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Thromb Haemost 2017; 117(03): 479-490
DOI: 10.1160/TH16-10-0750
Coagulation and Fibrinolysis
Schattauer GmbH

Paradoxical bleeding and thrombotic episodes of dysprothrombinaemia due to a homozygous Arg382His mutation

Qiulan Ding
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Likui Yang
2   Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA
,
Xiaoqing Zhao
3   Dermatology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Wenman Wu
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Xuefeng Wang
1   Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
,
Alireza R. Rezaie
2   Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA
› Author Affiliations Financial support: The research discussed herein was supported in part by grants awarded by the National Heart, Lung, and Blood Institute of the National Institutes of Health (Grants No. HL62565 and HL101917).
Further Information

Publication History

Received: 04 October 2016

Accepted after major revision: 20 November 2016

Publication Date:
28 November 2017 (online)

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Summary

We have characterised the pathogenic basis of dysprothrombinaemia in a patient exhibiting paradoxical bleeding and thrombotic defects during pregnancy and postpartum. Genetic analysis revealed that the proband is homozygous for the prothrombin Arg382His mutation, possessing only ~1 % clotting activity. The proband experienced severe bleeding episodes during her pregnancy, which required treatment with prothrombin complex concentrates, and then pulmonary embolism and deep-vein thrombosis at 28 days postpartum, which required treatment with LMWH and fresh frozen plasma. Analysis of haemostatic parameters revealed that the subject had elevated FDP and DD and decreased fibrinogen levels, indicating the presence of hyperfibrinolysis. Thrombin generation and clotting assays with the proband’s plasma in the presence of soluble thrombomodulin and tissue-type plasminogen activator indicated a defect in activation of both protein C and thrombin activatable fibrinolysis inhibitor (TAFI). Unlike normal plasma, no TAFI activation could be detected in the patient’s plasma. The expression and characterisation of recombinant prothrombin Arg382His indicated that zymogen activation by prothrombinase was markedly impaired and the activation of protein C and TAFI by thrombin-Arg382His was impaired 600-fold and 2500-fold, respectively. The recombinant thrombin mutant exhibited impaired catalytic activity toward both fibrinogen and PAR1 as determined by clotting and signalling assays. However, the mutant activated factor XI normally in both the absence and presence of polyphosphates. Arg382 is a key residue on (pro)exosite-1 of prothrombin and kinetic analysis of substrate activation suggested that the poor zymogenic activity of the mutant is due to its inability to bind factor Va in the prothrombinase complex.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Prothrombin - protein C - fibrinogen - TAFI - fibrinolysis

 

  • References

  • 1 MacGillivray RT, Degen SJ, Chandra T. et al. Cloning and analysis of a cDNA coding for bovine prothrombin. Proc Natl Acad Sci USA 1980; 77: 5153-5157.
    CrossrefPubMedGoogle Scholar
  • 2 Furie B, Furie BC.. The molecular basis of blood coagulation. Cell 1988; 53: 505-518.
    CrossrefPubMedGoogle Scholar
  • 3 Esmon CT, Owen WG, Jackson CM.. The conversion of prothrombin to thrombin V. The activation of prothrombin by factor Xa in the presence of phospholipid. J Biol Chem 1974; 249: 7798-7807.
    PubMedGoogle Scholar
  • 4 Mann KG, Butenas S, Brummel K.. The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003; 23: 17-25.
    CrossrefPubMedGoogle Scholar
  • 5 Pedersen LC, Yee VC, Bishop PD. et al. Transglutaminase factor XIII uses proteinase-like catalytic triad to crosslink macromolecules. Protein Sci 1994; 03: 1131-1135.
    CrossrefPubMedGoogle Scholar
  • 6 Bajzar L, Morser J, Nesheim M.. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem 1996; 271: 16603-16608.
    CrossrefPubMedGoogle Scholar
  • 7 Mosnier LO, Meijers JC, Bouma BN.. Regulation of fibrinolysis in plasma by TAFI and protein C is dependent on the concentration of thrombomodulin. Thromb Haemost 2001; 85: 5-11.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 8 Esmon CT.. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 29-35.
    PubMedGoogle Scholar
  • 9 Dahlbäck B, Villoutreix BO.. Molecular recognition in the protein C anticoagulant pathway. J Thromb Haemost 2003; 01: 1525-1534.
    CrossrefPubMedGoogle Scholar
  • 10 Walker FJ, Fay PJ.. Regulation of blood coagulation by the protein C system. FASEB J 1992; 06: 2561-2567.
    CrossrefPubMedGoogle Scholar
  • 11 Coughlin SR.. Protease-activated receptors in haemostasis, thrombosis and vascular biology. J Thromb Haemost 2005; 03: 1800-1814.
    CrossrefPubMedGoogle Scholar
  • 12 Estevez B, Kim K, Delaney MK. et al. Signalling-mediated cooperativity between glycoprotein Ib-IX and protease-activated receptors in thrombin-induced platelet activation. Blood 2016; 127: 626-636.
    CrossrefPubMedGoogle Scholar
  • 13 Mosnier LO, Griffin JH.. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
    CrossrefPubMedGoogle Scholar
  • 14 Benezra M, Vlodavsky I, Ishai-Michaeli R. et al. Thrombin-induced release of active basic fibroblast growth factor-heparan sulfate complexes from subendothelial extracellular matrix. Blood 1993; 81: 3324-3331.
    PubMedGoogle Scholar
  • 15 Peyvandi F, Mannucci PM.. Rare coagulation disorders. Thromb Haemost 1999; 82: 1207-1214.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 16 Sun WY, Coleman MJ, Witte DP. et al. Rescue of prothrombin-deficiency by transgene expression in mice. Thromb Haemost 2002; 88: 984-991.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Sun WY, Witte DP, Degen JL. et al. Prothrombin deficiency results in embryonic and neonatal lethality in mice. Proc Natl Acad Sci USA 1998; 95: 7597-7602.
    CrossrefPubMedGoogle Scholar
  • 18 Akhavan S, De Cristofaro R, Peyvandi F. et al. Molecular and functional characterisation of a natural homozygous Arg67His mutation in the prothrombin gene of a patient with a severe procoagulant defect contrasting with a mild hemorrhagic phenotype. Blood 2002; 100: 1347-1353.
    CrossrefPubMedGoogle Scholar
  • 19 Acharya SS, Coughlin A, Dimichele DM. North American Rare Bleeding Disorder Study Group. Rare Bleeding Disorder Registry: deficiencies of factors II, V, VII, X, XIII, fibrinogen and dysfibrinogenemias. J Thromb Haemost 2004; 02: 248-256.
    CrossrefPubMedGoogle Scholar
  • 20 Peyvandi F, Duga S, Akhavan S. et al. Rare coagulation deficiencies. Haemophilia 2002; 08: 308-321.
    CrossrefPubMedGoogle Scholar
  • 21 Strijks E, Poort SR, Renier WO. et al. Hereditary prothrombin deficiency presenting as intracranial haematoma in infancy. Neuropediatrics 1999; 30: 320-324.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 22 Girolami A, Scarano L, Saggiorato G. et al. Congenital deficiencies and abnormalities of prothrombin. Blood Coagul Fibrinolysis 1998; 09: 557-569.
    CrossrefPubMedGoogle Scholar
  • 23 Morishita E, Saito M, Kumabashiri I. et al. Prothrombin Himi: a compound heterozygote for two dysfunctional prothrombin molecules (Met-337-->Thr and Arg-388-->His). Blood 1992; 80: 2275-2280.
    PubMedGoogle Scholar
  • 24 Henriksen RA, Owen WG, Nesheim ME. et al. Identification of a congenital dysthrombin, thrombin Quick. J Clin Invest 1980; 66: 934-940.
    CrossrefPubMedGoogle Scholar
  • 25 O’Marcaigh AS, Nichols WL, Hassinger NL. et al. Genetic analysis and functional characterisation of prothrombins Corpus Christi (Arg382-Cys), Dhahran (Arg271-His), and hypoprothrombinaemia. Blood 1996; 88: 2611-2618.
    PubMedGoogle Scholar
  • 26 Sekine O, Sugo T, Ebisawa K. et al. Substitution of Gly-548 to Ala in the substrate binding pocket of prothrombin Perijá leads to the loss of thrombin proteolytic activity. Thromb Haemost 2002; 87: 282-287.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 27 Miyawaki Y, Suzuki A, Fujita J. et al. (2012) Thrombosis from a prothrombin mutation conveying antithrombin resistance. N Engl J Med 2012; 366: 2390-2396.
    CrossrefPubMedGoogle Scholar
  • 28 Djordjevic V, Kovac M, Miljic P. et al. A novel prothrombin mutation in two families with prominent thrombophilia--the first cases of antithrombin resistance in a Caucasian population. J Thromb Haemost 2013; 11: 1936-1939.
    CrossrefPubMedGoogle Scholar
  • 29 Sivasundar S, Oommen AT, Prakash O. et al. Molecular defect of ’Prothrombin Amrita’: substitution of arginine by glutamine (Arg553 to Gln) near the Na(+) binding loop of prothrombin. Blood Cells Mol Dis 2013; 50: 182-183.
    CrossrefPubMedGoogle Scholar
  • 30 Bulato C, Radu CM, Campello E. et al. New Prothrombin Mutation (Arg596Trp, Prothrombin Padua 2) Associated With Venous Thromboembolism. Arterioscler Thromb Vasc Biol 2016; 36: 1022-1029.
    CrossrefPubMedGoogle Scholar
  • 31 Bode W, Mayr I, Baumann U. et al. The refined 1. 9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment EMBO J 1989; 08: 3467-3475.
    PubMedGoogle Scholar
  • 32 Lane DA, Philippou H, Huntington JA.. Directing thrombin. Blood 2005; 106: 2605-2612.
    CrossrefPubMedGoogle Scholar
  • 33 Rezaie AR.. Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin. Protein Sci 1998; 07: 349-357.
    PubMedGoogle Scholar
  • 34 Yang L, Manithody C, Rezaie AR.. Activation of protein C by the thrombin-thrombomodulin complex: cooperative roles of Arg-35 of thrombin and Arg-67 of protein C. Proc Natl Acad Sci USA 2006; 103: 879-884.
    CrossrefPubMedGoogle Scholar
  • 35 Chen L, Yang L, Rezaie AR.. Proexosite-1 on prothrombin is a factor Va-dependent recognition site for the prothrombinase complex. J Biol Chem 2003; 278: 27564-27569.
    CrossrefPubMedGoogle Scholar
  • 36 Mosnier LO, von dem Borne PA, Meijers JC. et al. Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation. Thromb Haemost 1998; 80: 829-835.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Geng Y, Verhamme IM, Smith SA. et al. Factor XI anion-binding sites are required for productive interactions with polyphosphate. J Thromb Haemost 2013; 11: 2020-2028.
    CrossrefPubMedGoogle Scholar
  • 38 Ding Q, Yang L, Dinarvand P. et al. Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays. Blood 2015; 125: 2428-2434.
    CrossrefPubMedGoogle Scholar
  • 39 Wang W, Nagashima M, Schneider M. et al. Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation. J Biol Chem 2000; 275: 22942-22947.
    CrossrefPubMedGoogle Scholar
  • 40 Anderson PJ, Nesset A, Dharmawardana KR. et al. Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation. J Biol Chem 2000; 275: 16435-16442.
    CrossrefPubMedGoogle Scholar
  • 41 Rezaie AR, Cooper ST, Church FC. et al. Protein C inhibitor is a potent inhibitor of the thrombin-thrombomodulin complex. J Biol Chem 1995; 270: 25336-25339.
    CrossrefPubMedGoogle Scholar
  • 42 Yang L, Manithody C, Qureshi SH, Rezaie AR.. Factor Va alters the conformation of the Na+-binding loop of factor Xa in the prothrombinase complex. Biochemistry 2008; 47: 5976-5985.
    CrossrefPubMedGoogle Scholar
  • 43 Müller F, Mutch NJ, Schenk WA. et al. Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell 2009; 139: 1143-1156.
    CrossrefPubMedGoogle Scholar
  • 44 Choi SH, Smith SA, Morrissey JH.. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood 2011; 118: 6963-6970.
    CrossrefPubMedGoogle Scholar
  • 45 Von dem Borne PA, Bajzar L. et al. Thrombin-mediated activation of factor XI results in a thrombin-activatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis. J Clin Invest 1997; 99: 2323-2327.
    CrossrefPubMedGoogle Scholar
  • 46 Tollefsen DM.. Vascular dermatan sulfate and heparin cofactor II. Prog Mol Biol Transl Sci 2010; 93: 351-372.
    CrossrefPubMedGoogle Scholar
  • 47 Bae JS, Kim YU, Park MK. et al. Concentration dependent dual effect of thrombin in endothelial cells via PAR-1 and PI3 Kinase. J Cell Physiol 2009; 219: 744-751.
    CrossrefPubMedGoogle Scholar