Download PDF
Thromb Haemost 1998; 80(01): 92-98
DOI: 10.1055/s-0037-1615145
Rapid Communication
Schattauer GmbH

Cleavage Requirements of Factor V in Tissue-factor Induced Thrombin Generation

Elisabeth Thorelli
1   Department of Clinical Chemistry, Lund University, University Hospital Malmö, Sweden
,
Randal J. Kaufman
2   Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, USA
,
Björn Dahlbäck
1   Department of Clinical Chemistry, Lund University, University Hospital Malmö, Sweden
› Author Affiliations
Further Information

Publication History

Received 20 November 1997

Accepted after revision 06 March 1998

Publication Date:
08 December 2017 (online)

    Permissions and Reprints

Summary

Factor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components. Surprisingly, in the standard FV clotting assay, all mutants gave similar clotting activities and the thrombin generation curves obtained with wild-type and thrombin-resistant FV were similar. Differences in clotting activities and thrombin generation patterns between wild-type and thrombin-resistant FV were only observed when lower TF concentrations were used. The thrombin generation curve obtained in plasma containing wt FV was characterized by a short lag phase and a subsequent phase of rapid thrombin generation (propagation phase). The Arg709 to Gln mutation yielded a slightly prolonged lag phase and the rate of thrombin generation during the propagation phase was approximately 5-fold lower than that observed with wt FV. The Arg1018 to Ile mutation only slightly affected the thrombin generation curve, whereas the Arg1545 to Gln mutation yielded a prolonged lag phase and decreased maximum thrombin activity. Thrombin-resistant FV (mutated at all three sites) yielded a prolonged lag phase and poor thrombin generation during the propagation phase. The purified system further demonstrated the importance of the three cleavage sites for rapid and sustained thrombin generation. The results demonstrate that cleavages at positions 709, 1018 and 1545 are not required for assembly of a FXa-FV complex expressing low but significant prothrombinase activity but that all three sites in different ways are important for the creation of a FVa which maximally supports the FXa-mediated activation of prothrombin.

 

  • References

  • 1 Nemerson Y. Tissue factor: now and then. Thromb Haemost 1995; 74: 180-4.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 2 Edgington TS, Mackman N, Brand K, Ruf W. The structural biology of expression and function of tissue factor. Thromb Haemost 1991; 66: 67-79.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Butenas S, Mann KG. Kinetics of human factor VII activation. Biochemistry 1996; 35: 1904-9.
    CrossrefPubMedGoogle Scholar
  • 4 Kane WH, Davie EW. Blood coagulation factors V and VIII: Structural and functional similarities and their relationship to hemorrhagic and trombotic disorders. Blood 1988; 71: 539-55.
    PubMedGoogle Scholar
  • 5 Jenny RJ, Tracy PB, Mann KG. The physiology and biochemistry of factor V. In: Haemostasis and Thrombosis. Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD. eds. New York: Churchill Livingstone Inc; 1994: 465-76.
    Google Scholar
  • 6 Kane WH, Davie EW. Cloning of cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1986; 83: 6800-4.
    CrossrefPubMedGoogle Scholar
  • 7 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM, Kaufman RJ, Mann KG. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84: 4846-50.
    CrossrefPubMedGoogle Scholar
  • 8 Foster WB, Nesheim ME, Mann KG. The factor Xa-catalyzed activation of factor V. J Biol Chem 1983; 258: 13970-7.
    PubMedGoogle Scholar
  • 9 Moncovic DD, Tracy PB. Activation of human factor V by factor Xa and thrombin. Biochemistry 1990; 29: 1118-28.
    CrossrefPubMedGoogle Scholar
  • 10 Thorelli E, Kaufman RJ, Dahlbäck B. Cleavage requirements for Activation of factor V by factor Xa. Eur J Biochem 1997; 247: 12-20.
    CrossrefPubMedGoogle Scholar
  • 11 Esmon CT. The subunit structure of thrombin-activated factor V. Isolation of activated factor V, separation of subunits, and reconstitution of biologival activity. J Biol Chem 1979; 254: 964-73.
    PubMedGoogle Scholar
  • 12 Nesheim ME, Mann KG. Thrombin-catalyzed activation of single chain bovine factor V. J Biol Chem 1979; 254: 1326-34.
    PubMedGoogle Scholar
  • 13 Suzuki K, Dahlbäck B, Stenflo J. Thrombin-catalyzed activation of human coagulation factor V. J Biol Chem 1982; 257: 1210-4.
    PubMedGoogle Scholar
  • 14 Keller FG, Ortel TL, Quinn-Allen MA, Kane WH. Thrombin catalyzed activation of recombinant human factor V. Biochemistry 1995; 34: 4118-24.
    CrossrefPubMedGoogle Scholar
  • 15 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: 10952-62.
    PubMedGoogle Scholar
  • 16 Willems GM, Lindhout T, Hermens WT, Hemker HC. Simulation model for thrombin generation in plasma. Haemostasis 1991; 21: 197-207.
    PubMedGoogle Scholar
  • 17 Béguin S, Lindhout T, Hemker HC. The effect of trace amounts of tissue Factor on thrombin generation in platelet rich plasma its inhibition by heparin. Thromb Haemost 1989; 61: 25-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 18 Kumar R, Béguin S, Hemker HC. The influence of fibrinogen and fibrin on thrombin generation-evidence for feedback activation of the clotting system by clot bound thrombin. Thromb Haemost 1994; 72: 713-21.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Rand MD, Lock JB, van’t Veer C, Gaffney DP, Mann KG. Blood clotting in minimally altered whole blood. Blood 1996; 88: 3432-45.
    PubMedGoogle Scholar
  • 20 van’t Veer, Golden NJ, Kalafatis M, Mann KG. Inhibitory mechanism of the protein C pathway on Tissue factor-induced thrombin generation. Synergistic effect in combination with tissue factor pathway inhibitor. J Biol Chem 1997; 272: 7983-94.
    CrossrefPubMedGoogle Scholar
  • 21 Hemker HC, Willems GM, Béguin S. A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemost 1986; 56: 9-17.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 22 Hemker HC, Béguin S. Thrombin generation in plasma: Its Assessment via the endogenous thrombin potential. Thromb Haemost 1995; 74: 134-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 23 Hemker HC. Thrombin generation, an essential step in hamostasis and thrombosis. In: »Haemostasis and Thrombosis«. Bloom A. et al., eds. Edingburgh: Churchill Livingstone; 1993: 477-90.
    Google Scholar
  • 24 Wielders S, Mukherjee M, Michiels J, Rijkers DTS, Cambus J-P, Kneber RWC, Kakkar V, Hemker HC. The routine determination of the endogenous thrombin potential, first results in different forms of hyper- and hypocoagulability. Thromb Haemost 1997; 77: 629-36.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 25 Travis J, Salvesen GS. Human plasma proteinase inhibitors. Ann Rev Biochem 1983; 52: 655-709.
    CrossrefPubMedGoogle Scholar
  • 26 Rosenberg RD, Harpel PC. α2-macroglobulin and antithrombin-heparin cofactor: modulators of hemostatic and inflammatory reactions. Prog Haemost Thromb 1976; 3: 145-98.
    PubMedGoogle Scholar
  • 27 Wencel-Drake JD, Dahlbäck B, Ginsburg MH. Ultrastructural localization of coagulation factor V in human platelets. Blood 1986; 68: 244-9.
    PubMedGoogle Scholar
  • 28 Kaufman RJ. Vectors used for expression in mammalian cells. Methods of enzymology 1989; 185: 487-509.
    PubMedGoogle Scholar
  • 29 Lawson JH, Kalafatis M, Stram S, Mann K. A Model for the Tissue Factor Pathway to Thrombin. An empirical study. J Biol Chem 1994; 269: 23357-66.
    PubMedGoogle Scholar
  • 30 Marquette KA, Pittman DD, Kaufman RJ. The factor V B-domain provides two functions to facilitate thrombin cleavage and release of the light chain. Blood 1995; 86: 3026-34.
    PubMedGoogle Scholar
  • 31 Pieters J, Lindhout T, Hemker C. In situ generated thrombin is the only enzyme that effectively activates factor V in thromboplastin activated plasma. Blood 1989; 74: 1021-4.
    PubMedGoogle Scholar
  • 32 Yang XJ, Blajchman MA, Craven S, Smith LM, Anvari N, Ofuso FA. Activation of Factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg- CH2Cl. Biochem J 1990; 272: 399-406.
    CrossrefPubMedGoogle Scholar
  • 33 Butenas S, van’t Veer C, Mann K. Evaluation of the initiation phase of blood coagulation using ultrasensitive assays for serine proteases. J Biol Chem 1997; 272: 21527-33.
    CrossrefPubMedGoogle Scholar
  • 34 Ofuso FA, Lormeau JC, Craven S, Dewar L, Anvari N. Heparin and low molecular weight heparins inhibit prothrombinase formation but not its activity in plasma. Thromb Haemost 1994; 72: 862-8.
    Article in Thieme ConnectPubMedGoogle Scholar