiopoietin-2 production by early recruited myofibrocytes critically influences the development of IH after vascular injury and suggest new therapeutic avenues for exploration.
Introduction: Advances in genomic sequencing have facilitated the sequencing of genes associated with disorders of haemostasis. The identification of variants within genes, access to curated data incorporating structural, functional, evolutionary as well as phenotypic data has become increasingly important in order to ascribe pathogenicity.
Aim: The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Database Project aims to provide a single port of entry to a web accessible resource for variants in genes involved in clinical bleeding disorders.
Results: New databases have evolved from previously developed single gene variant coagulation database projects, incorporating new data, new analysis tools and a new common database architecture with new interfaces and filters. These new databases currently present information about the genotype, phenotype (laboratory and clinical) and structural and functional effects of variants described in the genes of factor (F) VII (F7), FVIII (F8), FIX (F9) and von Willebrand factor (VWF).
Conclusion: The project has improved the quality and quantity of information available to the haemostasis research and clinical communities, thereby enabling accurate classification of disease severity in order to make assessments of likely pathogenicity.
Major surgery generates a stress response which increases oxygen demand and consumption post-operatively. Patients with low cardiopulmonary reserve may not be able to meet the increased oxygen demand and are at risk of increased morbidity. Dietary nitrate reduces the amount of oxygen required to perform a set amount of exercise and enhances exercise tolerance and performance in athletes by improving oxygen utilisation.
Cardiopulmonary exercise testing (CPX) is an established method of assessing patients? cardiopulmonary reserve prior to surgery. The anaerobic threshold (AT) is the oxygen uptake at which anaerobic metabolism supplements aerobic metabolism; is calculated during CPX and can predict short and long term outcomes after surgery and postoperative complications.
We conducted a single centre, randomised, double-blind, placebo controlled trial to determine if dietary organic nitrate supplementation with beetroot juice (BRJ) improves pre-operative performance in CPX in patients with colorectal cancer (CRC). Eligible patients were adults undergoing elective laparoscopic resection for CRC. Patients were randomised to receive BRJ or nitrate-depleted BRJ (Placebo). Initial CPX was performed to a standard ramped incremental exercise protocol. Patients then received BRJ or placebo every day for 7 days followed by a second CPX. The primary outcome measure was the change in AT.
There was a statistically significant increase in the mean AT in the nitrate group (+0.706, 95% CI 0.130 to 1.281; p=0.018) but not in the placebo group. There was a significantly lower length of stay in nitrates group. There was no significant difference in rate of complications between the two groups.
Just 7 days of dietary nitrate supplementation results in a significant improvement in oxygen utilisation in an elderly population with colorectal cancer. This is a novel finding and this study is the first in patients undergoing surgery. This trial provides evidence that dietary nitrate supplementation is beneficial in a preoperative setting.
Chowdhary Pratima, Lancaster Simon, Hamid Colleen, Slatter David, Morris Richard, Foley Jonathan H., Gomez Keith, Brodkin Edgar, Fox Thomas, Gatt Alex, McVey John (2020) Impaired platelet dependent thrombin generation
associated with thrombocytopenia is improved by
prothrombin complex concentrates in vitro,Research and Practice in Thrombosis and Haemostasis 4 (2) pp. 334-342
Impaired thrombin generation (TG) is seen in patients with acquired coagulopathy, due to a
combination of low coagulation factors and decreased platelet count (thrombocytopenia).
Low coagulation factors seen in acquired coagulopathy are typically treated with plasma and
occasionally with prothrombin complex concentrates (PCCs), and the decreased platelet
count can be treated with platelet transfusions. Our study investigated the influence of PCCs
on impaired generation of thrombin in the above context. Generation of adequate thrombin
is crucial for the formation of a stable blood clot. We conclude that further clinical studies
would be of interest to investigate the role of PCCs in the management of complex
coagulopathy characterized by abnormal coagulation and mild-to-moderate
Hereditary blood coagulation factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder resulting from variants in the gene encoding FVII (F7). Integration of genetic variation with functional consequences on protein function is essential for the interpretation of the pathogenicity of novel variants. Here, we describe the integration of previous locus?specific databases for F7 into a single curated database with enhanced features. The database provides access to in silico analyses that may be useful in the prediction of variant pathogenicity as well as cross?species sequence alignments, structural information, and functional and clinical severity described for each variant, where appropriate. The variant data is shared with the F7 Leiden Open Variation Database. The updated database now includes 221 unique variants, representing gene variants identified in 728 individuals. Single nucleotide variants are the most common type (88%) with missense representing 74% of these variants. A number of variants are found with relatively high minor allele frequencies that are not pathogenic but contribute significantly to the likely pathogenicity of coinherited variants due to their effect on FVII plasma levels. This comprehensive collection of curated information significantly aids the assessment of pathogenicity.
Chen Daxin, Li Ke, Festenstein Sam, Karegli Julieta, Wilkinson Hannah, Leonard Hugh, Lin-Lin Wei, Ma Ning, Min Xia, Tam Henry, Wang Jian-an, Xu Qingbo, McVey John, Smith Richard A.G, Dorling Anthony (2020) Regression of atherosclerosis in ApoE-/- mice via modulation of monocyte recruitment and phenotype, induced by weekly dosing of a novel ?cytotopic? anti thrombin without prolonged anticoagulation.,Journal of the American Heart Association
American Heart Association
Background: Coagulation proteases play an important role in atherogenesis. Accordingly,
anticoagulants can induce regression in animal models of atherosclerosis, but exploiting this
clinically has been limited by major bleeding events that occur after systemic anticoagulation.
Here we test a novel thrombin inhibitor, PTL060, that comprises hirulog covalently linked to a
synthetic myristoyl electrostatic switch to tether it to cell membranes.
Methods and Results: ApoE-/- mice, fed either chow or high fat diets were used.
Transplantation of congenic aortic segments was used to demonstrate the impact of
expressing anticoagulants on endothelium. PTL060, parental hirulog or controls were tested
to assess suppression of vessel wall chemokine gradients, impact on plaque development
and regression of existing plaques. Adoptive transfer of labelled CD11b positive cells was
used to assess recruitment of monocytes and inform on how PTL060 influenced monocyte
Transgenic expression of anticoagulant fusion proteins based on TFPI or hirudin on EC led
to complete suppression of MIF and CCL2 expression throughout the vessel wall and
segments of aorta transplanted into ApoE-/- mice did not develop atherosclerosis. A single IV
injection of PTL060, but not parental (unmanipulated) hirulog inhibited the same chemokines
for >1 week and atheroma formation was reduced by >50% compared to controls when
assessed 4 weeks later. Mice had prolonged bleeding times for only 1/7th of the time that
PTL060 was biologically active. Repeated weekly injections of PTL060 but not parental
hirulog caused regression of atheroma in ApoE-/- mice fed either chow or high fat diets.
Mechanistically, 100% of circulating monocytes quickly became coated with PTL060 after the
first dose, following which >70% of CCR2+ monocytes recruited into plaques expressed
CCR7, ABCA1 and IL-10, a phenotype associated with regression, compared to
CCR2+ recruits in control mice.. Multiple doses caused a significant reduction in the number
of monocytes recruited, and a switch to recruitment of CCR2-negative cells, the majority of which (>90%) had a similar regression-associated phenotype. The impact of PTL060 on circulating monocytes appeared 1 dominant, as regression equivalent to that induced by IV PTL060 was induced by adoptive transfer of CD11b+ cells pre-coated with PTL060.
Conclusions: PTL060, a novel tethered direct thrombin inhibitor causes regression of atherosclerosis in ApoE-/- mice, via an effect at the endothelial surface but also through a direct effect on monocytes, causing differentiation into macrophages capable of plaque
regression. Covalent linkage of a myristoyl electrostatic switch onto hirulog uncouples the pharmacodynamic effects on haemostasis and atherosclerosis, such that regression is
accompanied by only transient anticoagulation.