Factor Xa Dimerization and Prothrombinase Complex Formation Are Competitive Process On a Membrane Surface.

Abstract 2123

Poster Board II-100

Exposure of phosphatidylserine (PS) molecules on activated platelet membranes is a crucial event in blood coagulation. Binding of PS to a specific site on factors Xa and on Va promotes prothrombinase assembly and enhances proteolytic activity of factor Xa. PS binding produces significant structural changes in both proteins. Recent studies demonstrate formation of inactive Xa dimer by both soluble, short-chain PS and PS-containing membranes at high Ca⁺² concentration (Chattopadhyay et al.,2009, Biophys. J, 974). Association of factor Xa with factor Va in the presence of soluble PS relieves this inactivation. From kinetic measurements, we found that apparent Kd's for the Xa-Va interaction in the presence of 50 mM PS/PC (25/75) membranes increased in with increasing factor Xa concentrations, suggesting that factor Va must compete with factor Xa dimer in order to form the Xa-Va complex on the membrane surface. The fluorescence anisotropy of active site labeled factor Xa, FEGR (Fluorescein-GLU-GLY-ARG-chloromethylketone)-Xa, is lowered in the presence of PS membrane on which it forms dimer (Koklic et al., October 2009, Biophys. J., V-97, Issue 8) We report now that addition of factor Va to FEGR-Xa/membrane samples produced a saturating increase in anisotropy, clearly indicating that factor Va competed with factor Xa dimer formation. This experiment was repeated for several Ca²⁺ concentrations and yielded increasing apparent K_d's for Xa-Va interaction with increasing Ca²⁺concentration. FEGR-Xa fluorescence anisotropy as well as DEGR-Xa (factor Xa labeled at the active site with Dansyl-GLU-GLY-ARG-chloromethylketone) fluorescence intensity at varying membrane and factor Va concentrations for both 23 nm and 120 nm vesicles were analyzed globally to obtain surface K_d's of dimerization and factor Va-factor Xa interaction. This showed that membrane discreteness (slow distribution of factor Xa between discrete membrane vesicles) had almost no effect on the competition between factor Xa dimer formation and Xa-Va complex formation. We conclude that prothrombinase complex formation can overcome PS-induced factor Xa inhibition through factor Xa dimer formation at Ca²⁺ concentrations just above plasma concentration. In this way, one can imagine that factor Va is required to overcome factor Xa dimerization and amplify thrombin production in the early platelet plug.

Supported by USPHS grant HL072827 to BRL.