John McVey

Professor John McVey

Head of Department of Biochemical Sciences, Professor of Cardiovascular Biology
+44 (0)1483 686390
29 AY 04

Academic and research departments

School of Biosciences.



John McVey is Professor of Cardiovascular Biology at the University of Surrey and holds an Honorary Professorship at University College London (UCL). His research focuses on the regulation of blood coagulation and the role that coagulation factors play in normal physiology and in the pathophysiology of disease.Professor McVey has published in excess of 120 research articles, reviews, and books. He has received major grant funding from the MRC, Wellcome Trust, BBSRC and BHF. He is a named inventor on 3 patents. He is a fellow of the Royal College of Pathologists.

Visiting Professor: Kings College London; 2011-2013Weston Professor of Molecular Medicine: Thrombosis Research Institute; 2008 to 2011Group Leader: MRC Clinical Sciences Centre, Haemostasis and Thrombosis; 2006 to 2008Honorary Reader in Haemostasis & Thrombosis: Imperial College London; 2006-2008Postdoctoral Research Scientist: MRC CSC, Haemostasis and Thrombosis; 1987 to 2006Postdoctoral Research Scientist: National Institute for Medical Research, London; 1984 1987Postdoctoral Research Scientist: Courtauld Institute of Biochemistry, London; 1982 1984Postgraduate Research Student: Beatson Institute for Cancer Research, Glasgow; 1979-1982

Research interests

• Studies on the initiation of blood coagulation in disease models• Development of novel anticoagulant strategies• Gene therapy for inherited coagulation factor disorders• Genetic and functional studies in inherited coagulation disorders• Role of coagulation factors in modulating delivery of adenoviral gene therapy vectors

Research collaborations

Professor Andy Baker, University of GlasgowProfessor Tony Dorling, Kings College LondonDr Keith Gomez, University College LondonProfessor Amit Nathwani, University College LondonDr Simon Waddington, University College London

Group Website


Mariam Bapir, Gavrielle R. Untracht, Julie E. A. Hunt, John H. McVey, Jenny Harris, Simon S. Skene, Paola Campagnolo, Nikolaos Dikaios, Ana Rodriguez-Mateos, David D Sampson, Danuta M. Sampson, Christian Heiss (2022)Age-Dependent Decline in Common Femoral Artery Flow-Mediated Dilation and Wall Shear Stress in Healthy Subjects, In: Life12(12)2023 MDPI

Femoral artery (FA) endothelial function is a promising biomarker of lower extremity vascular health for peripheral artery disease (PAD) prevention and treatment; however, the impact of age on FA endothelial function has not been reported in healthy adults. Therefore, we evaluated the reproducibility and acceptability of flow-mediated dilation (FMD) in the FA and brachial artery (BA) (n = 20) and performed cross-sectional FA- and BA-FMD measurements in healthy non-smokers aged 22–76 years (n = 50). FMD protocols demonstrated similar good reproducibility. Leg occlusion was deemed more uncomfortable than arm occlusion; thigh occlusion was less tolerated than forearm and calf occlusion. FA-FMD with calf occlusion was lower than BA-FMD (6.0 ± 1.1% vs 6.4 ± 1.3%, p = 0.030). Multivariate linear regression analysis indicated that age (−0.4%/decade) was a significant independent predictor of FA-FMD (R2 = 0.35, p = 0.002). The age-dependent decline in FMD did not significantly differ between FA and BA (pinteraction agexlocation = 0.388). In older participants, 40% of baseline FA wall shear stress (WSS) values were

MARIAM BAPIR, Gabrielle Untracht, Debbie Cooke, John H. McVey, Simon S. SKENE, Paola Campagnolo, Martin B. Whyte, Nikolaos Dikaios, Ana Rodriguez-Mateos, David D Sampson, Danuta M. Sampson, Christian Heiss (2022)Cocoa flavanol consumption improves lower extremity endothelial function in healthy individuals and people with type 2 diabetes, In: Food & Function13(20)pp. 10439-10448 Royal Society of Chemistry

Background: Diabetes and age are major risk factors for the development of lower extremity peripheral artery disease (PAD). Cocoa flavanol (CF) consumption is associated with lower risk for PAD and improves brachial artery (BA) endothelial function. Objectives: to assess if femoral artery (FA) endothelial function and dermal microcirculation are impaired in individuals with type 2 diabetes mellitus (T2DM) and evaluate the acute effect of CF consumption on FA endothelial function. Methods: In a randomised, controlled, double-blind, cross-over study, 22 individuals (n = 11 healthy, n = 11 T2DM) without cardiovascular disease were recruited. Participants received either 1350 mg CF or placebo capsules on 2 separate days in random order. Endothelial function was measured as flow-mediated dilation (FMD) using ultrasound of the common FA and the BA before and 2 hours after interventions. The cutaneous microvasculature was assessed using optical coherence tomography angiography. Results: Baseline FA-FMD and BA-FMD were significantly lower in T2DM (FA: 3.2 ± 1.1% [SD], BA: 4.8 ± 0.8%) compared to healthy (FA: 5.5 ± 0.7%, BA: 6.0 ± 0.8%); each p < 0.001. Whereas in healthy individuals FA-FMD did not significantly differ from BA-FMD (p = 0.144), FA-FMD was significantly lower than BA-FMD in T2DM (p = 0.003) indicating pronounced and additional endothelial dysfunction of lower limb arteries (FA-FMD/BA-FMD: 94 ± 14% [healthy] vs. 68 ± 22% [T2DM], p = 0.007). The baseline FA blood flow rate (0.42 ± 0.23 vs. 0.73 ± 0.35 l min−1, p = 0.037) and microvascular dilation in response to occlusion in hands and feet were significantly lower in T2DM subjects than in healthy ones. CF increased both FA- and BA-FMD at 2 hours, compared to placebo, in both healthy and T2DM subgroups (FA-FMD effect: 2.9 ± 1.4%, BA-FMD effect 3.0 ± 3.5%, each pintervention< 0.001). In parallel, baseline FA blood flow and microvascular diameter significantly increased in feet (3.5 ± 3.5 μm, pintervention< 0.001) but not hands. Systolic blood pressure and pulse wave velocity significantly decreased after CF in both subgroups (−7.2 ± 9.6 mmHg, pintervention = 0.004; −1.3 ± 1.3 m s−1, pintervention = 0.002). Conclusions: Individuals with T2DM exhibit decreased endothelial function that is more pronounced in the femoral than in the brachial artery. CFs increase endothelial function not only in the BA but also the FA both in healthy individuals and in those with T2DM who are at increased risk of developing lower extremity PAD and foot ulcers.

VALERIA MASTRULLO, Daan R. van der Veen, ROLANDO SZILVESZTER MATOS, JONATHAN DAVID JOHNSTON, JOHN HENDERSON MCVEY, Paolo Madeddu, EIRINI VELLIOU, PAOLA CAMPAGNOLO, PRIYANKA GUPTA (2022)Pericytes’ Circadian Clock Affects Endothelial Cells’ Synchronization and Angiogenesis in a 3D Tissue Engineered Scaffold, In: Frontiers in pharmacology Frontiers
Fenella K S Welsh, Caoimhe M Walsh, Kandiah Chandrakumaran, Wasula S Rathnaweera, Ashok Roy, Jane Needham, Adrian B Cresswell, John H McVey, Myrddin Rees (2023)Peri-operative thrombophilia in patients undergoing liver resection for colorectal metastases, In: HPB (Oxford, England)25(1)pp. 63-72

Routine chemical venous thromboembolism (VTE) prophylaxis for liver surgery remains controversial, and often delayed post-operatively due to perceived bleeding risk. This study asked whether patients undergoing hepatectomy for colorectal metastases (CRM) were at risk from VTE pre-operatively, and the impact of hepatectomy on that risk. Single-centre prospective observational cohort study of patients undergoing open hepatectomy for CRM, comparing pre-, peri- and post-operative haemostatic variables. Of 336 hepatectomies performed October 2017-December 2019, 60 resections in 57 patients were recruited. There were 28 (46.7%) major resections, with median (interquartile range [IQR]) blood loss 150.0 (76.3-263.7) mls, no blood transfusions, post-operative VTE events or deaths. Patients were prothrombotic pre-operatively (high median factor VIIIC and increased thrombin generation velocity index), an effect exacerbated post-hepatectomy. Major hepatectomies had a significantly greater median drop in Protein C, rise in Factor VIIIC and von Willebrand Factor, versus minor resections (p = 0.001, 0.005, 0.001 respectively). Patients with parenchymal transection times greater than median (40 min), had significantly increased median (IQR) PMBC-TFmRNA expression [1.65(0.93-2.70)2ddCt], versus quicker transections [0.99(0.69-1.28)2ddCt, p = 0.020]. Patients with CRM are prothrombotic pre-operatively, an effect exacerbated by hepatectomy, particularly longer, complex resections, suggesting chemical thromboprophylaxis be considered early in the patient pathway.

Daxin Chen, Ke Li, Sam Festenstein, Julieta Karegli, Hannah Wilkinson, Hugh Leonard, Wei Lin-Lin, Ning Ma, Xia Min, Henry Tam, Jian-an Wang, Qingbo Xu, John McVey, Richard A.G Smith, Anthony Dorling (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, In: 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 phenotype. 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 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.

Hannah Wilkinson, Hugh Leonard, Michael G. Robson, Richard Smith, ElLi Tam, John H. McVey, Daniel Kirckhofer, Daxin Chen, Anthony Dorling (2022)Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH, In: Frontiers in immunology13999871pp. 999871-999871 Frontiers Media Sa

BackgroundTissue factor (TF) generates proteases that can signal through PAR-1 and PAR-2. We have previously demonstrated PAR-1 signalling primes innate myeloid cells to be exquisitely sensitive to interferon-gamma (IFN gamma). In this work we explored how TF mediated PAR-2 signalling modulated responsiveness to IFN gamma and investigated the interplay between PAR-1/-2 signalling on macrophages. MethodologyWe characterised how TF through PAR-2 influenced IFN gamma sensitivity in vitro using PCR and flow cytometry. and how it influenced oxazolone-induced delayed type hypersensitivity (DTH) responses in vivo. We investigated how basal signalling through PAR-2 influenced PAR-1 signalling using a combination of TF-inhibitors and PAR-1 &-2 agonists and antagonists. Finally, we investigated whether this system could be targeted therapeutically using 3-mercaptopropionyl-F-Cha-Cha-RKPNDK (3-MP), which has actions on both PAR-1 and -2. ResultsTF delivered a basal signal through PAR-2 that upregulated SOCS3 expression and blunted M1 polarisation after IFN gamma stimulation, opposing the priming achieved by signalling through PAR-1. PAR-1 and -2 agonists or antagonists could be used in combination to modify this basal signal in vitro and in vivo. 3-MP, by virtue of its PAR-2 agonist properties was superior to agents with only PAR-1 antagonist properties at reducing M1 polarisation induced by IFN gamma and suppressing DTH. Tethering a myristoyl electrostatic switch almost completely abolished the DTH response. ConclusionsTF-mediated signalling through PARs-1 and -2 act in a homeostatic way to determine how myeloid cells respond to IFN gamma. 3-MP, an agent that simultaneously inhibits PAR-1 whilst delivering a PAR-2 signal, can almost completely abolish immune responses dependent on M1 polarisation, particularly if potency is enhanced by targeting to cell membranes; this has potential therapeutic potential in multiple diseases.

Robert D. Johnson, Ming Lei, John H. Mcvey, Patrizia Camelliti (2023)Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell‑derived cardiomyocytes, In: Cellular and Molecular Life Sciences80276 Springer

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to remuscularize infarcted hearts but their arrhythmogenicity remains an obstacle to safe transplantation. Myofbroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defned. Here, we investigate the efect of myofbroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell–cell interaction modalities. Human myofbroblasts altered the electrophysiology and Ca2+ handling of hiPSC-CMs and downregulated mRNAs encoding voltage channels (KV4.3, KV11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 was elevated in the presence of myofbroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine efects of myofbroblasts. Blocking interleukin-6 reduced the efects of myofbroblasts only in the absence of physical contact between cell-types. Myofbroblast-specifc connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the frst in-depth investigation into how human myofbroblasts modulate hiPSC-CMs function, identifying interleukin-6 and connexin43 as paracrine- and contact-mediators respectively, and highlighting their potential as targets for reducing arrhythmic risk in cardiac cell therapy

AB Kritz, CG Nicol, KL Dishart, R Nelson, S Holbeck, DJ Von Seggern, LM Work, JH McVey, SA Nicklin, AH Baker (2007)Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop, In: MOLECULAR THERAPY15(4)pp. 741-749 NATURE PUBLISHING GROUP
DP Barlow, JH McVey, BLM Hogan (1987)[26] Molecular cloning of laminin, In: Methods in Enzymology144(C)pp. 464-474
JA Greig, SMK Buckley, SN Waddington, AL Parker, D Bhella, R Pink, AA Rahim, T Morita, SA Nicklin, JH McVey, AH Baker (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), In: Molecular Therapy17(10)pp. 1830-?
KM Johnson, JH McVey (1999)Molecular Analysis in Factor XI Deficiency.31pp. 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 μg/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.

M Christian, P Marangos, I Mak, J McVey, F Barker, J White, JJ Brosens (2001)Interferon-gamma modulates prolactin and tissue factor expression in differentiating human endometrial stromal cells., In: Endocrinology142(7)pp. 3142-3151

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.

JH McVey (1994)Staden Plus., In: Methods Mol Biol25pp. 171-179
K Takacs, C Du Roure, S Nabarro, N Dillon, JH McVey, Z Webster, A MacNeil, L Bartok, C Higgins, D Gray, M Merkenschlager, AG Fisher (2004)The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes, In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA101(46)pp. 16298-16303 NATL ACAD SCIENCES
J McIntosh, PJ Lenting, C Rosales, D Lee, S Rabbanian, D Raj, N Patel, EGD Tuddenham, OD Christophe, JH McVey, S Waddington, AW Nienhuis, JT Gray, P Fagone, F Mingozzi, S-Z Zhou, KA High, M Cancio, CYC Ng, J Zhou, CL Morton, AM Davidoff, AC Nathwani (2013)Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant, In: BLOOD121(17)pp. 3335-3344 AMER SOC HEMATOLOGY
DX Chen, K Riesbeck, JH McVey, G Kemball-Cook, EGD Tuddenham, RI Lechler, A Dorling (2001)Human thrombin and FXa mediate porcine endothelial cell activation; modulation by expression of TFPI-CD4 and hirudin-CD4 fusion proteins, In: XENOTRANSPLANTATION8(4)pp. 258-265 MUNKSGAARD INT PUBL LTD
J Karegli, T Melchionna, CA Farrar, R Greenlaw, D Smolarek, C Horsfield, R Charif, John McVey, A Dorling, SH Sacks, RAG Smith (2017)Thrombalexins: Cell-Localized Inhibition of Thrombin and its Effects in a Model of HighRisk Renal Transplantation, In: American Journal of Transplantation17(1)pp. 272-280 Wiley

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.

D Chen, JM Abrahams, LM Smith, JH Mcvey, RI Lechler, A Dorling (2008)Regenerative repair after endoluminal injury in mice with specific antagonism of protease activated receptors on CD34(+) vascular progenitors, In: BLOOD111(8)pp. 4155-4164 AMER SOC HEMATOLOGY
John McVey (2016)The role of the tissue factor pathway in hemostasis and beyond, In: Current Opinion in Hematology23(5)pp. 453-461 Lippincott, Williams & Wilkins

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.

AD Mumford, JH McVey, CV Morse, K Gomez, M Steen, EA Norstrom, EGD Tuddenham, B Dahlback, PHB Bolton-Maggs (2003)Factor VI359T: a novel mutation associated with thrombosis and resistance to activated protein C, In: BRITISH JOURNAL OF HAEMATOLOGY123(3)pp. 496-501 BLACKWELL PUBLISHING LTD
D Chen, A Carpenter, J Abrahams, RC Chambers, RI Lechler, JH McVey, A Dorling (2008)Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1-dependent leukocyte recruitment in vivo, In: JOURNAL OF EXPERIMENTAL MEDICINE205(8)pp. 1739-1746 ROCKEFELLER UNIV PRESS
S Morichika, M Shima, S Kamisue, I Tanaka, Y Imanaka, H Suzuki, H Shibata, S Pemberton, K Gale, J McVey, EGD Tuddenham, A Yoshioka (1997)Factor VIII gene analysis in Japanese CRM-positive and CRM-reduced haemophilia A patients by single-strand conformation polymorphism, In: BRITISH JOURNAL OF HAEMATOLOGY98(4)pp. 901-906 BLACKWELL SCIENCE LTD
D David, EL Saenko, IMA Santos, DJD Johnson, EGD Tuddenham, JH McVey, G Kemball-Cook (2001)Stable recombinant expression and characterization of the two haemophilic factor VIII variants C329S (CRM-) and G1948D (CRMr), In: BRITISH JOURNAL OF HAEMATOLOGY113(3)pp. 604-615 BLACKWELL SCIENCE LTD
M Di Matteo, E Samara-Kuko, NJ Ward, SN Waddingon, JH McVey, MKL Chuah, T Vanden Driessche (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), In: Molecular Therapy23(10)pp. 1671-?
DS Millar, G Kemball-Cook, JH McVey, EGD Tuddenham, AD Mumford, GB Attock, JC Reverter, N Lanir, LA Parapia, J Reynaud, E Meili, A von Felton, U Martinowitz, DR Prangnell, M Krawczak, DN Cooper (2000)Molecular analysis of the genotype-phenotype relationship in factor VII deficiency, In: HUMAN GENETICS107(4)pp. 327-342 SPRINGER-VERLAG
EB Bachli, CM Pech, KM Johnson, DJD Johnson, EGD Tuddenham, JH McVey (2003)Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS1(9)pp. 1935-1944 BLACKWELL PUBL LTD
G Kemball-Cook, EGD Tuddenham, JH McVey (2007)Normal Haemostasis, In: Postgraduate Haematology: Fifth Editionpp. 783-807
DX Chen, K Riesbeck, G Kemball-Cook, JH McVey, EGD Tuddenham, RI Lechler, A Dorling (1999)Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins, In: TRANSPLANTATION67(3)pp. 467-474 LIPPINCOTT WILLIAMS & WILKINS
CJ Davidson, EG Tuddenham, JH McVey (2003)450 million years of hemostasis, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS1(7)pp. 1487-1494 BLACKWELL PUBL LTD
JH McVEY (2012)Factor seven-activating protease: does it do what it says on the tin?, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS10(5)pp. 857-858 WILEY-BLACKWELL
B Zhang, MA Cunningham, WC Nichols, JA Bernat, U Seligsohn, SW Pipe, JH McVey, U Schulte-Overberg, NB de Bosch, A Ruiz-Saez, GC White, EGD Tuddenham, RJ Kaufman, D Ginsburg (2003)Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex, In: NATURE GENETICS34(2)pp. 220-225 NATURE PUBLISHING GROUP
AB Kritz, CG Nicol, KL Dishart, R Nelson, S Holbeck, DJ Von Seggern, LM Work, JH McVey, SA Nicklin, AH Baker (2007)Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop., In: Mol Ther15(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.

S Shrivastava, L Ma, E-L Tham, JH McVey, D Chen, A Dorling (2013)Protease-activated receptor-2 signalling by tissue factor on dendritic cells suppresses antigen-specific CD4(+) T-cell priming, In: IMMUNOLOGY139(2)pp. 219-226 WILEY-BLACKWELL
DX Chen, K Giannopoulos, PG Shiels, Z Webster, JH McVey, G Kemball-Cook, E Tuddenham, M Moore, R Lechler, A Dorling (2004)Inhibition of intravascular thrombosis in murine endotoxemia by targeted expression of hirudin and tissue factor pathway inhibitor analogs to activated endothelium, In: BLOOD104(5)pp. 1344-1349 AMER SOC HEMATOLOGY
M Steen, EA Norstrom, AL Tholander, PHB Bolton-Maggs, A Mumford, JH McVey, EGD Tuddenham, B Dahlback (2004)Functional characterization of factor V-Ile359Thr: a novel mutation associated with thrombosis, In: BLOOD103(9)pp. 3381-3387 AMER SOC HEMATOLOGY
AL Parker, JH McVey, JH Doctor, O Lopez-Franco, SN Waddington, MJE Havenga, SA Nicklin, AH Baker (2007)Influence of coagulation factor zymogens on the infectivity of adenoviruses pseudotyped with fibers from subgroup D, In: JOURNAL OF VIROLOGY81(7)pp. 3627-3631 AMER SOC MICROBIOLOGY
D Chen, M Weber, PG Shiels, R Dong, Z Webster, JH Mcvey, G Kemball-Cook, EGD Tuddenham, RI Lechler, A Dorling (2006)Postinjury vascular intimal hyperplasia in mice is completely inhibited by CD34+bone marrow-derived progenitor cells expressing membrane-tethered anticoagulant fusion proteins, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS4(10)pp. 2191-2198 BLACKWELL PUBLISHING
PHB Bolton-Maggs, CRM Hay, D Shanks, MJ Mitchel, JH Mcvey (2007)The importance of tissue factor source in the management of Factor VII deficiency, In: THROMBOSIS AND HAEMOSTASIS97(1)pp. 151-152 SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN
M Christian, P Marangos, I Mak, J McVey, F Barker, J White, JJ Brosens (2001)Interferon-gamma modulates prolactin and tissue factor expression in differentiating human endometrial stromal cells, In: ENDOCRINOLOGY142(7)pp. 3142-3151 ENDOCRINE SOC
K Gomez, JH McVey (2006)Tissue factor initiated blood coagulation, In: FRONTIERS IN BIOSCIENCE11pp. 1349-1359 FRONTIERS IN BIOSCIENCE INC
Pratima Chowdhary, Simon Lancaster, Colleen Hamid, David Slatter, Richard Morris, Jonathan H. Foley, Keith Gomez, Edgar Brodkin, Thomas Fox, Alex Gatt, John McVey (2020)Impaired platelet dependent thrombin generation associated with thrombocytopenia is improved by prothrombin complex concentrates in vitro, In: Research and Practice in Thrombosis and Haemostasis4(2)pp. 334-342 Wiley

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 thrombocytopenia.

C Binny, J McIntosh, M Della Peruta, H Kymalainen, EG Tuddenham, SM Buckley, SN Waddington, JH McVey, Y Spence, CL Morton, AJ Thrasher, JT Gray, FJ Castellino, AF Tarantal, AM Davidoff, AC Nathwani (2012)AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage., In: Blood119(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% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration 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.

S Shrivastava, JH McVey, A Dorling (2007)The interface between coagulation and immunity, In: AMERICAN JOURNAL OF TRANSPLANTATION7(3)pp. 499-506 BLACKWELL PUBLISHING
SA Maroney, JP Ferrel, S Pan, TA White, RD Simari, JH McVey, AE Mast (2009)Temporal expression of alternatively spliced forms of tissue factor pathway inhibitor in mice., In: J Thromb Haemost7(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 TFPIalpha existed over 430 Ma while TFPIbeta and TFPIgamma 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 TFPIalpha mRNA is more prevalent than TFPIbeta or TFPIgamma mRNA in mouse tissues, western blot studies demonstrated that TFPIbeta is the primary protein isoform produced in adult tissues, while TFPIalpha is expressed during embryonic development and in placenta. Consistent with TFPIbeta as the primary isoform produced within adult vascular beds, the TFPI isoform in mouse plasma migrates like TFPIbeta in SDS-PAGE and mice have a much smaller heparin-releasable pool of plasma TFPIalpha than humans. CONCLUSIONS: The data demonstrate that alternatively spliced isoforms of TFPI are temporally expressed in mouse tissues at the level of protein production. TFPIalpha and TFPIbeta are produced in embryonic tissues and in placenta while adult tissues produce almost exclusively TFPIbeta.

AA Rahim, AM Wong, S Ahmadi, K Hoefer, SMK Buckley, DA Hughes, AN Nathwani, AH Baker, JH McVey, JD Cooper, SN Waddington (2012)In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression, In: GENE THERAPY19(9)pp. 936-946 NATURE PUBLISHING GROUP
JA Greig, SMK Buckley, SN Waddington, AL Parker, D Bhella, R Pink, AA Rahim, T Morita, SA Nicklin, JH McVey, AH Baker (2009)Influence of Coagulation Factor X on In Vitro and In Vivo Gene Delivery by Adenovirus (Ad) 5, Ad35, and Chimeric Ad5/Ad35 Vectors, In: MOLECULAR THERAPY17(10)pp. 1683-1691 NATURE PUBLISHING GROUP
Irving Donadon, John McVey, Isabella Garagiola, Alessio Branchini, Mimosa Mortarino, Flora Peyvandi, Francesco Bernardi, Mirko Pinotti (2017)Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis/activity, In: Haematologica103(2)pp. 344-350 Ferrata Storti Foundation

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.

D Chen, K Li, E-L Tham, L-L Wei, N Ma, P Dodd, D Kirchhofer, John McVey, A Dorling (2018)Inhibition of angiopoietin-2 production by myofibrocytes inhibits neointimal hyperplasia after endoluminal injury in mice, In: Frontiers in Immunology91517 Frontiers Media

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.

M Di Matteo, E Samara-Kuko, NJ Ward, SN Waddingon, JH McVey, MKL Chuah, T VandenDriessche (2014)Hyperactive PiggyBac Transposons for Sustained and Robust Liver-targeted Gene Therapy, In: MOLECULAR THERAPY22(9)pp. 1614-1624 NATURE PUBLISHING GROUP
John McVey, Pavithra M. Rallapalli, Geoffrey Kemball-Cook, Daniel J. Hampshire, Muriel Giansily-Blaizot, Keith Gomez, Stephen J. Perkins, Christopher A. Ludlam (2020)The EAHAD Coagulation Factor Variant Databases: important resources for haemostasis clinicians and researchers, In: HAEMOPHILIA BLACKWELL SCIENCE LTD

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.

D Chen, L Ma, E-L Tham, S Maresh, RI Lechler, JH Mcvey, A Dorling (2013)Fibrocytes mediate intimal hyperplasia post-vascular injury and are regulated by two tissue factor-dependent mechanisms, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS11(5)pp. 963-974 WILEY-BLACKWELL
JI Elliott, JH McVey, CF Higgins (2005)The P2X(7) receptor is a candidate product of murine and human lupus susceptibility loci: a hypothesis and comparison of murine allelic products, In: ARTHRITIS RESEARCH & THERAPY7(3)pp. R468-R475 BIOMED CENTRAL LTD
AD Mumford, D Chen, A Dorling, G Kemball-Cook, JH McVey (2005)Generation of a polyclonal rabbit anti-mouse tissue factor antibody by nucleic acid immunisation., In: Thromb Haemost93(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.

J Ma, MR Duffy, L Deng, RS Dakin, T Uil, J Custers, SM Kelly, JH McVey, SA Nicklin, AH Baker (2015)Manipulating Adenovirus Hexon Hypervariable Loops Dictates Immune Neutralisation and Coagulation Factor X-dependent Cell Interaction In Vitro and In Vivo, In: PLOS PATHOGENS11(2)ARTN e PUBLIC LIBRARY SCIENCE
G Kemball-Cook, DJD Johnson, O Takamiya, DW Banner, JH McVey, EGD Tuddenham (1998)Coagulation factor VII Gln(100)-> Arg - Amino acid substitution at the epidermal growth factor 2-protease domain interface results in severely reduced tissue factor binding and procoagulant function, In: JOURNAL OF BIOLOGICAL CHEMISTRY273(14)pp. 8516-8521 AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DX Chen, K Riesbeck, JH McVey, G Kemball-Cook, EGD Tuddenham, RI Lechler, A Dorling (1999)Regulated inhibition of coagulation by porcine endothelial cells expressing: P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins, In: TRANSPLANTATION68(6)pp. 832-839 LIPPINCOTT WILLIAMS & WILKINS
DX Chen, M Weber, JH McVey, G Kemball-Cook, EGD Tuddenham, RI Lechler, A Dorling (2004)Complete inhibition of acute humoral rejection using regulated expression of membrane-tethered anticoagulants on xenograft endothelium, In: AMERICAN JOURNAL OF TRANSPLANTATION4(12)pp. 1958-1963 BLACKWELL MUNKSGAARD
L Lenaerts, JH McVey, AH Baker, L Denby, S Nicklin, E Verbeken, L Naesens (2009)Mouse adenovirus type 1 and human adenovirus type 5 differ in endothelial cell tropism and liver targeting., In: J Gene Med11(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.

AD Mumford, M Laffan, J O'Donnell, JH McVey, DJD Johnson, RA Manning, G Kemball-Cook (2002)A Tyr346 -> Cys substitution in the interdomain acidic region a1 of factor VIII in an individual with factor VIII : C assay discrepancy, In: BRITISH JOURNAL OF HAEMATOLOGY118(2)pp. 589-594 BLACKWELL PUBLISHING LTD
K White, H Buening, A Kritz, H Janicki, J McVey, L Perabo, G Murphy, M Odenthal, LM Work, M Hallek, SA Nicklin, AH Baker (2008)Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions, In: GENE THERAPY15(6)pp. 443-451 NATURE PUBLISHING GROUP
JH McVey, M-C Tsai, K-D Chen, C-C Wang (2016)Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling, In: CELL DEATH DISCOVERY1 NATURE PUBLISHING GROUP

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.

JH McVey (2003)Your bleeding heart: lessons from low tissue factor expression in mice, In: TRENDS IN PHARMACOLOGICAL SCIENCES24(6)pp. 269-272 ELSEVIER SCIENCE LONDON
L Denby, LM Work, DJ Von Seggern, E Wu, JH Mcvey, SA Nicklin, AH Baker (2007)Development of renal-targeted vectors through combined in vivo phage display and capsid engineering of adenoviral fibers from serotype 19p, In: MOLECULAR THERAPY15(9)pp. 1647-1654 NATURE PUBLISHING GROUP
C Albrecht, JH McVey, JI Elliott, A Sardini, I Kasza, AD Mumford, RP Naoumova, EGD Tuddenham, K Szabo, CF Higgins (2005)A novel missense mutation in ABCA1 results in altered protein trafficking and reduced phosphatidylserine translocation in a patient with Scott syndrome, In: BLOOD106(2)pp. 542-549 AMER SOC HEMATOLOGY
SA Shestopal, J-J Hao, E Karnaukhova, Y Liang, MV Ovanesov, M Lin, JH Kurasawa, TK Lee, John McVey, AG Sarafanov (2017)Expression and characterization of a codonoptimized blood coagulation factor VIII, In: Journal of Thrombosis and Haemostasis15(4)pp. 709-720 Wiley

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.

EGD Tuddenham, JH McVey (1998)The genetic basis of inhibitor development in haemophilia A, In: HAEMOPHILIA4(4)pp. 543-545 BLACKWELL SCIENCE LTD
AL Parker, SN Waddington, CG Nicol, DM Shayakhmetov, SM Buckley, L Denby, G Kemball-Cook, S Ni, A Lieber, JH McVey, SA Nicklin, AH Baker (2006)Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes, In: BLOOD108(8)pp. 2554-2561 AMER SOC HEMATOLOGY
SN Waddington, JH McVey, D Bhella, AL Parker, K Barker, H Atoda, R Pink, SMK Buckley, JA Greig, L Denby, J Custers, T Morita, IMB Francischetti, RQ Monteiro, DH Barouch, N van Rooijen, C Napoli, MJE Hlavenga, SA Nicklin, AH Baker (2008)Adenovirus serotype 5 hexon mediates liver gene transfer, In: CELL132(3)pp. 397-409 CELL PRESS
R Alba, AC Bradshaw, L Coughlan, L Denby, RA McDonald, SN Waddington, SM Buckley, JA Greig, AL Parker, AM Miller, H Wang, A Lieber, N van Rooijen, JH McVey, SA Nicklin, AH Baker (2010)Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors., In: Blood116(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 macrophage-depleted 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.

AH Baker, JH Mcvey, SN Waddington, NC Di Paolo, DM Shayakhmetov (2007)The influence of blood on in vivo adenovirus bio-distribution and transduction, In: MOLECULAR THERAPY15(8)pp. 1410-1416 NATURE PUBLISHING GROUP
SN Waddington, AL Parker, M Havenga, SA Nicklin, SMK Buckley, JH Mcvey, AH Baker (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, In: JOURNAL OF VIROLOGY81(17)pp. 9568-9571 AMER SOC MICROBIOLOGY
D Chen, S Shrivastava, L Ma, ELL Tham, J Abrahams, JD Coe, D Scott, RI Lechler, JH McVey, A Dorling (2012)Inhibition of thrombin receptor signaling on α-smooth muscle actin(+) CD34(+) progenitors leads to repair after murine immune vascular injury., In: Arterioscler Thromb Vasc Biol32(1)pp. 42-49

OBJECTIVE: The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-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 α-TFPI-transgenic (Tg) mice (H-2(b)) 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 α-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 α-SMA and were recruited to the neointima. In contrast, the α-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. CONCLUSIONS: Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.

R Alba, AC Bradshaw, AL Parker, D Bhella, SN Waddington, SA Nicklin, N van Rooijen, J Custers, J Goudsmit, DH Barouch, JH Mcvey, AH Baker (2009)Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer, In: BLOOD114(5)pp. 965-971 AMER SOC HEMATOLOGY
K Gomez, MA Laffan, G Kemball-Cook, J Pasi, M Layton, JD Singer, EGD Tuddenham, JH McVey (2004)Two novel mutations in severe factor VII deficiency, In: BRITISH JOURNAL OF HAEMATOLOGY126(1)pp. 105-110 BLACKWELL PUBLISHING LTD
AL Parker, SN Waddington, SM Buckley, J Custers, MJ Havenga, N van Rooijen, J Goudsmit, JH McVey, SA Nicklin, AH Baker (2009)Effect of neutralizing sera on factor x-mediated adenovirus serotype 5 gene transfer., In: J Virol83(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.

JH McVey, K Lal, Y Imanaka, G Kemball-Cook, PH Bolton-Maggs, EG Tuddenham (2005)Characterisation of blood coagulation factor XI T475I., In: Thromb Haemost93(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.

H Wang, Y Liu, Z Li, S Tuve, D Stone, O Kalyushniy, D Shayakhmetov, CL Verlinde, T Stehle, J McVey, A Baker, KW Peng, S Roffler, A Lieber (2008)In vitro and in vivo properties of adenovirus vectors with increased affinity to CD46., In: J Virol82(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 CD46(high) 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.

Muriel Giansily‐Blaizot, Pavithra M. Rallapalli, Stephen J. Perkins, Geoffrey Kemball‐Cook, Daniel J. Hampshire, Keith Gomez, Christopher A. Ludlam, John H. McVey (2020)The EAHAD blood coagulation factor VII variant database, In: Human Mutation Wiley

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.

MR Duffy, AC Bradshaw, AL Parker, JH McVey, AH Baker (2011)A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes., In: J Virol85(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.

D Chen, M Xia, C Hayford, E-L Tham, V Semik, S Hurst, Y Chen, HH Tam, J Pan, Y Wang, X Tan, H-Y Lan, H Shen, VV Kakkar, Q Xu, JH McVey, A Dorling (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, In: CIRCULATION131(15)pp. 1350-U101 LIPPINCOTT WILLIAMS & WILKINS
NJ Ward, SM Buckley, SN Waddington, T Vandendriessche, MK Chuah, AC Nathwani, J McIntosh, EG Tuddenham, C Kinnon, AJ Thrasher, JH McVey (2011)Codon optimization of human factor VIII cDNAs leads to high-level expression., In: Blood117(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.

CC Lin, D Chen, JH McVey, DKC Cooper, A Dorling (2008)Expression of tissue factor and initiation of clotting by human platelets and monocytes after incubation with porcine endothelial cells, In: TRANSPLANTATION86(5)pp. 702-709 LIPPINCOTT WILLIAMS & WILKINS
JH McVey, E Boswell, AD Mumford, G Kemball-Cook, EGD Tuddenham (2001)Factor VII deficiency and the FVII mutation database, In: HUMAN MUTATION17(1)pp. 3-17 WILEY-LISS
D David, IMA Santos, K Johnson, EGD Tuddenham, JH McVey (2003)Analysis of the consequences of premature termination codons within factor VIII coding sequences, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS1(1)pp. 139-146 BLACKWELL PUBL LTD
Hatem Maamoun, Matshediso Zachariah, John McVey, Fiona Green, A Agouni (2016)Heme Oxygenase (HO)-1 Induction Prevents Endoplasmic Reticulum Stress-Mediated Endothelial Cell Death and Impaired Angiogenic Capacity, In: Biochemical Pharmacology127pp. 46-59 Elsevier

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)-κB 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.

Rolando S. Matos, Davide Maselli, Christian Heiss, Paola Campagnolo, John Henderson Mcvey (2022)3D printed bioreactor enabling the pulsatile culture of native and angioplastied large arteries, In: Frontiers in Cardiovascular Medicine9864580 Frontiers Media

Routine interventions such as balloon angioplasty, result in vascular activation and remodeling, often requiring re-intervention. 2D in vitro models and small animal experiments have enabled the discovery of important mechanisms involved in this process, however the clinical translation is often underwhelming. There is a critical need for an ex vivo model representative of the human vascular physiology and encompassing the complexity of the vascular wall and the physical forces regulating its function. Vascular bioreactors for ex vivo culture of large vessels are viable alternatives, but their custom-made design and insufficient characterization often hinders the reproducibility of the experiments. The objective of the study was to design and validate a novel 3D printed cost-efficient and versatile perfusion system, capable of sustaining the viability and functionality of large porcine arteries for 7 days and enabling early post-injury evaluations. MultiJet Fusion 3D printing was used to engineer the EasyFlow insert, converting a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine carotid arteries either left untreated or injured with an angioplasty balloon, were cultured under pulsatile flow for up to 7 days. Pressure, heart rate, medium viscosity and shear conditions were adjusted to resemble arterial in vivo hemodynamics. Tissue viability, cell activation and matrix remodeling were analyzed by immunohistochemistry, and vascular function was monitored by duplex ultrasound. Culture conditions in the EasyFlow bioreactor preserved endothelial coverage and smooth muscle organization and extracellular matrix structure in the vessel wall, as compared to static culture. Injured arteries presented hallmarks of early remodeling, such as intimal denudation, smooth muscle cell disarray and media/adventitia activation in flow culture. Duplex ultrasound confirmed continuous pulsatile blood flow conditions, dose-dependent vasodilator response to nitroglycerin in untreated vessels and impaired dilator response in angioplastied vessels. The scope of this work is to validate a low-cost, robust and reproducible system to explore the culture of native and injured large arteries under pulsatile flow. While the study of vascular pathology is beyond the scope of the present paper, our system enables future investigations and provides a platform to test novel therapies and devices ex vivo, in a patient relevant system.

Angelika B Kritz, Campbell G Nicol, Kate L Dishart, Ruth Nelson, Susan Holbeck, Dan J Von Seggern, Lorraine M Work, John H Mcvey, Stuart A Nicklin, Andrew H Baker (2007)Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop Nature Publishing Group

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.

Rolando Matos, Davide Maselli, John Mcvey, Christian Heiss, Paola Campagnolo 3D printed bioreactor enabling the physiological culture and the study of pathological processes in large vessels, In: bioRxiv Cold Spring Harbor Laboratory Press

Routine cardiovascular interventions such as balloon angioplasty, result in vascular activation and remodeling, often requiring re-interventions. 2D in vitro culture models and small animal experiments have enabled the discovery of important molecular and cellular pathways involved in this process, however the clinical translation of these results is often underwhelming. There is a critical need for an ex vivo model representative of the human vascular physiology and encompassing the complexity of the vascular wall and the physical forces regulating its function. Vascular bioreactors for ex vivo culture of large vessels are viable alternatives, but their custom-made design and insufficient characterization often hinders the reproducibility of the experiments. The objective of the study was to design and validate a novel 3D printed cost-efficient and versatile perfusion system, capable of sustaining the viability and functionality of large porcine arteries for 7 days and enabling monitoring of post-injury remodeling. MultiJet Fusion 3D printing technology was used to engineer the EasyFlow insert, converting a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine carotid arteries either left untreated or injured with a conventional angioplasty balloon, were cultured under pulsatile flow for up to 7 days. Pressure, heart rate, medium viscosity and shear conditions were adjusted to represent the typical arterial physiology. Tissue viability, cell activation and matrix remodeling were analyzed by immunohistochemistry, and vascular function was monitored by duplex ultrasound. Physiological blood flow conditions in the EasyFlow bioreactor preserved endothelial coverage and smooth muscle organization and extracellular matrix structure in the vessel wall, as compared to static culture. Injured arteries presented hallmarks of early remodeling, such as intimal denudation, smooth muscle cell disarray and media/adventitia activation. Duplex ultrasound confirmed physiological hemodynamic conditions, dose-dependent vasodilator response to nitroglycerin in untreated vessels and impaired dilator response in angioplastied vessels. We here validate a low-cost, robust and reproducible system to study large blood vessels physiopathology, laying the basis for future investigations into the pathological remodeling of blood vessels and creating a platform to test novel therapies and devices ex vivo in a patient relevant system. Competing Interest Statement The authors have declared no competing interest.

Silvia Lombardi, Gabriele Leo, Simone Merlin, Antonia Follenzi, JOHN HENDERSON MCVEY, Iva Maestri, Francesco Bernardi, Mirko Pinotti, Dario Balestra (2021)Dissection of pleiotropic effects of variants in and adjacent to F8 exon 19 and rescue of mRNA splicing and protein function, In: American Journal of Human Genetics Cell Press

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on Hemophilia A, in which the definition of F8 variants’ causative role and association to bleeding phenotypes is crucial for proper patients’ classification, genetic counseling, and management. We extensively characterized a large panel of Hemophilia A-causing variants (N=30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise HA patients’ classification and favored the development of personalized therapeutic approaches.

Rolando Szilveszter Matos, Akram Jassim Jawad Jawad, Davide Maselli, John H. McVey, Christian Heiss, Paola Campagnolo (2023)Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor, In: JoVE MyJove Corporation

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Hannah Wilkinson, Hugh Leonard, Daxin Chen, Toby Lawrence, Michael Robson, Pieter Goossens, John H McVey, Anthony Dorling (2021)PAR-1 signaling on macrophages is required for effective in vivo delayed-type hypersensitivity responses, In: iScience24(1)101981 Elsevier Inc

Delayed-type hypersensitivity (DTH) responses underpin chronic inflammation. Using a model of oxazolone-induced dermatitis and a combination of transgenic mice, adoptive cell transfer, and selective agonists/antagonists against protease activated receptors, we show that that PAR-1 signaling on macrophages by thrombin is required for effective granuloma formation. Using BM-derived macrophages (BMMs) in vitro, we show that thrombin signaling induced (a) downregulation of cell membrane reverse cholesterol transporter ABCA1 and (b) increased expression of IFNγ receptor and enhanced co-localization within increased areas of cholesterol-rich membrane microdomains. These two key phenotypic changes combined to make thrombin-primed BMMs sensitive to M1 polarization by 1000-fold less IFNγ, compared to resting BMMs. We confirm that changes in ABCA1 expression were directly responsible for the exquisite sensitivity to IFNγ in vitro and for the impact on granuloma formation in vivo. These data indicate that PAR-1 signaling plays a hitherto unrecognized and critical role in DTH responses. [Display omitted] •Inhibiting thrombin signaling on monocytes is protective in type IV hypersensitivity•PTL060, a thrombin inhibitor, reduces inflammation in type IV hypersensitivity•Thrombin primes macrophages to be highly sensitive to IFNγ and LPS•Thrombin increases lipid rafts on macrophages in an ABCA1-dependent manner Molecular Biology; Immunology

G Kemball-Cook, I Garner, Y Imanaka, T Nishimura, DP O'Brien, EG Tuddenham, JH McVey (1994)High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector., In: Gene139(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.

DC Crossman, DP Carr, EG Tuddenham, JD Pearson, JH McVey (1990)The regulation of tissue factor mRNA in human endothelial cells in response to endotoxin or phorbol ester., In: J Biol Chem265(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.

SN Waddington, JH Mcvey, D Bhella, AL Parker, K Barker, K Atoda, R Pink, SMK Buckley, JA Greig, L Denby, J Custers, T Morita, IMB Francischetti, RQ Monteiro, DH Barouch, N van Rooijen, C Napoli, M Havenga, SA Nicklin, AH Baker (2008)A critical role for the adenovirus serotype 5 hexon in liver gene transfer, In: HUMAN GENE THERAPY19(4)pp. 409-409
CA Ludlam, A Cairo, G Dolan, K Gomez, AC Goodeve, DJ Hampshire, G Kemball-Cook, JH Mcvey, J Oldenburg, SJ Perkins, F Peyvandi, PM Rallapalli (2015)The European Association for Haemophilia and Allied Disorders Coagulation Factor Variant Databases, In: HAEMOPHILIA21pp. 37-37
DJ Connolly, LA Cotterill, RA Hederer, CJ Thorpe, PJ Travers, JH McVey, J Dyson, PJ Robinson (1993)A cDNA clone encoding the mouse Qa-1a histocompatibility antigen and proposed structure of the putative peptide binding site., In: J Immunol151(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.

N Salooja, J McVey, P Carmeliet, EGD Tuddenham (1997)Construction of a mouse model of factor IX deficiency., In: BLOOD90(10)pp. 4647-4647
D David, R Schwaab, AIM Santos, J McVey, EGD Tuddenham (1998)Impact of premature stop codons on factor VIII and recombinant re-FVIIISQ pre-mRNA processing, correlation with inhibitor formation, In: EUROPEAN JOURNAL OF HUMAN GENETICS6pp. 125-125
DP Barlow, JH McVey, BL Hogan (1987)Molecular cloning of laminin., In: Methods Enzymol144pp. 464-474

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.

C Daxin, K Riesbeck, J McVey, G Kembell-Cook, EGD Tuddenham, RI Lechler, A Dorling (2000)Expression of novel anticoagulant fusion proteins inhibits factor Xa- and thrombin-induced activation of porcine vascular endothelial cells., In: TRANSPLANTATION69(8)pp. S383-S383
EB Bachli, EGD Tuddenham, JH McVey (1998)Tissue factor expression in a transformed endothelial cell line., In: BLOOD92(10)pp. 92B-92B
A Graham, N Papalopulu, J Lorimer, JH McVey, EG Tuddenham, R Krumlauf (1988)Characterization of a murine homeo box gene, Hox-2.6, related to the Drosophila Deformed gene., In: Genes Dev2(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.

C Ventura, AIM Santos, A Tavares, T Gago, J Lavinha, JH McVey, D David (2000)Molecular genetic analysis of factor XI deficiency: Identification of five novel gene alterations and the origin of type II mutation in Portuguese families, In: THROMBOSIS AND HAEMOSTASIS84(5)pp. 833-840 F K SCHATTAUER VERLAG GMBH
P Houston, MC Dickson, V Ludbrook, B White, JL Schwachtgen, JH McVey, N Mackman, JM Reese, DG Gorman, C Campbell, M Braddock (1999)Fluid shear stress induction of the tissue factor promoter in vitro and in vivo is mediated by Egr-1, In: ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY19(2)pp. 281-289 LIPPINCOTT WILLIAMS & WILKINS
M Neerman-Arbez, KM Johnson, MA Morris, JH McVey, F Peyvandi, WC Nichols, D Ginsburg, C Rossier, SE Antonarakis, EGD Tuddenham (1999)Molecular analysis of the ERGIC-53 gene in 35 families with combined factor V factor VIII deficiency, In: BLOOD93(7)pp. 2253-2260 AMER SOC HEMATOLOGY
S Nomura, S Hashmi, JH McVey, J Ham, M Parker, BL Hogan (1989)Evidence for positive and negative regulatory elements in the 5'-flanking sequence of the mouse sparc (osteonectin) gene., In: J Biol Chem264(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.

AD Mumford, JH McVey (2004)Tissue Factor in the myocardium: Evidence of roles in haemostasis and inflammation, In: DISEASE MARKERS20(6)pp. 353-358 IOS PRESS
SW Pipe, HZ Miao, PF Kucab, JH McVey, RJ Kaufman (2005)The secretion efficiency of factor VIII can be regulated by the size and oligosaccharide content of the B domain., In: BLOOD106(11)pp. 203A-203A
DB Palmer, JH McVey, PJ Robinson, PJ Dyson (1992)The chromatin structure of the mouse beta-2-microglobulin locus., In: Differentiation51(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.

K Gomez, JH McVey, E Tuddenham (2005)Inhibition of coagulation by macromolecular complexes, In: HAEMATOLOGICA-THE HEMATOLOGY JOURNAL90(11)pp. 1570-1576 FERRATA STORTI FOUNDATION
D David, AIM Santos, K Johnson, EGD Tuddenham, JH McVey (1999)mRNA processing, translation and protein secretion from factor VIII genes containing premature stop codons: No correlation with inhibitor development, In: THROMBOSIS AND HAEMOSTASISpp. 238-239 F K SCHATTAUER VERLAG GMBH
PW Holland, SJ Harper, JH McVey, BL Hogan (1987)In vivo expression of mRNA for the Ca++-binding protein SPARC (osteonectin) revealed by in situ hybridization., In: J Cell Biol105(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.

JH McVey, S Nomura, P Kelly, IJ Mason, BL Hogan (1988)Characterization of the mouse SPARC/osteonectin gene. Intron/exon organization and an unusual promoter region., In: J Biol Chem263(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.

R Krumlauf, PW Holland, JH McVey, BL Hogan (1987)Developmental and spatial patterns of expression of the mouse homeobox gene, Hox 2.1., In: Development99(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.

SA Shestopal, JH Kurasawa, E Karnaukhova, J McVey, TK Lee, AG Sarafanov (2015)Expression, purification and characterization of codon optimized coagulation factor VIII, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS13pp. 822-823 WILEY-BLACKWELL
K Riesbeck, DX Chen, G Kemball-Cook, JH McVey, AJT George, EGD Tuddenham, A Dorling, RI Lechler (1998)Expression of hirudin fusion proteins in mammalian cells - A strategy for prevention of intravascular thrombosis, In: CIRCULATION98(24)pp. 2744-2752 LIPPINCOTT WILLIAMS & WILKINS
JA Greig, SMK Buckley, SN Waddington, AL Parker, D Bhella, R Pink, T Morita, J Custers, J Goudsmit, SA Nicklin, JH McVey, AH Baker (2009)Influence of Factor X on In Vitro and In Vivo Gene Delivery by Ad5 and Ad35 Vectors, In: MOLECULAR THERAPY17pp. S325-S325
JH McVey, K Gomez, EGD Tuddenham (2010)Normal Haemostasis, In: AR Green, AV Hoffbrand, D Catovsky, EGD Tuddenham (eds.), Postgraduate Haematology(39)pp. 746-771 Wiley-Blackwell
JA Greig, JH McVey, SN Waddington, AL Parker, SMK Buckley, MJE Havenga, SA Nicklin, AH Baker (2008)Factor X enhances binding and transduction of human cancer cell lines by adenovirus (Ad) serotype 5 vectors but not by Ad35, In: HUMAN GENE THERAPY19(4)pp. 398-398
R Schwaab, M Ludwig, L Kochhan, J Oldenburg, JH McVey, H Egli, HH Brackmann, K Olek (1991)Detection and characterisation of two missense mutations at a cleavage site in the factor VIII light chain., In: Thromb Res61(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.

D David, AI Santos, D Jonson, EGD Tuddenham, JH McVey, G Kemball-Cook (1999)Stable expression and characterization of CRM- (C329S) and a CRM+ (G1948D) factor VIII mutants using the B-domain deleted FVIIISQ variant, In: THROMBOSIS AND HAEMOSTASISpp. 3-3
L Kochhan, MR Lalloz, J Oldenburg, JH McVey, K Olek, HH Brackmann, EG Tuddenham, R Schwaab (1994)Haemophilia A diagnosis by automated fluorescent DNA detection of ten factor VIII intron 13 dinucleotide repeat alleles., In: Blood Coagul Fibrinolysis5(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%.

JI Elliott, AD Mumford, C Albrecht, PW Collins, JC Giddings, CF Higgins, EGD Tuddenham, JH McVey (2004)Characterisation of lymphocyte responses to Ca2+ in Scott syndrome, In: THROMBOSIS AND HAEMOSTASIS91(2)pp. 412-415 SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN
JH McVey, EJ Boswell, O Takamiya, G Tamagnini, V Valente, T Fidalgo, M Layton, EGD Tuddenham (1998)Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency, In: BLOOD92(3)pp. 920-926 W B SAUNDERS CO
M Shibata, M Shima, S Morichika, J McVey, EGD Tuddenham, I Tanaka, H Suzuki, K Nogami, Y Minamoto, T Hato, EL Saenko, D Scandella, A Yoshioka (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, In: THROMBOSIS AND HAEMOSTASIS84(3)pp. 442-448 SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN
ME Griffith, AN Turner, J McVey, CD Pusey (1996)Production of recombinant proteinase 3 recognised by human autoantibodies., In: JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY7(9)pp. A2271-A2271
K White, H Buening, J McVey, G Murphy, L Work, M Hallek, S Nicklin, A Baker (2007)Development and characterisation of viral vectors targeted to atherosclerotic plaques, In: HEART93pp. A99-A99
RL Bold, JH McVey, EGD Tuddenham, G Kemball-Cook (1999)Recombinant expression and characterization of site-directed variants of the human factor VIIa active site catalytic triad, In: THROMBOSIS AND HAEMOSTASISpp. 98-99
MR Lalloz, R Schwaab, JH McVey, K Michaelides, EG Tuddenham (1994)Haemophilia A diagnosis by simultaneous analysis of two variable dinucleotide tandem repeats within the factor VIII gene., In: Br J Haematol86(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.

A Dorling, D Chen, K Riesbeck, J McVey, G Kemball-Cook, EG Tuddenham, RI Lechler (2000)Regulated endothelial cell expression of novel anticoagulants: a strategy for the prevention and therapy of intravascular thrombosis., In: Transplant Proc32(5)pp. 971-?
JH McVey, K Michaelides, LP Hansen, M Ferguson-Smith, S Tilghman, R Krumlauf, EG Tuddenham (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)., In: Hum Mol Genet2(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.

AL Parker, CG Nicol, SN Waddington, SM Buckley, DM Shayakhmetov, SH Ni, A Lieber, JH McVey, L Denby, SA Nicklin, AH Baker (2006)Hepatic Tropism of Adenoviral Type 5 Vectors Can Be Mediated by Multiple Coagulation Factors, In: MOLECULAR THERAPY13pp. S143-S143 NATURE PUBLISHING GROUP
K Michaelides, JH McVey, K Lal, EGD Tuddenham (1999)The cloning and characterisation of the mouse platelet membrane glycoprotein IX gene, In: THROMBOSIS AND HAEMOSTASISpp. 47-48
AC Goodeve, PH Reitsma, JH McVey, Working Group on Nomenclature of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis (2011)Nomenclature of genetic variants in hemostasis., In: J Thromb Haemost9(4)pp. 852-855
DJ Hampshire, A Cairo, G Dolan, M Giansily-Blaizot, K Gomez, AC Goodeve, G Kemball-Cook, CA Ludlam, JH Mcvey, J Oldenburg, SJ Perkins, F Peyvandi, PM Rallapalli (2015)Eahad-DB: a combined coagulation factor variant databases resource for the clinical and scientificcommunities, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS13pp. 997-997 WILEY-BLACKWELL
ME Griffith, AN Turner, J McVey, CD Pusey (1997)Expression of intracellular and secreted recombinant proteinase 3 recognized by ANCA, In: KIDNEY INTERNATIONAL52(1)pp. 277-277
JK Pattinson, JH McVey, M Boon, A Ajani, EG Tuddenham (1990)CRM+ haemophilia A due to a missense mutation (372----Cys) at the internal heavy chain thrombin cleavage site., In: Br J Haematol75(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.

EJ Boswell, EGD Tuddenham, JH McVey (1997)Expression and characterisation of site-directed variants of the EGF-1 domain of human factor VII, In: THROMBOSIS AND HAEMOSTASISpp. PS41-PS41
EPG Mansvelt, M Laffan, JH McVey, EGD Tuddenham (1998)Analysis of the F8 gene in individuals with high plasma factor VIII : C levels and associated venous thrombosis, In: THROMBOSIS AND HAEMOSTASIS80(4)pp. 561-565 F K SCHATTAUER VERLAG GMBH
AL Parker, JH McVey, SN Waddington, SMK Buckley, IMB Francischetti, RQ Monteiro, SA Nicklin, AH Baker (2008)An exosite within the human FX serine protease domain mediates cell transduction of AD5: FX complexes, In: HUMAN GENE THERAPY19(4)pp. 407-407
DP O'Brien, G Kemball-Cook, AM Hutchinson, DM Martin, DJ Johnson, PG Byfield, O Takamiya, EG Tuddenham, JH McVey (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)., In: Biochemistry33(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)

DP OBrien, KM Gale, JS Anderson, John McVey, G Miller, TW Meade, EGD Tuddenham (1991)Purification and characterization of factor-vii 304-gln - a variant molecule with reduced activity isolated from a clinically unaffected male, In: Blood78(1)pp. 132-140 W B Saunders Co.
O Takamiya, S Abe, A Yoshioka, K Nakajima, JH McVey, EG Tuddenham (1995)Factor VIIShinjo: a dysfunctional factor VII variant homozygous for the substitution Gln for Arg at position 79., In: Haemostasis25(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.

E Bachli, CM Pech, KM Johnson, EGD Tuddenham, JH McVey (2000)Coagulation factors Xa and IIa but not VIIa induce a proinflammatory response in dermal fibroblasts., In: BLOOD96(11)pp. 636A-+
F Owen, M Poulter, T Shah, J Collinge, R Lofthouse, H Baker, R Ridley, J McVey, TJ Crow (1990)An in-frame insertion in the prion protein gene in familial Creutzfeldt-Jakob disease., In: Brain Res Mol Brain Res7(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.

D Chen, K Riesbeck, JH McVey, G Kemball-Cook, EGD Tuddenham, RI Lechler, A Dorling (1999)Regulated endothelial cell expression of novel molecules with anticoagulant properties: A strategy for the prevention and therapy of vascular rejection, In: THROMBOSIS AND HAEMOSTASISpp. 12-12
K Riesbeck, A Dorling, G KemballCook, JH McVey, M Jones, EGD Tuddenham, RI Lechler (1997)Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface, In: THROMBOSIS AND HAEMOSTASIS78(6)pp. 1488-1494 F K SCHATTAUER VERLAG GMBH
T Melchionna, J Karegli, CA Farrar, JH McVey, A Dorling, SH Sacks, RAG Smith (2010)Integrating cytotopic strategy in transplantation: (1) Design of locally-acting coagulation inhibitors for use with anti-complement therapeutics, In: MOLECULAR IMMUNOLOGY47(13)pp. 2203-2203
EB Bachli, AC Nathwani, C Pech, EGD Tuddenham, JH McVey (1998)Characterisation of nuclear factors binding to the 5 ' untranslated region of the human tissue factor gene., In: BLOOD92(10)pp. 92B-92B
CR Alshinawi, EJ Boswell, AE Felice, EGD Tuddenham, JH McVey (1997)Characterization of two naturally occurring variants of human factor VII, In: THROMBOSIS AND HAEMOSTASISpp. P1689-P1689
DJ Hampshire, K Gomez, AC Goodeve, G Kemball-Cook, CA Ludlam, JH McVey, J Oldenburg, SJ Perkins, F Peyvandi, PM Rallapalli (2014)The EAHAD coagulation factor variant databases, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS12pp. 105-106 WILEY-BLACKWELL
AC Nathwani, KD Pemberton, EB Bachli, EGD Tuddenham, JH McVey (1997)Expression of the human tissue factor gene in endothelial cells is downregulated by sequences in the 5' untranslated region., In: BLOOD90(10)pp. 1295-1295
C Hamid, J McVey, A Riddell, K Gomez, D Slatter, P Chowdary (2015)Prothrombin complex concentrate can increase thrombin generation in the presence of low platelet number, In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS13pp. 880-880 WILEY-BLACKWELL
AC Nathwani, KM Gale, KD Pemberton, DC Crossman, EG Tuddenham, JH McVey (1994)Efficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter., In: Br J Haematol88(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.

CJ Davidson, RP Hirt, K Lal, P Snell, G Elgar, EGD Tuddenham, JH McVey (2003)Molecular evolution of the vertebrate blood coagulation network, In: THROMBOSIS AND HAEMOSTASIS89(3)pp. 420-428 SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN
MR Lalloz, JH McVey, JK Pattinson, EG Tuddenham (1991)Haemophilia A diagnosis by analysis of a hypervariable dinucleotide repeat within the factor VIII gene., In: Lancet338(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.

RE Pugh, JH McVey, EG Tuddenham, JF Hancock (1995)Six point mutations that cause factor XI deficiency., In: Blood85(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.

RAG Smith, J Karegli, T Melchionna, CA Farrar, JH McVey, A Dorling, SH Sacks (2009)Towards an integrated cytotopic strategy for graft modification in transplantation-Complement and coagulation, In: MOLECULAR IMMUNOLOGY46(14)pp. 2833-2833
JH McVey, K Lal, Y Imanaka, T Nishimura, G Kemball-Cook, PHB Bolton-Maggs, EGD Tuddenham (1999)Identification and characterization of the molecular defect in blood coagulation factor Xi T475I, In: THROMBOSIS AND HAEMOSTASISpp. 285-285
L Lenaerts, AH Baker, JH McVey, S Waddington, E Verbeken, L Naesens (2007)Effect of coagulation factors on liver targeting of mouse adenovirus type 1, In: HUMAN GENE THERAPY18(10)pp. 1032-1032
N Salooja, J McVey, EGD Tuddenham (1998)Towards gene therapy for haemophilia B: Two sensitive ELISAS for mouse FIX antigen., In: BRITISH JOURNAL OF HAEMATOLOGY101pp. 83-83
S Morichika, M Shima, I Tanaka, B Nakai, H Suzuki, K Nogami, T Hato, J McVey, EGD Tuddenham, E Saenko, D Scandella, A Yoshioka (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., In: BLOOD88(10)pp. 2616-2616