Mutation of the Hydrophobic Residues in Factor Va2 C1 Domain Affects the Phosphatidylserine Mediated Prothrombin Activation.

Tightly associated factors V_a and X_a serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. The binding of factor V_a to PS membranes regulates assembly of the prothrombinase complex. The C-terminal domain (C2) of factor V_a (residues 2037–2196 in human factor V_a) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp²⁰⁶³/Trp²⁰⁶⁴ (Srivastava A, Quinn-Allen MA, Kim SW, Kane WH, Lentz BR. Biochemistry, 2001, 40(28): 8246–55). Our recent results have shown that mutating these Trp residues abolishes FV_a2-membrane binding, but does not affect the assembly and activity of the prothrombinase in the presence of 25% PS membranes or soluble C6PS. Our data also indicates that there is another site on factor V_a2 that might be specific for PS or C6PS and might serve as a regulatory site for assembly or activity of the FV_a2-FX_a complex. A pair of solvent exposed amino acids, Tyr¹⁹⁵⁶/Leu¹⁹⁵⁷ in the C1 domain is located analogously to the critical Trp residues in C2. Recently, we showed that prothrombin activation in the presence of the factor V_a mutant (Y1956, L1957) A was markedly impaired on phospholipid vesicles containing 10% or less PS but was essentially normal on vesicles containing 25% PS (Saleh, M., Peng, W., Quinn-Allen, MA., Macedo-Ribeiro, S., Fuentes-Prior, P., Bode,W. and Kane, WH. Thromb. Haemost. 2004,91:16–27). Our work aims to test the hypothesis that the PS regulatory site in V_a2 is located analogously to the C6PS binding site in the C2 domain. We have used factor V_a mutants with mutations in either the C1 domain, (Y1956, L1957) A or in both the C1 and C2 domains, (Y1956, L1957, W2063, W2064) A. We determine the rate of thrombin formation in the presence of 400 mM C6PS and wild type, C1 and C1C2 mutated factor V_a2 to be 170, 12 and 11 nM/S⁻¹/M⁻¹, respectively. Mutations in the C1 and C1C2 domains of factor V_a2 reduced the rate of activation of prothrombin to thrombin by 14–15 fold. We have also determined the effect of these mutations on the assembly of factors X_a–V_a2 complex by monitoring the change in fluorescence of dansyl-glutamyl-glycyl-arginyl-chloromethylketone (DEGR-CK)-X_a with the addition of wild type, C1 and C1C2 mutated factor V_a2 in the presence of 400 mM C6PS. Our data shows that the K_d’s of factor X_a with factor Va2 (wild type, C1 mutant and C1C2 mutant) are 3, 564, 624 nM respectively. Our results support the hypothesis that a PS regulatory site is located in the C1 domain of factor V_a and includes residues Tyr¹⁹⁵⁶ and Leu¹⁹⁵⁷.