Localization of Stereo-Specific Phosphatidylserine Binding Sites within the Factor V C2 Domain.

Binding of activated factor V (Fva) to phosphatidylserine (PS) is required for formation of the prothrombinase complex on the surface of activated platelets. The interaction between Fva and PS has been shown to be stereo-specific with bovine Fva favoring _L-PS over _D-PS. Our previous low resolution alanine scan data localized PS binding sites to the FV-C2 domain and identified two tryptophan residues, W2063 and W2064, which are required for high affinity binding. Two putative PS binding sites were also predicted based on modeling studies using the FV-C2 structure. In this study, the stereo-specific interaction of Fva with PS was evaluated with plasma derived human Fva (pd-HFVa), recombinant human Fva (rHFVa), rHFVa-C2 domain and rHFVa mutants. Quantitative fluorescence binding assays and functional prothrombinase assays were conducted using phospholipid vesicles containing 20 mol% dioleoyl phosphatidyl-_L-serine (_L-PS) or dioleoyl phosphatidyl-_D-serine (_D-PS). Direct binding experiments demonstrated that pd-HFVa and rHFVa bound to membranes containing _L-PS with 6 to 9 fold higher affinity than to membranes containing _D-PS. Analysis of prothrombinase activity on these membranes demonstrated the difference in V_max/K_1/2Va between _L-PS and _D-PS was 15-fold and 23-fold higher for pd-HFVa and rHFVa, respectively. These data confirm that HFVa, similar to bovine FVa, prefers _L-PS over _D-PS. The rHFV-C2 domain bound to _L-PS membranes with an affinity of 5.3 ± 1.7 nM. In contrast, the binding of C2 domain to _D-PS membranes was undetectable. This indicates that the rHFV-C2 domain contains a stereo-specific binding site for PS. We next analyzed the membrane binding of four rHFVa mutants containing alanine substitutions within the FV-C2 domain. rHFVa mutants W2063A and W2064A both bound to membranes containing 20% _L-PS with high affinity similar to native rHFVa with K_d’s of 0.97 ± 0.30 nM and 0.38 ± 0.18 nM, respectively. In contrast no detectable binding of W2063A or W2064A could be detected on membranes containing 20% _D-PS. Mutants were also prepared containing alanine substitutions for amino acid side chains predicted to participate in two stereospecific PS binding sites based on modeling studies using the rHFV-C2 structure. The binding of (K2060, Q2085, S2115)A and (Q2078, N2082, R2187)A to _L-PS and _D-PS were characterized as described above. These mutants bound to both _L-PS and _D-PS membranes with relatively high affinity. The K_d values for binding of (K2060, Q2085, S2115)A and (Q2078, N2082, R2187)A to 20% _D-PS membranes were 1.19 ± 0.31 nM and 1.11 ± 0.32 nM, respectively. These results suggest that these two sites play little or no role in the binding of FVa to PS membranes. In conclusion, the HFV-C2 domain contains stereo-specific binding sites for _L-PS which require the indole side chains of W2063 and W2064. These findings provide new insights into the regulation of prothrombinase activity by PS.