Mutation of hydrophobic residues in the factor Va C1 and C2 domains blocks membrane-dependent prothrombin activation

The binding of factor (FVa) to phosphatidylserine (PS) membranes regulates assembly of the prothrombinase complex. Two pairs of solvent-exposed amino acids, Tyr(1956) /Leu(1957) in the C1 domain and Trp(2063) /TrP2064 in the C2 domain, each make significant contributions to the affinity of FVa for PS membranes, but individually neither pair of amino acids is required for prothrombinase assembly on 25% PS membranes. In this study we characterize a FVa mutant with alanine substitutions in both the Cl and C2 domains: (Y1956,LI957,W2063,W2064)A. We conclude that: (i) prothrombinase assembly on PS membranes requires Trp(2063) /TrP2064 and/or Tyr(1956) /Leul(1957); (ii) combined mutation of TrP2063 /Trp(2064) and Tyr(1916) /Leu(1957) results in only a modest 4-fold decrease in the rate of thrombin generation in the absence of membranes; (iii) the present data provide experimental support for the joint participation of the C1 and C2 domains in the binding of FVa to phospholipid membranes as suggested by the recently solved structure for FVai (A1/A3-C1-C2).