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Activation of human factor V by meizothrombin
G Tans1, G A Nicolaes, M C Thomassen
1Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Abstract:
A recombinant human prothrombin was prepared in which Arg155 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 microM 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 > or = 50 microM 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.
Insights
A modified prothrombin, R155A meizothrombin, potently activates factor V, especially on phospholipid surfaces. This suggests meizothrombin plays a key role in factor V activation during blood coagulation.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Prothrombin activation is a critical step in blood coagulation.
- Meizothrombin, an intermediate in prothrombin activation, has been investigated for its enzymatic activities.
- The role of meizothrombin in activating factor V, particularly in the presence of phospholipids, requires further elucidation.
Purpose of the Study:
- To investigate the factor V activating potential of a modified meizothrombin derivative (R155A meizothrombin).
- To compare the kinetics of factor V activation by R155A meizothrombin with that of native thrombin.
- To determine the influence of negatively charged phospholipid vesicles on factor V activation by R155A meizothrombin.
Main Methods:
- Preparation of recombinant human prothrombin with an Arg155 to Ala substitution.
- Conversion of recombinant prothrombin to R155A meizothrombin.
- Assay of factor V activation using R155A meizothrombin and thrombin in the presence and absence of phosphatidylserine/phosphatidylcholine vesicles.
- Kinetic analysis using second-order rate constants.
- Gel electrophoretic analysis of factor V cleavage products.
Main Results:
- R155A meizothrombin demonstrated significantly enhanced factor V activation compared to thrombin, particularly on phospholipid vesicles (18 x 10^6 M^-1 S^-1 vs. 4.0 x 10^6 M^-1 S^-1).
- Factor V activation by R155A meizothrombin was highly dependent on the concentration of phosphatidylserine/phosphatidylcholine vesicles, reaching a plateau at approximately 50 microM.
- Both R155A meizothrombin and thrombin generated similar factor Va heavy and light chains, although cleavage rates differed.
Conclusions:
- R155A meizothrombin is a potent activator of factor V, with activity significantly enhanced by negatively charged phospholipids.
- The findings suggest that meizothrombin, a major product of initial prothrombin activation, likely contributes significantly to in vivo factor V activation.
- This study highlights a crucial role for meizothrombin in the hemostatic pathway beyond its role as a prothrombin intermediate.