Department of Medicine

Haemostasis & Thrombosis

The haemostasis and thrombosis research within the Centre for Haematology is driven by the collaborative interests of Prof David Lane, Prof Mike Laffan, Dr Jim Crawley and Dr Carolyn Millar. Thrombosis, bleeding disorders and diseases of blood vessels are among the most prevalent causes of premature death in Western society and a major cause of morbidity. Advances in the basic science underlying the function of the enzymes, cofactors and inhibitory components of haemostasis are urgently needed to find ways of preventing the onset and progression of disease, and to improve the treatment of affected individuals.

Prof David Lane, Prof Mike Laffan, Dr Jim Crawley and Dr Carolyn Millar have all had long-standing interests in haemostatic mechanisms.

The major physiological anticoagulant pathways, involving tissue factor pathway inhibitor (TFPI), protein C and antithrombin pathways, are important for normal blood fluidity. Inherited deficiency of components of these pathways caused by gene defects can predispose individuals towards thrombosis.

Previous studies have involved how protein C interacts with the endothelial cell protein C receptor (EPCR), and its cofactor, protein S, and also the study of the gene regulation of the EPCR, and how its expression is specifically directed to large vessel endothelial cells.

Current work includes projects that focus on the characterisation of the cytoprotective signalling function of APC. Activated protein C (APC) is a natural anticoagulant protein that in recent years has been found to transduce cytoprotective signals to endothelial cells. This signalling function has potentially major therapeutic implications for the treatment of patients with severe sepsis, or that have suffered from a stroke. Other anticoagulant research interests include structure/function studies on protein S and tissue factor pathway inhibitor (TFPI) and the physiological importance of their anticoagulant function.

Protein S Gla domain

Figure 1: Model of the N-terminal domains of protein S ( Gla domain, thrombin sensitive region - TSR, and epidermal growth factor-like domain 1 - EGF1).

 

Highlighted in red is Asp95 in EGF1 that we have shown is critical to the APC cofactor function of protein S. The gla domain with associated calcium ions is also important for  phospholipid binding and APC cofactor function. Highlighted in blue and purple are Face 1 & 2 of the Gla domain that others have suggested to contribute to protein S function (Andersson et al; Blood 2010)

 

The Haemostasis group also conducts research projects on von Willebrand factor (VWF) - a key protein that mediates platelet tethering at sites of vascular injury, and ADAMTS13 - a metalloprotease that cleaves VWF and thus modulates its function.

Abnormalities or deficiencies of these important proteins may result in thrombotic or haemorrhagic disorders such as thrombotic thrombocytopenic purpura and von Willebrand disease, respectively and are also risk factors for cardiovascular disease.
Current ADAMTS13 research activity includes structure/function studies that aim to delineate how ADAMTS13 interacts with and proteolyses VWF, how calcium modulates the activity of ADAMTS13, how the high degree of substrate specificity of ADAMTS13 is conferred, how plasma ADAMTS13 levels/activity influence thrombotic risk and the influence of anti-ADAMTS13 autoantibodies in acquired TTP patients.

S3S1

Figure 2 Model of the ADAMTS13 active centre aligned with VWF.

ADAMTS13 metalloprotease (MP - light blue) and disintegrin-like (Dis - grey) domains is shown with the area in the box enlarged to illustrate specific positioning of the scissile bond and potential docking points for the VWF peptide around the scissile bond. ADAMTS13 residues implicated in VWF proteolysis derived from the present and other reports are highlighted; Leu198, Leu232, Leu274 (dark blue), Val195, Leu151 (dark brown), Arg349 (green), active centre (light blue, with Zn2+ shown in brown) with catalytic Glu225 and residues flanking the S1’ pocket (purple). The S1 pocket that harbours the P1 residue Tyr1605 is predicted to lie around the Val195/Leu151 cluster in this model. We propose that the cluster of Leu198, Leu232 and Leu274 provides the S3 subsite for VWF Leu1603. Beneath the figure is the VWF1603-1614 polypeptide (shown to scale), with identified interacting residues (P3, P1, P1’ and P9’) highlighted in complementary colours. (Xiang et al PNAS (2011)

 

ADAMTS13 MP S1

Figure 3: Location of the S1' pocket in the ADAMTS13 metalloprotease domain.

 

Homology model of the ADAMTS13 MP-Dis. The metalloprotease domain (MP) is shown in grey with the three active site His (dark blue) that coordinate a catalytic zinc ion (light blue) highlighted. The Disintegrin-like domain (Dis) is depicted in light green. Adjacent to the catalytic centre is a deep pocket (circled) that is lined by amino acids Asp252 to Pro256 (red) in a region termed VR3B(I). This pocket specifically accomodates the R-group of the P1' residue in VWF (Met1606). In this way, this pocket influences both ADAMTS13 activity and cleavage site specificity (de Groot et al; Blood 2010).

 

 

 

ADAMTS13 MP-Dis

Figure 4: Model depicting the proposed role of the ADAMTS13 Disintegrin-like domain.

 

Homology model of the ADAMTS13 MP-Dis. The metalloprotease domain (MP) is shown in light blue with the three active site His (dark blue) that coordinate a catalytic zinc ion (pink) highlighted. The Disintegrin-like domain (Dis) is depicted in light green, light pink and red. The hypervariable region (HVR) is highlighted in light pink with Arg 349 and Leu350 highlighted in red. These two amino acids lie adjacent to the active site cleft in the MP domain. Arg349 is located ~26Å from the active site zinc ion. The catalytic efficiency of ADAMTS13 is reduced 10-20 fold when either Arg349 or Leu350 are mutated. We propose a model in which Asp1614 in the VWF A2 domain (located ~26Å from the Tyr1605-Met1606 scissile bond) interacts with Arg 349 in the ADAMTS13 Dis domain. This interaction assists in positioning the scissile bond into the the active site of ADAMTS13 (de Groot et al; Blood 2009)

 

ADAMTS13 Spacer

Figure 5: Model of the ADAMTS13 Metalloprotease domain (MP), Disintegrin-like domain (Dis), TSP1, Cystein-rich domain (Cys) and Spacer domain.

 

The active site zinc ion is highlighted (purple). The critical residues involved in the Dis exosite (Fig 3) are shown in dark green (de Groot et al; Blood 2009). Residues Arg660, Tyr661 and Tyr665 in the Spacer domain are central to both the binding and proteolysis of VWF by ADAMTS13 (Pos et al; Blood 2010).

 

 

Plasma levels of VWF are closely dependent on its numerous O- and N-linked sugar chains. Current research projects are exploring the functional consequences of N- and O-linked glycosylation on VWF conformation and on its many interactions with other molecules. Collaborative projects are producing a detailed glycan map of VWF and exploring the role of VWF in endothelial cell function.

Platelets are central to normal haemostasis and defects in both platelet number and function are causes of both bleeding and thrombotic diseases in humans. Platelets are formed by megakaryocytes in the bone marrow and thereafter released into the blood. The formation and function of platelets is influenced by multiple factors among those are cell surface receptors that transduce molecular signals. Transforming growth factor ß (TGF-ß) and bone morphogenic protein (Bmp) signalling are transduced through type I and type II TGF-ß superfamily receptors. Receptor signalling is modulated by BAMBI - a TGF-ß pseudoreceptor that inhibits receptor function. As part of the Bloodomics consortium effort to discover novel genes linked to the development/severity of coronary artery disease and myocardial infraction, BAMBI, along with other novel genes has been associated with platelet function. BAMBI has been implicated in several cancers however its role in thrombosis and haemostasis remains unknown. The Haemostasis group is currently examining how BAMBI modulates both platelet production and platelet function and its contribution to thrombosis.

BAMBI function

Figure 6: Proposed role of BAMBI in cell signalling.

 

TGF-β signalling is mediated through binding to type I and type II receptor complexes that in turn transduce the signal (A). BAMBI specifically modulates TGF-β signalling by complexing with and inhibiting type I and type II receptor function (B).

 

Biographical sketch

Prof David Lane is a Professor of Molecular Haematology in the Department of Haematology. He obtained his PhD in 1974 from the University of London. He joined Imperial College (then Charing Cross and Westminster Medical School) in 1979. He is Editor-in-Chief of the Journal of Thrombosis & Haemostasis.

Prof Mike Laffan a Professor of Haemostasis and Thrombosis in the Department of Haematology. He qualified in medicine from Oxford University in 1981 and obtained his DM in 1993. Prof Mike Laffan joined Imperial College in 1992 and is also director of the Hammersmith Hospital Haemophilia Centre.

Dr Jim Crawley is a non-clinical senior lecturer. He graduated from Durham University in with a BSc in Molecular Biology and Biochemistry, and obtained his PhD from Imperial College London in 2001. He joined the Haematology Department in 2002. Dr Jim Crawley is Scientific Editor for the Journal of Thrombosis & Haemostasis.

Dr Carolyn Millar  is a clinical senior lecturer. She joined Imperial College in 2008.

Haemostasis Group Members

Group Leaders

 Prof David Lane, Prof Mike Laffan, Dr Jim Crawley, Dr Carolyn Millar

Post-doctoral Scientists

 Dr Brenda Luken, Dr Rens de Groot, Dr Isabelle Salles, Dr Josefin Ahnstrom, Dr Anna Andreou, Dr Helena Andersson,

 Dr Tom McKinnon, Dr Ayesha Khan, Dr Agata Nowak

Clinical Fellows

 Dr Susan Shapiro, Dr Sarah Mangles, Dr Mari Thomas (UCL)

PhD Students

Yu (Jessica) Yao, Yaozu Xiang

Selected Publications

  1. Crawley JT, de Groot R, Xiang Y, Luken BM, Lane DA. Unravelling the scissile bond: how ADAMTS13 recognises and cleaves von Willebrand factor. Blood 2011 (in press)
  2. Xiang Y, de Groot R, Crawley JT, Lane DA. Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13 (ADAMTS13). Proc Natl Acad Sci U S A. 2011;(in press).
  3. Ahnström J, Andersson HM, Canis K, Norstrøm E, Yu Y, Dahlbäck B, Panico M, Morris HR, Crawley JT, Lane DA. Activated protein C cofactor function of protein S: a novel role for a {gamma}-carboxyglutamic acid residue. Blood. 2011;117:6685-93.
  4. Starke RD, Ferraro F, Paschalaki KE, Dryden NH, McKinnon TA, Sutton RE, Payne EM, Haskard DO, Hughes AD, Cutler DF, Laffan MA, Randi AM. Endothelial von Willebrand factor regulates angiogenesis. Blood. 2011;117:1071-80.
  5. de Groot R, Lane DA, Crawley JT. The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity. Blood. 2010;116:3064-72.
  6. McKinnon TA, Goode EC, Birdsey GM, Nowak AA, Chan AC, Lane DA, Laffan MA. Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor. Blood. 2010;116:640-8.
  7. Goodall AH, Burns P, Salles I, Macaulay IC, Jones CI, Ardissino D, de Bono B, Bray SL, Deckmyn H, Dudbridge F, Fitzgerald DJ, Garner SF, Gusnanto A, Koch K, Langford C, O'Connor MN, Rice CM, Stemple DL, Stephens J, Trip MD, Zwaginga JJ, Samani NJ, Watkins NA, Maguire PB, Ouwehand WH. Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function. Blood. 2010;116:4646-56
  8. Luken BM, Winn LY, Emsley J, Lane DA, Crawley JT. The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function. Blood. 2010;115:4910-3.
  9. Andersson HM, Arantes MJ, Crawley JT, Luken BM, Tran S, Dahlbäck B, Lane DA, Rezende SM. Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain. Blood. 2010;115:4878-85.
  10. Pos W, Crawley JT, Fijnheer R, Voorberg J, Lane DA, Luken BM. An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF. Blood. 2010;115:1640-9.
  11. McGrath RT, McKinnon TA, Byrne B, O'Kennedy R, Terraube V, McRae E, Preston RJ, Laffan MA, O'Donnell JS. Expression of terminal alpha2-6-linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13. Blood. 2010;115:2666-73.
  12. Salles, II, Thijs T, Brunaud C, De Meyer SF, Thys J, Vanhoorelbeke K, Deckmyn H. Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34(+) cells are functionally active in an ex vivo flow model of thrombosis. Blood. 2009;114:5044-5051.
  13. Riddell AF, Gomez K, Millar CM, Mellars G, Gill S, Brown SA, Sutherland M, Laffan MA, McKinnon TA. Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor. Blood. 2009;114:3489-96.
  14. Zanardelli S, Chion AC, Groot E, Lenting PJ, McKinnon TA, Laffan MA, Tseng M, Lane DA. A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF. Blood. 2009;114:2819-28.
  15. de Groot R, Bardhan A, Ramroop N, Lane DA, Crawley JT. Essential role of the disintegrin-like domain in ADAMTS13 function. Blood. 2009;113:5609-16.
  16. Gardner MD, Chion CK, de Groot R, Shah A, Crawley JT, Lane DA. A functional calcium-binding site in the metalloprotease domain of ADAMTS13. Blood. 2009;113:1149-57.
  17. O'Connor MN, Salles, II, Cvejic A, Watkins NA, Walker A, Garner SF, Jones CI, Macaulay IC, Steward M, Zwaginga JJ, Bray SL, Dudbridge F, de Bono B, Goodall AH, Deckmyn H, Stemple DL, Ouwehand WH. Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins. Blood. 2009;113:4754-4762.
  18. McKinnon TA, Chion AC, Millington AJ, Lane DA, Laffan MA. N-linked glycosylation of VWF modulates its interaction with ADAMTS13. Blood. 2008; 111: 3042-9.
  19. Ghevaert C, Salsmann A, Watkins NA, Schaffner-Reckinger E, Rankin A, Garner SF, Stephens J, Smith GA, Debili N, Vainchenker W, de Groot PG, Huntington JA, Laffan M, Kieffer N, Ouwehand WH. A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the alphaIIbbeta3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia. Blood. 2008; 111: 3407-14.
  20. Crawley JT, Lane DA. The haemostatic role of tissue factor pathway inhibitor. Arterioscler Thromb Vasc Biol. 2008; 28: 233-42.
  21. Chion CK, Doggen CJ, Crawley JT, Lane DA, Rosendaal FR. ADAMTS13 and von Willebrand factor and the risk of myocardial infarction in men. Blood. 2007; 109: 1998-2000.
  22. Zanardelli S, Crawley JT, Chion CK, Lam JK, Preston RJ, Lane DA. ADAMTS13 substrate recognition of von Willebrand factor A2 domain. J Biol Chem. 2006; 281: 1555-63.
  23. Preston RJ, Ajzner E, Razzari C, Karageorgi S, Dua S, Dahlback B, Lane DA. Multifunctional specificity of the protein C/activated protein C Gla domain. J Biol Chem. 2006; 281: 28850-7.
  24. Mollica LR, Crawley JT, Liu K, Rance JB, Cockerill PN, Follows GA, Landry JR, Wells DJ, Lane DA. Role of a 5'-enhancer in the transcriptional regulation of the human endothelial cell protein C receptor gene. Blood. 2006; 108: 1251-9.
  25. O'Donnell JS, McKinnon TA, Crawley JT, Lane DA, Laffan MA. Bombay phenotype is associated with reduced plasma-VWF levels and an increased susceptibility to ADAMTS13 proteolysis. Blood. 2005; 106: 1988-91.
  26. Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood. 2005; 106: 2605-12.
  27. Crawley JT, Lam JK, Rance JB, Mollica LR, O'Donnell JS, Lane DA. Proteolytic inactivation of ADAMTS13 by thrombin and plasmin. Blood. 2005; 105: 1085-93.
  28. Rezende SM, Simmonds RE, Lane DA. Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S-C4b binding protein complex. Blood. 2004; 103: 1192-201.
  29. Hurtado V, Montes R, Gris JC, Bertolaccini ML, Alonso A, Martinez-Gonzalez MA, Khamashta MA, Fukudome K, Lane DA, Hermida J. Autoantibodies against EPCR are found in antiphospholipid syndrome and are a risk factor for fetal death. Blood. 2004; 104: 1369-74.
  30. Rance JB, Follows GA, Cockerill PN, Bonifer C, Lane DA, Simmonds RE. Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites. Blood. 2003; 101: 4393-401.
  31. Raja SM, Chhablani N, Swanson R, Thompson E, Laffan M, Lane DA, Olson ST. Deletion of P1 arginine in a novel antithrombin variant (antithrombin London) abolishes inhibitory activity but enhances heparin affinity and is associated with early onset thrombosis. J Biol Chem. 2003; 278: 13688-95.
  32. Philippou H, Rance J, Myles T, Hall SW, Ariens RA, Grant PJ, Leung L, Lane DA. Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation. Competition for cofactor sites on thrombin determines its fate. J Biol Chem. 2003; 278: 32020-6.
  33. Mille-Baker B, Rezende SM, Simmonds RE, Mason PJ, Lane DA, Laffan MA. Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity. Blood. 2003; 101: 1416-8.
  34. Rezende SM, Lane DA, Mille-Baker B, Samama MM, Conard J, Simmonds RE. Protein S Gla-domain mutations causing impaired Ca(2+)-induced phospholipid binding and severe functional protein S deficiency. Blood. 2002; 100: 2812-9.
  35. Ouyang YB, Crawley JT, Aston CE, Moore KL. Reduced body weight and increased postimplantation fetal death in tyrosylprotein sulfotransferase-1-deficient mice. J Biol Chem. 2002; 277: 23781-7.
  36. O'Donnell J, Boulton FE, Manning RA, Laffan MA. Amount of H antigen expressed on circulating von Willebrand factor is modified by ABO blood group genotype and is a major determinant of plasma von Willebrand factor antigen levels. Arterioscler Thromb Vasc Biol. 2002; 22: 335-41.
  37. Luttun A, Lupu F, Storkebaum E, Hoylaerts MF, Moons L, Crawley J, Bono F, Poole AR, Tipping P, Herbert JM, Collen D, Carmeliet P. Lack of plasminogen activator inhibitor-1 promotes growth and abnormal matrix remodeling of advanced atherosclerotic plaques in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2002; 22: 499-505.
  38. Kunz G, Ohlin AK, Adami A, Zoller B, Svensson P, Lane DA. Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function. Blood. 2002; 99: 3646-53.
  39. Gu JM, Crawley JT, Ferrell G, Zhang F, Li W, Esmon NL, Esmon CT. Disruption of the endothelial cell protein C receptor gene in mice causes placental thrombosis and early embryonic lethality. J Biol Chem. 2002; 277: 43335-43.
  40. Crawley JT, Goulding DA, Ferreira V, Severs NJ, Lupu F. Expression and localization of tissue factor pathway inhibitor-2 in normal and atherosclerotic human vessels. Arterioscler Thromb Vasc Biol. 2002; 22: 218-24.
  41. Lane DA, Grant PJ. Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease. Blood. 2000; 95: 1517-32.
  42. Kunz G, Ireland HA, Stubbs PJ, Kahan M, Coulton GC, Lane DA. Identification and characterization of a thrombomodulin gene mutation coding for an elongated protein with reduced expression in a kindred with myocardial infarction. Blood. 2000; 95: 569-76.
  43. Crawley J, Lupu F, Westmuckett AD, Severs NJ, Kakkar VV, Lupu C. Expression, localization, and activity of tissue factor pathway inhibitor in normal and atherosclerotic human vessels. Arterioscler Thromb Vasc Biol. 2000; 20: 1362-73.
  44. Ariens RA, Philippou H, Nagaswami C, Weisel JW, Lane DA, Grant PJ. The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure. Blood. 2000; 96: 988-95.
  45. Al-Obaidi MK, Philippou H, Stubbs PJ, Adami A, Amersey R, Noble MM, Lane DA. Relationships between homocysteine, factor VIIa, and thrombin generation in acute coronary syndromes. Circulation. 2000; 101: 372-7.
  46. Simmonds RE, Lane DA. Structural and functional implications of the intron/exon organization of the human endothelial cell protein C/activated protein C receptor (EPCR) gene: comparison with the structure of CD1/major histocompatibility complex alpha1 and alpha2 domains. Blood. 1999; 94: 632-41.
  47. Bayston TA, Tripodi A, Mannucci PM, Thompson E, Ireland H, Fitches AC, Hananeia L, Olds RJ, Lane DA. Familial overexpression of beta antithrombin caused by an Asn135Thr substitution. Blood. 1999; 93: 4242-7.

 

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Academics

Researchers

Clinical Fellows

  • Dr Sarah Mangles
    (PhD student)
  • Dr Susie Shapiro
    (PhD student)
  • Dr Mari Thomas
    (PhD student)

PhD Students

  • Yaozu Xiang
    (PhD student)
  • Yao Yu
    (PhD Student)