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Thromb Haemost 1990; 64(04): 542-547
DOI: 10.1055/s-0038-1647354
Original Article
Schattauer GmbH Stuttgart

Continuous Flow and the Prothrombinase-Catalyzed Activation of Prothrombin

Pieter Schoen
1   The Department of Biochemistry, University of Limburg, Maastricht, The Netherlands
,
Theo Lindhout
1   The Department of Biochemistry, University of Limburg, Maastricht, The Netherlands
,
George Willems
2   The Department of Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands
,
H Coenraad Hemker
1   The Department of Biochemistry, University of Limburg, Maastricht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 29 December 1989

Accepted after revision23 July 1990

Publication Date:
25 July 2018 (online)

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Summary

The activation of prothrombin by prothrombinase was investigated in a continuous flow system at 25 °C. A glass capillary, containing a continuous phospholipid bilayer attached to the interior surface, was first perfused with factor Va. The factor Va bound to the phospholipid surface functioned as sites for the formation of prothrombinase, when subsequently a factor Xa and prothrombin containing solution was perfused. Under the conditions used, steady-state rates of prothrombin activation were attained after 4 to 15 min. The rates of prothrombinase formation increased with increasing factor Xa concentrations and flow rates, which is compatible with the assembly of prothrombinase being dependent on the flux of factor Xa to the phospholipid-bound factor Va. As long as factor Xa and prothrombin were present in the fluid phase the assembly of prothrombinase was apparently irreversible; during at least 20 min no loss of activity occurred. The steady-state rate of prothrombin activation was dependent on the surface concentration of prothrombinase, at 1.0 ΜM prothrombin and a shear rate of 82 s−1 the average rate was 870 mol thrombin/min per mol prothrombinase. In contrast to test tube experiments it was observed that in this flow system, the formation of Α-thrombin is favoured above the formation of meizothrombin (des fragment 1).

 

  • References

  • 1 Goldsmith HL, Turitto VT. Rheological aspects of thrombosis and haemostasis: basic principles and applications. Thromb Haemostas 1986; 55: 415-435
    PubMedSearch in Google Scholar
  • 2 Leonard EF. Rheology of thrombosis. In: Hemostasis and Thrombosis; Basic Principles and Clinical Practice, 2nd edition Colman RW, Hirsh J, Marder VJ, Salzman EW. (eds). JB Lippincott Company; Philadelphia: 1987: 1111-1122
  • 3 Gemmell CH, Turitto VT, Nemerson Y. Flow as a regulator of the activation of factor X by tissue factor. Blood 1988; 72: 1404-1406
    PubMedSearch in Google Scholar
  • 4 Rosing J, Tans G. Meizothrombin, a major product of factor Xa-catalyzed prothrombin activation. Thromb Haemostas 1988; 60: 355-360
    PubMedSearch in Google Scholar
  • 5 Tracy PB. Regulation of thrombin generation at cell surfaces. Sem Thromb Hemostas 1988; 14: 227-233
    Thieme ConnectPubMedSearch in Google Scholar
  • 6 Lindhout T, Govers-Riemslag JW P, vd Waart P, Hemker HC, Rosing J. Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition. Biochemistry 1982; 21: 5494-5502
    CrossrefPubMedSearch in Google Scholar
  • 7 Rosing J, Tans G, Govers-Riemslag JW P, Zwaal RF A, Hemker HC. The role of phospholipid and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-283
    PubMedSearch in Google Scholar
  • 8 Schoen P, Lindhout T. The in situ inhibition of prothrombinase-formed human a-thrombin and meizothrombin (des FI) by antithrombin III and heparin. J Biol Chem 1987; 262: 11268-11274
    PubMedSearch in Google Scholar
  • 9 Hendrix H, Lindhout T, Mertens K, Engels W, Hemker HC. Activation of human prothrombin by stoichiometric levels of staphy-locoagulase. J Biol Chem 1983; 258: 3637-3644
    CrossrefPubMedSearch in Google Scholar
  • 10 Chase T, Shaw E. Comparison of the esterase activities of trypsin, plasmin and thrombin on guanidinobenzoate esters. Titration of the enzymes. Biochemistry 1969 8. 2212-2224
    PubMedSearch in Google Scholar
  • 11 Mertens K, Bertina RM. Pathways in the activation of human coagulation factor X. Biochem J 1980; 185: 647-658
    CrossrefPubMedSearch in Google Scholar
  • 12 Lindhout T, Kop-Klaassen BH M, Hemker HC. Activation of decarboxyfactor X by a protein from Russell’s viper venom purification and partial characterization of activated decarboxyfactor X. Biochim Biophys Acta 1978; 533: 327-341
    CrossrefPubMedSearch in Google Scholar
  • 13 Smith RL. Titration of activated bovine factor X. J Biol Chem 1973; 248: 2418-2423
    PubMedSearch in Google Scholar
  • 14 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedSearch in Google Scholar
  • 15 Miller-Andersson M, Gaffney PJ, Seghatchian MJ. Preparation and stability of a highly purified human thrombin standard. Thromb Res 1980; 20: 109-122
    CrossrefPubMedSearch in Google Scholar
  • 16 Bird RB, Stewart WE, Lightfoot EN. Transport Phenomena. . John Wiley & Sons, Inc., New York 1960; 46-363
    PubMedSearch in Google Scholar
  • 17 Brian AA, McConnell HM. Allogenic stimulation of cytotoxic T cells by supported planar membranes. Proc Natl Acad Sci USA 1984; 81: 6159-6163
    CrossrefPubMedSearch in Google Scholar
  • 18 Higgins DL, Mann KG. The interaction of bovine factor V and factor V-derived peptides with phospholipid vesicles. J Biol Chem 1983; 258: 6503-6508
    PubMedSearch in Google Scholar
  • 19 vd Waart P, Bruls H, Hemker HC, Lindhout T. Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles. Biochemistry 1983; 22: 2427-2432
    CrossrefPubMedSearch in Google Scholar
  • 20 Hermens WT, Kop JM M, Willems GM. Adsorption of coagulation factors II, V, and X at phospholipid membranes. In: Coagulation and Lipids Zwaal RF A. (ed). CRC Press, Inc.,; Boca Raton: 1988: 73-97
  • 21 Subramaniam S, Seul M, McConnell HM. Lateral diffusion of specific antibodies bound to lipid monolayers on alkylated substrates. Proc Natl Acad Sci USA 1986; 83: 1169-1173
    CrossrefPubMedSearch in Google Scholar