Activation of human factor V by Meizothrombin

Guido Tans, Gerry A.F. Nicolaes, M. Christella L.G.D. Thomassen, H. Coenraad Hemker, Anton-Jan van Zonneveld, Hans Pannekoek, Jan Rosing

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    Abstract

    A recombinant human prothrombin was prepared in which Arg(155) was replaced by Ala. The recombinant prothrombin was converted into a meizothrombin derivative (R155A meizothrombin) that was resistant to autocatalytic removal of the fragment 1 domain. R155A meizothrombin appeared to be a potent factor V activator in reaction mixtures that contained negatively charged phospholipid vesicles. Factor V activation by R155A meizothrombin was characterized by second-order rate constants of 0.06 x 10(6) M(-1) S-1 in the absence of phospholipid and 18 x 10(6) M(-1) S-1 in the presence of 60 mu M phospholipid vesicles composed of a 10:90 mol/mol mixture of phosphatidylserine (PS) and phosphatidylcholine (PC). The rate constant for thrombin-catalyzed activation of factor V was hardly affected by the presence of phospholipid vesicles and was 4.0 x 10(6) M(-1) S-1. The initial rate of activation of 3 nM factor V by R155A meizothrombin was a function of the concentration of PS/PC vesicles present in the reaction mixture, and the calculated rate constant reached a plateau value at greater than or equal to 50 mu M PS/PC. Gel electrophoretic analysis of factor V activation showed that R155A meizothrombin and thrombin cleaved the susceptible peptide bonds in factor V at different rates. However, both activators finally generated a factor Va molecule composed of a heavy chain with an M(r) of 104,000 and a light chain doublet with M(r) values of 74,000 and 71,000. Since meizothrombin is one of the major reaction products formed during the initial phase of prothrombin activation, these findings are indicative of a significant contribution of meizothrombin to in vivo factor V activation.
    Original languageEnglish
    Pages (from-to)15969-15972
    Number of pages4
    JournalJournal of Biological Chemistry
    Volume269
    Issue number23
    DOIs
    Publication statusPublished - 1994

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