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Thromb Haemost 1998; 80(04): 578-584
DOI: 10.1055/s-0037-1615424
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Schattauer GmbH

Active Site-inactivated Factors VIIa, Xa, and IXa Inhibit Individual Steps in a Cell-based Model of Tissue Factor-initiated Coagulation

Marianne Kjalke
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, NC
3   From the TF/FVIIa Research, Novo Nordisk, Maaloev, Denmark
,
Dougald M. Monroe
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, NC
,
Maureane Hoffman
2   From the Department of Pathology, Duke University, Durham, NC, USA
,
Julie A. Oliver
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, NC
2   From the Department of Pathology, Duke University, Durham, NC, USA
,
Mirella Ezban
3   From the TF/FVIIa Research, Novo Nordisk, Maaloev, Denmark
,
Harold R. Roberts
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, NC
› Author AffiliationsSupported in part by grants R01 HL48320 and T32 HL07149 from the National Institutes of Health
Further Information

Publication History

Received 09 December 1997

Accepted after revision 29 June 1998

Publication Date:
08 December 2017 (online)

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Summary

Factors VIIa, Xa, and IXa play different roles in the initiation of tissue factor-dependent coagulation. The consequences of competing with the different enzymes were investigated, thereby examining the effects of inhibiting the initiation process at different steps. Active site-inactivated factors VIIa, Xa, and IXa (FVIIai, FXai, and FIXai, respectively) were added to various cell-based assays mimicking the individual steps in tissue factor-initiated coagulation. In an assay involving tissue factor-expressing monocytes, coagulation proteins and unactivated platelets, FVIIai and FXai inhibited platelet activation and thrombin generation while FIXai only inhibited thrombin generation. FVIIai inhibited factor Xa generation and subsequent thrombin generation on monocytes, while FXai inhibited thrombin generation on the monocytes as well as on the activated platelets. FIXai had no effect on factor Xa or thrombin generation on the monocytes, but inhibited factor Xa and subsequent thrombin generation on the activated platelets. FVIIai had no effect on the reactions taking place on the activated platelets. The data confirm a model where tissue factor/factor VIIa mediates factor Xa generation and subsequent prothrombin activation on the tissue factor-bearing cells. Thrombin then activates platelets, which serve as the physiologically important surface for large-scale thrombin generation.

 

  • References

  • 1 Camerer E, Kolstø A-B, Prydz H. Cell biology of tissue factor, the principal initiator of blood coagulation.. Thromb Res 1996; 81: 1-41.
    CrossrefPubMedGoogle Scholar
  • 2 Østerud B. Tissue factor: A complex biological role.. Thromb Haemost 1997; 78: 755-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues.. Am J Pathol 1989; 134: 1087-97.
    PubMedGoogle Scholar
  • 4 Fleck RA, Rao LVM, Rapaport SI, Varki N. Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody.. Thromb Res 1990; 59: 421-37.
    CrossrefPubMedGoogle Scholar
  • 5 Flössel C, Luther T, Müller M, Albrecht S, Kasper M. Immunohistochemi cal detection of tissue factor (TF) on paraffin sections of routinely fixed human tissue.. Histochemistry 1994; 101: 449-53.
    CrossrefPubMedGoogle Scholar
  • 6 Wildgoose P, Nemerson Y, Hansen LL, Nielsen FE, Glazer S, Hedner U. Measurements of basal levels of factor VIIa in hemophilia A and B patients.. Blood 1992; 80: 25-8.
    PubMedGoogle Scholar
  • 7 Morrissey JH, Macik BG, Neuenschwander PF, Comp PC. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation.. Blood 1993; 81: 734-44.
    PubMedGoogle Scholar
  • 8 Monroe DM, Roberts HR, Hoffman M. Platelet procoagulant complex assembly in a tissue factor-initiated system.. Br J Haematol 1994; 88: 364-71.
    CrossrefPubMedGoogle Scholar
  • 9 Hoffman M, Monroe DM, Oliver JA, Roberts HR. Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation.. Blood 1995; 86: 1794-801.
    PubMedGoogle Scholar
  • 10 Monroe DM, Hoffman M, Roberts HR. Transmission of a procoagulant signal from tissue factor-bearing cells to platelets.. Blood Coag Fibrinol 1996; 7: 459-64.
    CrossrefPubMedGoogle Scholar
  • 11 Berndt MC, Phillips DR. Platelet membrane proteins: Composition and receptor function.. In: Platelets in Biology and Pathology.. Gordon J (ed).. Amsterdam, Elsvier/North Holland:: Biomedical Press; 1981. pp 43-74.
    Google Scholar
  • 12 Coughlin SR. Platelet thrombin receptor.. In: Molecular basis of thrombosis and hemostasis.. High KA, Roberts HR. (eds). New York:: Marcel Dekker, Inc.; 1995. pp 639-41.
    Google Scholar
  • 13 Kurachi K, Fujikawa K, Schmer G, Davie EW. Inhibition of bovine factor IXa and factor Xaβ by antithrombin III.. Biochemistry 1976; 15: 373-7.
    CrossrefPubMedGoogle Scholar
  • 14 Østerud B, Miller-Andersson M, Abildgaard U, Prydz H. The effect of antithrombin III on the activity of the coagulation factors VII, IX, and X.. Thromb Haemost 1976; 35: 295-304.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Pieters J, Willems G, Hemker HC, Lindhout T. Inhibition of factor IXa and factor Xa by antithrombin III/heparin during factor X activation.. J Biol Chem 1988; 263: 15313-8.
    PubMedGoogle Scholar
  • 16 Walch PN. Platelets and coagulation proteins.. Federation Proc 1981; 40: 2086-91.
    PubMedGoogle Scholar
  • 17 Harker LA. The role of platelets in the early and late vascular responses initiated by mechanical vascular injury.. Fibrinol Proteolysis 1997; 11 (Suppl. 01) 97-103.
    PubMedGoogle Scholar
  • 18 Kjalke M, Oliver JA, Monroe DM, Hoffman M, Ezban M, Hedner U, Roberts HR. The effect of active site-inhibited factor VIIa on tissue factor-initiated coagulation using platelets before and after aspirin administration.. Thromb Haemost 1997; 78: 1202-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Bach R, Gentry R, Nemerson Y. Factor VII binding to tissue factor in reconstituted phospholipid vesicles: Induction of cooperativity by phosphatidylserine.. Biochemistry 1986; 25: 4007-20.
    CrossrefPubMedGoogle Scholar
  • 20 Søensen BB, Persson E, Freskgård P-O, Kjalke M, Ezban M, Williams T, Rao LVM. Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor.. J Biol Chem 1997; 272: 11863-8.
    CrossrefPubMedGoogle Scholar
  • 21 Nesheim ME, Kettner C, Shaw E, Mann KG. Cofactor dependence of factor Xa incorporation into the prothrombinase complex.. J Biol Chem 1981; 256: 6537-40.
    PubMedGoogle Scholar
  • 22 Lollar P, Fass DN. Inhibition of activated porcine factor IX by dansylglutamyl-glycyl-arginyl-chloromethylketone.. Arch Biochem Biophys 1984; 233: 438-46.
    CrossrefPubMedGoogle Scholar
  • 23 Monroe DM, Roberts HR. Purification of prothrombin on a copper chelate column.. Thromb Haemost. 1997 437 (abstract).
    PubMedGoogle Scholar
  • 24 Church FC, Meade JB, Treanor RE, Whinna HC. Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin.. J Biol Chem 1989; 264: 3618-23.
    PubMedGoogle Scholar
  • 25 Pedersen AH, Nordfang O, Norris F, Wiberg FC, Christensen PM, Moeller KB, Meidahl-Pedersen J, Beck TC, Norris K, Hedner U, Kisiel W. Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions.. J Biol Chem 1990; 265: 16786-93.
    PubMedGoogle Scholar
  • 26 Thim L, Bjoern S, Christensen M, Nicolaisen EM, Lund-Hansen T, Pedersen AH, Hedner U. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.. Biochemistry 1988; 27: 7785-93.
    CrossrefPubMedGoogle Scholar
  • 27 Hoffman M, Monroe DM, Roberts HR. Cellular interactions in hemostasis.. Haemostasis 1996; 26 (Suppl. 01) 12-6.
    PubMedGoogle Scholar
  • 28 van’t Veer C, Mann KG. Regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor, antithrombin-III, and heparin cofactor-II.. J Biol Chem 1997; 272: 4367-77.
    CrossrefPubMedGoogle Scholar
  • 29 Butenas S, van’t Veer C, Mann KG. Evaluation of the initiation phase of blood-coagulation using ultrasensitive assays for serine proteases.. J Biol Chem 1997; 272: 21527-33.
    CrossrefPubMedGoogle Scholar
  • 30 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor.. J Biol Chem 1973; 248: 6490-505.
    PubMedGoogle Scholar
  • 31 Rao LVM, Rapaport SI. Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation.. Blood 1987; 69: 645-51.
    PubMedGoogle Scholar
  • 32 Kirchhofer D, Tschopp TB, Baumgartner HR. Active site-blocked factors VIIa and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model.. Arterioscler Thromb Vasc Biol 1995; 15: 1098-106.
    CrossrefPubMedGoogle Scholar
  • 33 Ørvim U, Barstad RM, Örning L, Petersen LB, Ezban M, Hedner U, Sakariassen KS. Inactivated active site recombinant FVIIa is an efficient inhibitor of tissue factor/FVIIa induced arterial thrombus formation in human blood.. Thromb Haemost. 1995 73. 1185 (abstract).
    PubMedGoogle Scholar
  • 34 Tijburg PNM, Ryan J, Stern DM, Wollitzky B, Rimon S, Rimon A, Handley D, Nawroth P, Sixma JJ, de Groot PG. Activation of the coagulation mechanism on tumor necrosis factor-stimulated cultured endothelial cells and their extracellular matrix. The role of flow and factor IX/IXa.. J Biol Chem 1991; 266: 12067-74.
    PubMedGoogle Scholar
  • 35 Jang I-K, Gold HK, Leinbach RC, Fallon JT, Collen D, Wilcox JN. Anti-thrombotic effect of a monoclonal antibody against tissue factor in a rabbit model of platelet-mediated arterial thrombosis.. Arterioscler Thromb 1992; 12: 948-54.
    CrossrefPubMedGoogle Scholar
  • 36 Pawashe AB, Golino P, Ambrosio G, Migliaccio F, Ragni M, Pascucci I, Chiariello M, Bach R, Garen A, Konigsberg WK, Ezekowitz MD. A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries.. Circ Res 1994; 74: 56-63.
    CrossrefPubMedGoogle Scholar
  • 37 Golino P, Ragni M, Cirillo P, D’Andrea D, Scognamiglio A, Ravera A, Buono C, Ezban M, Corcione N, Vigorito F, Condorelli M, Chiariello M. Antithrombotic effects of recombinant human active site-blocked factor VIIa in a rabbit model of recurrent arterial thrombosis.. Circ Res 1998; 82: 39-46.
    CrossrefPubMedGoogle Scholar
  • 38 Jang Y, Guzman LA, Lincoff AM, Gottsauner-Wolf M, Forudi F, Hart CE, Courtman DW, Ezban M, Ellis SG, Topol EJ. Influence of blockade at specific levels of the coagulation cascade on restenosis in a rabbit arthero-sclerotic femoral artery injury model.. Circulation 1995; 92: 3041-50.
    CrossrefPubMedGoogle Scholar
  • 39 Arnljots B, Ezban M, Hedner U. Prevention of experimental arterial thrombosis by topical administration of active site-inactivated factor VIIa.. J Vasc Surg 1997; 25: 341-6.
    CrossrefPubMedGoogle Scholar
  • 40 Harker LA, Hanson SR, Wilcox JN, Kelly AB. Antithrombotic and anti-lesion benefits without hemorrhagic risks by inhibiting tissue factor pathway.. Haemostasis 1996; 26 (Suppl. 01) 76-82.
    PubMedGoogle Scholar
  • 41 Harker LA, Hanson SR, Kelly AB. Antithrombotic strategies targeting thrombin activities, thrombin receptors and thrombin generation.. Thromb Haemost 1997; 78: 736-41.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 42 Khouri RK, Koudsi B, Kaiding F, Ornberg RL, Wun T-C. Prevention of thrombosis by topical application of tissue factor pathway inhibitor in a rabbit model of vascular trauma.. Ann Plast Surg 1993; 30: 398-402.
    CrossrefPubMedGoogle Scholar
  • 43 Vlasuk GP, Ramjit D, Fujita T, Dunwiddie CT, Nutt EM, Smith DE, Shebuski RJ. Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis.. Thromb Haemost 1991; 65: 257-62.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 44 Harker LA, Hanson SR, Kelly AB. Antithrombotic benefits and hemorrhagic risks of direct thrombin antagonists.. Thromb Haemost 1995; 74: 464-72.
    Article in Thieme ConnectPubMedGoogle Scholar