Download PDF
Thromb Haemost 1994; 72(06): 947-956
DOI: 10.1055/s-0038-1648989
Original Article
Schattauer GmbH Stuttgart

Thrombin-stimulated Human Platelets Express Molecular Forms of α-Granule Factor V (FV), which Differ from FVa

Edward G Wyshock
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA
,
Gwendolyn J Stewart
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA
,
Robert W Colman
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA
› Author Affiliations
Further Information

Publication History

Received 06 June 1994

Accepted after revision 18 August 1994

Publication Date:
06 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

Binding of 125I-Fab fragments of chain-specific antibodies indicate that both heavy and light chains of a-granule factor Va (FVa) were externalized on the platelet membrane after stimulation with thrombin. Using a Mab against the activation peptide of factor V (FV), the epitope appears on the stimulated platelet surface. Half as much light chain and heavy chain (FVa) was expressed compared to the activation peptide, suggesting that expression of α-granule FV occurs after thrombin stimulation. Using an ELISA, we find that 32% of a-granule FV was released and 68% is retained in the platelet pellet. Immunoblots of platelets indicate that FV exists in 200 kDa und 150 kDa forms, representing incomplete cleavage, while the releasate demonstrates a more complete cleavage by proteases. We conclude that expression of α-granule FV is quantitatively greater than that released and exists in molecular forms which cannot be completely explained by the binding of FVa.

 

  • References

  • 1 Dahlbäck B. Human coagulation factor V purification and thrombin-catalyzed activation. J Clin Invest 1980; 66: 583-591
    CrossrefPubMedGoogle Scholar
  • 2 Suzuki K, Dahlbäck B, Stenflo J. Thrombin-catalyzed activation of human coagulation factor V. J Biol Chem 1982; 257: 6556-6564
    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 Kane WH, Majerus PW. The interaction of human coagulation factor Va with platelets. J Biol Chem 1982; 257: 3963-3969
    PubMedGoogle Scholar
  • 5 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 USA 1983; 80: 2380-2384
    CrossrefPubMedGoogle Scholar
  • 6 Annamalai AE, Rao AK, Chiu HC, Wang D, Dutta Roy AK, Walsh PN, Colman RW. Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium. Blood 1987; 70: 139-146
    PubMedGoogle Scholar
  • 7 Miletich JP, Jackson CM, Majerus PW. Properties of the factor Xa binding site on human platelets. J Biol Chem 1978; 253: 6908-6916
    PubMedGoogle Scholar
  • 8 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
  • 9 Tracy PB, Eide LL, Bowie EJ, Mann KG. Radioimmunoassay of factor V in human plasma and platelets. Blood 1982; 60: 59-63
    CrossrefPubMedGoogle Scholar
  • 10 Chesney CM, Pifer D, Colman RW. Subcellular localization and secretion of factor V from human platelets. Proc Natl Acad Sci USA 1981; 78: 5180-5184
    CrossrefPubMedGoogle Scholar
  • 11 Vicic WJ, Lages B, Weiss HJ. Release of human platelet factor V activity is induced by both collagen and ADP and is inhibited by aspirin. Blood 1980; 56: 448-455
    PubMedGoogle Scholar
  • 12 Kane WH, Lindhout MJ, Jackson CM, Majerus PW. Factor Va-dependent binding of factor Xa to human platelets. J Biol Chem 1980; 255: 1170-1174
    PubMedGoogle Scholar
  • 13 Baruch D, Hemker HC, Lindhout T. Kinetics of thrombin-induced release and activation of platelet factor V. Eur J Biochem 1986; 154: 213-218
    CrossrefPubMedGoogle Scholar
  • 14 Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express pro-thrombinase activity. J Biol Chem 1988; 263: 18205-18212
    PubMedGoogle Scholar
  • 15 Tuszynski GP, Walsh PN, Piperno JR, Koshy A. Association of coagulation factor V with the platelet cytoskeleton. J Biol Chem 1982; 257: 4557-4563
    PubMedGoogle Scholar
  • 16 Wang DL, Annamalai AE, Ghosh S, Gewirtz AM, Colman RW. Human platelet factor V is crosslinked to actin by FXIIIa during platelet activation by thrombin. Thromb Res 1990; 57: 39-57
    CrossrefPubMedGoogle Scholar
  • 17 Osterud B, Rapaport SI, Lavine KK. Factor V activity of platelets: evidence for an activated factor V molecule and for a platelet activator. Blood 1977; 49: 819-834
    PubMedGoogle Scholar
  • 18 Kane WH, Mruk JS, Majerus PW. Activation of coagulation factor V by a platelet protease. J Clin Invest 1982; 70: 1092-1100
    CrossrefPubMedGoogle Scholar
  • 19 Tracy PB, Nesheim ME, Mann KG. Proteolytic alterations of factor Va bound to platelets. J Biol Chem 1983; 258: 662-669
    PubMedGoogle Scholar
  • 20 Bradford HN, Annamalai A, Doshi K, Colman RW. Factor V is activated and cleaved by platelet calpain: comparison with thrombin proteolysis. Blood 1988; 71: 388-394
    PubMedGoogle Scholar
  • 21 Viskup RW, Tracy PB, Mann KG. The isolation of human platelet factor V. Blood 1987; 69: 1188-1195
    PubMedGoogle Scholar
  • 22 Gewirtz AM, Keefer M, Doshi K, Annamalai AE, Chiu HC, Colman RW. Biology of human megakaryocyte factor V. Blood 1986; 67: 1639-1648
    PubMedGoogle Scholar
  • 23 Goding JW. Use of staphylococcal protein A as an immunological reagent. J Immunol Methods 1978; 20: 241-253
    CrossrefPubMedGoogle Scholar
  • 24 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
    CrossrefPubMedGoogle Scholar
  • 25 Fraker PJ, Speck JC. Jr. Protein and cell membrane iodinations with a sparingly soluble chloroamide, l,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem Res Commun 1978; 80: 849-857
    CrossrefPubMedGoogle Scholar
  • 26 Chiu HC, Whitaker E, Colman RW. Heterogeneity of human factor V deficiency. Evidence for the existence of antigen-positive variants. J Clin Invest 1983; 72: 493-503
    CrossrefPubMedGoogle Scholar
  • 27 Ware AG, Murphy RC, Seegers WH. The function of Ac-globulin in blood clotting. Science 1947; 106: 618-619
    PubMedGoogle Scholar
  • 28 Wiedmer T, Esmon CT, Sims PJ. Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase. Blood 1986; 68: 875-880
    CrossrefPubMedGoogle Scholar
  • 29 Sandberg H, Andersson LO. A highly sensitive assay of platelet factor 3 using a chromogenic substrate. Thromb Res 1979; 14: 113-124
    CrossrefPubMedGoogle Scholar
  • 30 Schmaier AH, Colman RW. Crotalocytin: characterization of the timber rattlnesnake platelet activating protein. Blood 1980; 56: 1020-1028
    PubMedGoogle Scholar
  • 31 Timmons S, Hawiger J. Separation of human platelets from plasma proteins including factor VIII VWF by a combined albumin gradient-gel filtration method using HEPES buffer. Thromb Res 1978; 12: 297-306
    CrossrefPubMedGoogle Scholar
  • 32 Kornecki E, Niewiarowski S, Morinelli TA, Kloczewiak M. Effects of chy-motrypsin and adenosine diphosphate on the exposure of fibrinogen receptors on normal human and Glanzmann,s thrombasthenic platelets. J Biol Chem 1981; 256: 5696-5701
    PubMedGoogle Scholar
  • 33 Brass LF, Shattil SJ. Changes in surface-bound and exchangeable calcium during platelet activation. J Biol Chem 1982; 257: 14000-14005
    PubMedGoogle Scholar
  • 34 Klotz IM. Numbers of receptor sites from Scatchard graphs: Facts and fantasies. Science 1982; 217: 1247-1249
    CrossrefPubMedGoogle Scholar
  • 35 Scatchard G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660-672
    CrossrefPubMedGoogle Scholar
  • 36 Canellas PF, Karu AE. Statistical package for analysis of competition ELISA results. J Immunol Methods 1981; 47: 375-385
    CrossrefPubMedGoogle Scholar
  • 37 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1979; 227: 680-685
    PubMedGoogle Scholar
  • 38 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
    CrossrefPubMedGoogle Scholar
  • 39 Wencel Drake JD, Dahlbäck B, White JG, Ginsberg MH. Ultrastructural localization of coagulation factor V in human platelets. Blood 1986; 68: 244-249
    PubMedGoogle Scholar
  • 40 Chiu HC, Schick PK, Colman RW. Biosynthesis of factor V in isolated guinea pig megakaryocytes. J Clin Invest 1985; 75: 339-346
    CrossrefPubMedGoogle Scholar
  • 41 Ginsberg MH, Painter RG, Birdwell C, Plow EF. The detection, immuno-fluorescent localization, and thrombin induced release of human platelet-associated fibronectin antigen. J Supramol Struct 1979; 11: 167-174
    CrossrefPubMedGoogle Scholar
  • 42 Koutts J, Walsh PN, Plow EF, Fenton JW, Bouma BN, Zimmerman TS. Active release of human platelet factor VUI-related antigen by adenosine diphosphate, collagen, and thrombin. J Clin Invest 1978; 62: 1255-1263
    CrossrefPubMedGoogle Scholar
  • 43 Schmaier AH, Colman RW. Platelet high-molecular-weight kininogen. Methods Enzymol 1989; 169: 276-296
    PubMedGoogle Scholar
  • 44 Tracy PB, Eide LL, Mann KG. Human prothrombinase complex assembly and function on isolated peripheral blood cell populations. J Biol Chem 1985; 260: 2119-2124
    PubMedGoogle Scholar
  • 45 Swords NA, Mann KG. The assembly of the prothrombinase complex on adherent platelets. Arterioscler Thromb 1993; 13: 1602-1612
    CrossrefPubMedGoogle Scholar
  • 46 Legrand C, Dubernard V, Nurden AT. Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets. Blood 1989; 73: 1226-1234
    PubMedGoogle Scholar
  • 47 Fernandez MF, Ginsberg MH, Ruggeri ZM, Batlle FJ, Zimmerman TS. Multimeric structure of platelet factor VUI/von Willebrand factor: the presence of larger multimers and their reassociation with thrombin-stimulated platelets. Blood 1982; 60: 1132-1138
    PubMedGoogle Scholar
  • 48 Adelman B, Carlson P, Powers P. von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP. Blood 1987; 70: 1362-1366
    PubMedGoogle Scholar
  • 49 Monkovic DD, Tracy PB. Activation of human factor V by factor Xa and thrombin. Biochemistry 1990; 29: 1118-1128
    CrossrefPubMedGoogle Scholar
  • 50 Dahlbäck B. A new model for coagulation factor V suggesting a unique mechanism of activation. Scand J Clin Lab Invest Suppl 1988; 191: 47-61
    PubMedGoogle Scholar
  • 51 Cerveny TJ, Fass DN, Mann KG. Synthesis of coagulation factor V by cultured aortic endothelium. Blood 1984; 63: 1467-1474
    PubMedGoogle Scholar
  • 52 Rodgers GM, Shuman MA. Characterization of the interaction between factor Xa and bovine aortic endothelial cells. Biochim Biophys Acta 1985; 844: 320-329
    CrossrefPubMedGoogle Scholar
  • 53 Annamalai AE, Stewart GJ, Hansel B, Memoli M, Chiu HC, Manuel DW, Doshi K, Colman RW. Expression of factor V on human umbilical vein endothelial cells is modulated by cell injury. Arteriosclerosis 1986; 6: 196-202
    CrossrefPubMedGoogle Scholar
  • 54 Rodgers GM, Cong JY, Goll DE, Kane WH. Activation of coagulation factor V by calcium-dependent proteinase. Biochim Biophys Acta 1987; 929: 263-270
    CrossrefPubMedGoogle Scholar
  • 55 Wiedmer T, Esmon CT, Sims PJ. On the mechanism by which complement proteins C5b-9 increase platelet prothrombinase activity. J Biol Chem 1986; 261: 14587-14592
    PubMedGoogle Scholar
  • 56 Swords NA, Tracy PB, Mann KG. Intact platelet membranes, not platelet microvesicles, support the procoagulant activity of adherent platelets. Arterioscler Thromb 1993; 13: 1613-1622
    CrossrefPubMedGoogle Scholar
  • 57 Monkovic DD, Tracy PB. Functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin. J Biol Chem 1990; 265: 17132-17140
    PubMedGoogle Scholar