Factor Xa Dimerization and Prothrombinase Complex Formation are Competitive Process on a Membrane Surface

Abstract

Table of ContentsExposure of phosphatidylserine (PS) molecules on activated platelet membranes is a crucial event in blood coagulation. Binding of PS to a specific site on factor Xa (fXa) and factorVa (fVa) promotes their assembly into a complex that dramatically enhances proteolytic activity of fXa. Recent studies demonstrate that by both soluble PS and PS-containing membranespromote formation of inactive fXa dimer at 5mM Ca+2. The fluorescence anisotropy of active site labeled fXa, FEGR (Fluorescein-GLU-GLY-ARG-chloromethylketone)-Xa, is decreased in the presence of PS membrane on which it forms dimer. We report now the addition of fVa to membrane-bound FEGR-Xa produced fVa- FEGR-Xa complex formation with a Kd, surface approximately 60-fold lower than that characterizing FEGR-Xa surface dimerization, clearly indicating fVa strongly competed with fXa dimer formation in order to form active Xa-Va complex on the membrane surface. Analysis of FEGR-Xa fluorescence anisotropy yielded roughly constant Kd's for Xa-Va interaction with increasing Ca2+ concentration from 2 to 5 mM Ca2+ despite the fact that fXa dimer formation varied dramatically over this Ca2+ range. Experiments performed at varying membrane and fVa concentrations for both 23 nm and 120 nm confirmed that protein distribution between vesicles was sufficiently rapid as to overcome any possible effects of membrane discreteness. We conclude that PS-induced fXa dimerization on membrane strongly competes with fXa-fVa complex formation at high Ca2+ concentrations. Supported by USPHS grant HL072827 to BRL.