Role of the membrane surface in the activation of human coagulation factor X

S. Krishnaswamy, K. A. Field, T. S. Edgington, J. H. Morrissey, K. G. Mann

Research output: Contribution to journalArticlepeer-review

Abstract

Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS) or phosphatidylcholine alone (PC). Factor X activation was evaluated by discontinuous measurements of the amidolytic activity of the product, factor Xa, or continuously monitored using the fluorescent serine protease inhibitor 4-aminobenzamidine. The results of both techniques were verified by direct physical measurements of zymogen activation using SDS-polyacrylamide gel electrophoresis. The rate of factor X activation with PC vesicles was less than 5% of that observed with PCPS vesicles. Since factor X does not bind to vesicles containing only PC, these data suggested an important role for the substrate-membrane interaction in the catalytic cycle. The importance of the substrate-membrane interaction in the activation process was investigated by using membrane-binding proteins to compete with the substrate for combining sites on PCPS vesicles. Prothrombin fragment 1 was an inhibitor of factor X activation. The dependence of inhibition by fragment 1 on PCPS and factor X was consistent with a significant reduction in initial velocity due to the displacement of factor X from the membrane surface. The inhibition data also suggested that the membrane-bound pool of factor X was the preferred substrate for the human extrinsic Xase complex. The influence of PCPS concentrations on the rate of factor X activation was systematically investigated. Increasing concentrations of PCPS resulted in a modest change in the K(m,app) and a dramatic change in the V(max,app) for the reaction. The initial velocity data could be globally analyzed according to the preferential utilization of membrane-bound factor X with the intrinsic kinetic constants: K(m) ≃ 1 μM and k(cat) = 37 s-1 at saturating PCPS. In addition, the equilibrium parameters for the factor X-membrane interaction inferred from these studies were in excellent agreement with the directly determined values. Collectively, the data suggest that the substrate-membrane interaction must precede catalysis for the efficient activation of human factor X by the extrinsic Xase complex.

Original languageEnglish (US)
Pages (from-to)26110-26120
Number of pages11
JournalJournal of Biological Chemistry
Volume267
Issue number36
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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