Prothrombin amino terminal region helps protect coagulation factor Va from proteolytic inactivation by activated protein C

 

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Summary

The hypothesis that prothrombin (FII) protects coagulation factor Va (FVa) from proteolytic inactivation by activated protein C (APC) was tested using purified proteins. FII dose-dependently protected FVa from APC proteolysis under conditions where competition of proteins for binding to negatively-charged phospholipid surface was not relevant (i.e. either at high phospholipid vesicle concentrations or using soluble dicaproylphosphatidylserine at levels below its critical micellar concentration). Cleavages in FVa at both Arg⁵⁰⁶ and Arg³⁰⁶ by APC were inhibited by FII. FII did not alter the amidolytic activity of APC towards chromogenic oligopeptide substrates or inhibit FVIIIa inactivation by APC, implying that the FII-mediated protection of FVa from APC proteolysis was due to the ability of FII to inhibit protein-protein interactions between FVa and APC. FII also protected FVa from inactivation by Gla-domainless APC, ruling out a role for the APC Gla domain for these observations. To identify domains of FII responsible for the observed phenomenon, various forms or fragments of FII were employed. Biotin-PheProArg-CMK-inhibited meizothrombin and fII-fragment 1•2 protected FVa from proteolysis by APC. In contrast, no significant protection of FVa from APC cleavage was observed for Gladomainless-FII, prethrombin-1, prethrombin-2, FII fragment 1 or active site inhibited-thrombin (DEGR-thrombin). Overall, these data demonstrate that the Gla domain of FII linked to kringle 1 and 2 is necessary for the ability of FII to protect FVa from APC cleavage and support the general concept that assembly of the FII activation complex (FXa•FVa•FII•lipid surface) protects FVa from APC inactivation so that the procoagulant, thrombin generating pathway can act unhindered by APC. Only following FII activation and dissociation of the FII Gla domain fragments from the FII-ase complex, can APC inactivate FVa and down-regulate thrombin generation.

• References

• 1 Furie B, Furie BC. Molecular basis of blood coagulation. In: Hematology. Basic Principles and Practices. 3^rd ed. Philadelphia: Churchill Livingstone; 2000: 1783-1803.
  Google Scholar
• 2 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: 10952-10625.
  PubMedGoogle Scholar
• 3 Rosing J, Tans G, Grovers-Riemslag JW. et al. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-283.
  PubMedGoogle Scholar
• 4 Jackson CM, Nemerson Y. Blood coagulation. Ann Rev Biochem 1980; 49: 765-811.
  CrossrefPubMedGoogle Scholar
• 5 Mann KG. Prothrombin and thrombin. In: Hemostasis and Thrombosis. Basic Principles and Clinical Practices. 3^rd ed. Philadelphia: J. B. Lippincott Co.; 1994: 184-199.
  Google Scholar
• 6 Rosing J, Zwaal RF, Tans G. Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation. J Biol Chem 1986; 261: 4224-4228.
  PubMedGoogle Scholar
• 7 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
• 8 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: 3291-3299.
  PubMedGoogle Scholar
• 9 Boskovic DS, Bajzar LS, Nesheim ME. Channeling during prothrombin activation. J Biol Chem 2001; 276: 28686-28693.
  CrossrefPubMedGoogle Scholar
• 10 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
• 11 Bianchini EP, Orcutt SJ, Panizzi P. et al. Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase. Proc Natl Acad Sci USA 2005; 102: 10099-10104.
  CrossrefPubMedGoogle Scholar
• 12 Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem 1994; 269: 31869-31880.
  PubMedGoogle Scholar
• 13 Nicolaes GA, Tans G, Thomassen MC. et al. Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 21158-21166.
  CrossrefPubMedGoogle Scholar
• 14 Rosing J, Hoekema L, Nicolaes GA. et al. Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 27852-27858.
  CrossrefPubMedGoogle Scholar
• 15 Gale AJ, Xu X, Pellequer J-C. et al. Interdomain engineered disulfide bond permitting elucidation of mechanisms of inactivation of coagulation factor Va by activated protein C. Prot Sci 2002; 11: 2091-2101.
  PubMedGoogle Scholar
• 16 Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated Protein C through the selective inactivation of activated Factor V, Biochim. Biophys Acta 1979; 571: 333-342.
  PubMedGoogle Scholar
• 17 Nesheim ME, Canfield WM, Kisiel W. et al. Studies of the capacity of factor Xa to protect factor Va from inactivation by activated protein C. J Biol Chem 1982; 257: 1443-1447.
  PubMedGoogle Scholar
• 18 Suzuki K, Stenflo J, Dahlback B. et al. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-1920.
  PubMedGoogle Scholar
• 19 Norstrom EA, Steen M, Tran S. et al. Effects of factor Xa and protein S on the individual activated protein C-mediated cleavages of coagulation factor Va. J Biol Chem 2006; 278: 24904-24911.
  PubMedGoogle Scholar
• 20 Yegneswaran S, Kojima Y, Nguyen PM. et al. Factor Va residues 311–325 represent an activated protein C binding region. J Biol Chem 2007; 282: 28353-28361.
  CrossrefPubMedGoogle Scholar
• 21 Kojima Y, Heeb MJ, Gale AJ. et al. Binding site for blood coagulation factor Xa involving residues 311–325 in factor Va. J Biol Chem 1998; 273: 14900-14905.
  CrossrefPubMedGoogle Scholar
• 22 Smirnov MD, Safa O, Esmon NL. et al. Inhibition of activated protein C anticoagulant activity by prothrombin. Blood 1999; 94: 3839-3846.
  PubMedGoogle Scholar
• 23 Mitchell CA, Jane SM, Salem HH. Inhibition of the anticoagulant activity of protein S by prothrombin. J Clin Invest 1988; 82: 2142-2147.
  CrossrefPubMedGoogle Scholar
• 24 Tran S, Norstrom E, Dahlback B. Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va. J Biol Chem 2008; 283: 6648-6655.
  CrossrefPubMedGoogle Scholar
• 25 Yegneswaran S, Wood GM, Esmon CT. et al. Protein S alters the active site location of activated protein C above the membrane surface. A fluorescence resonance energy transfer study of topography. J Biol Chem 1997; 272: 25013-25021.
  CrossrefPubMedGoogle Scholar
• 26 Gale AJ, Cramer TJ, Rozenshteyn D. et al. Detailed mechanisms of the inactivation of factor VIIIa by activated protein C in the presence of its cofactors, protein S and factor V. J Biol Chem 2008; 283: 16355-16362.
  CrossrefPubMedGoogle Scholar
• 27 Koppaka V, Wang J, Banerjee M. et al. Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution. Biochemistry 1996; 35: 7482-7491.
  CrossrefPubMedGoogle Scholar
• 28 Orcutt SJ, Krishnaswamy S. Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin. J Biol Chem 2004; 279: 54927-54936.
  CrossrefPubMedGoogle Scholar
• 29 Bukys MA, Kim PY, Nesheim ME. et al. A control switch for prothrombinase: characterization of a hirudin-like pentapeptide from the COOH terminus of factor Va heavy chain that regulates the rate and pathway for prothrombin activation. J Biol Chem 2006; 281: 39194-39204.
  CrossrefPubMedGoogle Scholar
• 30 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
• 31 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
• 32 Yegneswaran S, Mesters RM, Fernandez JA. et al. Prothrombin residues 473–487 contribute to factor Va binding in the prothrombinase complex. J Biol Chem 2004; 279: 49019-49025.
  CrossrefPubMedGoogle Scholar
• 33 Kotkow KJ, Deitcher SR, Furie B. et al. The second kringle domain of prothrombin promotes factor Va-mediated prothrombin activation by prothrombinase. J Biol Chem 1995; 270: 4551-4557.
  CrossrefPubMedGoogle Scholar
• 34 Church WR, Quellette LA, Messier TL. Modulation of human prothrombin activation on phospholipid vesicles and platelets using monoclonal antibodies to prothrombin fragment 2. J Biol Chem 1991; 266: 8384-8391.
  PubMedGoogle Scholar
• 35 Deguchi H, Takeya H, Gabazza EC. et al. Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex. Biochem J 1997; 321: 729-735.
  CrossrefPubMedGoogle Scholar
• 36 Blostein MD, Rigby AC, Jacobs M. et al. The Gla domain of human prothrombin has a binding site for factor Va. J Biol Chem 2000; 275: 38120-38126.
  CrossrefPubMedGoogle Scholar
• 37 Poort SR, Rosendaal FR, Reitsma PH. et al. A common genetic variation in the 3‘-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996; 88: 3698-3703.
  PubMedGoogle Scholar
• 38 Bertina RM. The prothrombin 20210 G to A variation and thrombosis. Curr Opin Hematol 1998; 05: 339-342.
  CrossrefPubMedGoogle Scholar
• 39 Armstrong SA, Husten EJ, Esmon CT. et al. The active site of membrane-bound meizothrombin. J Biol Chem 1990; 265: 6210-6218.
  CrossrefPubMedGoogle Scholar
• 40 Boskovic DS, Troxler T, Krishnaswamy S. Active site-independent recognition of substrates and product by bovine prothrombinase. J Biol Chem 2004; 279: 20786-20793.
  CrossrefPubMedGoogle Scholar