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Thromb Haemost 2009; 102(04): 623-633
DOI: 10.1160/TH09-04-0238
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Differences in prethrombin-1 activation with human or bovine factor Va can be attributed to the heavy chain

Paul Y. Kim
1   Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada
,
Reginald Manuel
1   Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada
,
Michael E. Nesheim
1   Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada
2   Department of Medicine, Queen’s University, Kingston, Ontario, Canada
› Author AffiliationsFinancial support: This work was supported by grant MT-9781 from the Canadian Institutes of Health Research (to MEN) and by grant HS4441 from the Heart and Stroke Foundation of Canada (to MEN). This work was also supported by a Heart and Stroke Foundation of Canada Doctoral Research Award (to PYK).
Further Information

Publication History

Received: 09 April 2009

Accepted after major revision: 02 July 2009

Publication Date:
24 November 2017 (online)

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Summary

Human and bovine factor Va (FVa) function similarly in the activation of prothrombin but differently in the activation of prethrombin-1 (Pre-1). Pre-1 activation with human FVa proceeds at about 22 percent of the rate with bovine FVa. The dependencies of initial rates on the FVa and Pre-1 concentrations indicate that the differential activity is expressed in kcat differences, rather than differences in the assembly of prothrombinase or the Km value of the substrate. The heavy and light chains of both species of FVa were separated and interspecies hybrids were constructed in the presence of Ca++. Studies of the activation of Pre-1 with these hybrids indicate that the species difference can be attributed specifically to the heavy chain of FVa. Analyses of the reactions by SDS-PAGE indicated that cleavage at Arg271 occurs at about the same rate with both species of FVa, but cleavage at Arg320 with human FVa is specifically retarded. A major difference in primary structure between the human and bovine FVa heavy chains comprises 10 residues at COOH-terminus, adjacent to the negatively charged hirudin-like DYDYQ sequence. These residues have pI values of 12.5 and 4.26 in human and bovine FVa, respectively. The lower value would complement the negatively charged DYDYQ sequence but the higher value would counteract it. Thus, we suggest that the differences in the COOH-terminus of the heavy chain are responsible for the differences in Pre-1 activation, and that it specifically influences cleavage at Arg320 in Pre-1.

Keywords

Coagulation factors - factor Va - prethrombin-1 - prothrombin - activation
 

  • References

  • 1 Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Ann Rev Biochem 1988; 57: 915-956.
    CrossrefPubMedGoogle Scholar
  • 2 Mann KG, Nesheim ME, Church WR. et al. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16.
    PubMedGoogle Scholar
  • 3 Esmon CT. The subunit structure of thrombin-activated factor V isolation of activated factor V, separation of subunits, and reconstitution of biological activity. J Biol Chem 1979; 254: 964-973.
    PubMedGoogle Scholar
  • 4 Nesheim ME, Katzmann JA, Tracy PB. et al. Factor V. Methods Enzymol 1981; 80: 249-274.
    PubMedGoogle Scholar
  • 5 Nesheim ME, Mann KG. Thrombin-catalyzed activation of single chain bovine factor V. J Biol Chem 1979; 254: 1326-1334.
    PubMedGoogle Scholar
  • 6 Foster WB, Nesheim ME, Mann KG. The factor Xa-catalyzed activation of factor V. J Biol Chem 1983; 258: 13970-13977.
    PubMedGoogle Scholar
  • 7 Nesheim ME, Foster WB, Hewick R. et al. Characterization of factor V activation intermediates. J Biol Chem 1984; 259: 3187-3196.
    PubMedGoogle Scholar
  • 8 Jenny RJ, Pittman DD, Toole JJ. et al. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84: 4846-4850.
    CrossrefPubMedGoogle Scholar
  • 9 Guinto ER, Esmon CT, Mann KG. et al. The complete cDNA sequence of bovine coagulation factor V. J Biol Chem 1992; 267: 2971-2978.
    PubMedGoogle Scholar
  • 10 Mann KG, Kalafatis M. Factor V: a combination of Dr Jekyll and Mr Hyde. Blood 2003; 101: 20-30.
    CrossrefPubMedGoogle Scholar
  • 11 Krishnaswamy S, Russell GD, Mann KG. The reassociation of factor Va from its isolated subunits. J Biol Chem 1989; 264: 3160-3168.
    PubMedGoogle Scholar
  • 12 Mann KG. Prothrombin. Methods Enzymol 1976; 45: 123-156.
    PubMedGoogle Scholar
  • 13 Owen WG, Esmon CT, Jackson CM. The conversion of prothrombin to thrombin; 1. Characterization of the reaction products formed during the activation of bovine prothrombin. J Biol Chem 1974; 249: 594-605.
    PubMedGoogle Scholar
  • 14 Bukys MA, Orban T, Kim PY. et al. The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase. Thromb Haemost 2008; 99: 511-522.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Pryzdial EL, Mann KG. The association of coagulation factor Xa and factor Va. J Biol Chem 1991; 266: 8969-8977.
    PubMedGoogle Scholar
  • 16 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol Chem 1979; 254: 10952-10962.
    PubMedGoogle Scholar
  • 17 Tracy PB, Mann KG. Prothrombinase complex assembly on the platelet surface is mediated through the 74,000-dalton component of factor Va. Proc Natl Acad Sci 1983; 80: 2380-2384.
    CrossrefPubMedGoogle Scholar
  • 18 Tracy PB, Rohrbach MS, Mann KG. Functional prothrombinase complex assembly on isolated monocytes and lymphocytes. J Biol Chem 1983; 258: 7264-7267.
    PubMedGoogle Scholar
  • 19 Krishnaswamy S, Jones KC, Mann KG. Prothrombinase complex assembly kinetic mechanism of enzyme assembly on phospholipid vesicles. J Biol Chem 1988; 263: 3823-3834.
    PubMedGoogle Scholar
  • 20 Nesheim ME, Tracy RP, Mann KG. “Clotspeed”, a mathematical simulation of the functional properties of prothrombinase. J Biol Chem 1984; 259: 1447-1453.
    PubMedGoogle Scholar
  • 21 Luckow EA, Lyons DA, Ridgeway TM. et al. Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation. Biochemistry 1989; 28: 2348-2354.
    CrossrefPubMedGoogle Scholar
  • 22 Guinto ER, Esmon CT. Loss of prothrombin and of factor Xa-factor Va interactions upon inactivation of factor Va by activated protein C. J Biol Chem 1984; 259: 13986-13992.
    PubMedGoogle Scholar
  • 23 Boskovic DS, Giles AR, Nesheim ME. Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2 and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid. J Biol Chem 1990; 265: 10497-10505.
    PubMedGoogle Scholar
  • 24 Kalafatis M, Beck DO. Identification of a binding site for blood coagulation factor Xa on the heavy chain of factor Va. Amino acid residues 323–331 of factor V represent an interactive site for activated factor X. Biochemistry 2002; 41: 12715-12728.
    CrossrefPubMedGoogle Scholar
  • 25 Singh LS, Bukys MA, Beck DO. et al. Amino acids Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are essential for expression of cofactor activity. J Biol Chem 2003; 278: 28335-28345.
    CrossrefPubMedGoogle Scholar
  • 26 Kalafatis M, Beck DO, Mann KG. Structural requirements for expression of factor Va activity. J Biol Chem 2003; 278: 33550-33561.
    CrossrefPubMedGoogle Scholar
  • 27 Beck DO, Bukys MA, Singh LS. et al. The contribution of amino acid region Asp695-Tyr698 of factor V to procofactor activation and factor Va function. J Biol Chem 2004; 279: 3084-3095.
    CrossrefPubMedGoogle Scholar
  • 28 Gerads I, Tans G, Yukelson LY. et al. Activation of bovine factor V by an activator purified from the venom of Naja naja oxiana. Toxicon 1992; 30: 1065-1079.
    CrossrefPubMedGoogle Scholar
  • 29 Bakker HM, Tans G, Thomassen MC. et al. Functional properties of human factor Va lacking the Asp683 – Arg709 domain of the heavy chain. J Biol Chem 1994; 269: 20662-20667.
    PubMedGoogle Scholar
  • 30 Camire RM, Kalafatis M, Tracy PB. Proteolysis of factor V by cathepsin G and elastase indicates that cleavage at Arg1545 optimizes cofactor function by facilitating factor Xa binding. Biochemistry 1998; 37: 11896-11906.
    CrossrefPubMedGoogle Scholar
  • 31 Hirbawi J, Bukys MA, Barhoover MA. et al. Role of the acidic hirudin-like COOH-terminal amino acid region of factor Va heavy chain in the enhanced function of prothrombinase. Biochemistry 2008; 47: 7963-7974.
    CrossrefPubMedGoogle Scholar
  • 32 Toso R, Camire RM. Role of hirudin-like factor Va heavy chain sequences in prothrombinase function. J Biol Chem 2006; 281: 8773-8779.
    CrossrefPubMedGoogle Scholar
  • 33 Bajaj SP, Rapaport SI, Maki SL. et al. A procedure for isolation of human protein C and protein S as byproducts of the purification of factors VII, IX, X and prothrombin. Prep Biochem 1983; 13: 191-214.
    PubMedGoogle Scholar
  • 34 Bajzar L, Fredenburgh JC, Nesheim ME. The activated protein C-mediated enhancement of tissue-type plasminogen activator-induced fibrinolysis in a cellfree system. J Biol Chem 1990; 265: 16948-16954.
    PubMedGoogle Scholar
  • 35 Krishnaswamy S, Mann KG, Nesheim ME. The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J Biol Chem 1986; 261: 8977-8984.
    PubMedGoogle Scholar
  • 36 Nesheim ME. A simple rate law that describes the kinetics of the heparin-catalyzed reaction between antithrombin III and thrombin. J Biol Chem 1983; 258: 14708-14717.
    PubMedGoogle Scholar
  • 37 Nesheim ME, Prendergast FG, Mann KG. Interactions of a fluorescent active-site-directed inhibitor of thrombin: dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. Biochemistry 1979; 18: 996-1003.
    CrossrefPubMedGoogle Scholar
  • 38 Barenholz Y, Gibbes D, Litman BJ. et al. A simple method for the preparation of homogeneous phospholipid vesicle. Biochemistry 1977; 16: 2806-2810.
    CrossrefPubMedGoogle Scholar
  • 39 Fujikawa K, Legaz ME, Davie EW. Bovine factors X1 and X2 (Stuart Factor). Isolation and characterization. Biochemistry 1972; 11: 4882-4891.
    CrossrefPubMedGoogle Scholar
  • 40 Butkowski RJ, Elion J, Downing MR. et al. Primary structure of human prethrombin 2 and alpha-thrombin. J Biol Chem 1977; 252: 4942-4957.
    PubMedGoogle Scholar
  • 41 Fenton II JW, Fasco MJ, Stackrow AB. Human thrombins. Production, evaluation, and properties of alpha-thrombin. J Biol Chem 1977; 252: 3587-3598.
    PubMedGoogle Scholar
  • 42 Krishnaswamy S, Church WR, Nesheim ME. et al. Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism. J Biol Chem 1987; 262: 3291-3219.
    PubMedGoogle Scholar
  • 43 Kim PY, Nesheim ME. Further evidence for two functional forms of prothrombinase each specific for either of the two prothrombin activation cleavages. J Biol Chem 2007; 282: 32568-32581.
    CrossrefPubMedGoogle Scholar
  • 44 Church WR, Jernigan RL, Toole J. et al. Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins. Proc Natl Acad Sci USA 1984; 81: 6934-6937.
    CrossrefPubMedGoogle Scholar