Activated protein C cleavage of factor Va leads to dissociation of the A2 domain

The products of cleavage of bovine factor Va by activated protein C (APC) in the presence and absence of phospholipid (25% phosphatidylserine, 75% phosphatidylcholine, PCPS) were evaluated using sedimentation velocity/equilibrium methods in the analytical ultracentrifuge and by immunoprecipitation using an antibody directed against the light chain of the factor Va molecule. The molecular weight and sedimentation coefficient of the associated heavy and light chains of factor Va, 173,000 (7.9 S) is reduced to 132,000 (7.1 S) by APC cleavage at Arg505 and Arg662. Complete cleavage of the factor Va heavy chain (with APC-PCPS) at Arg505, Arg662 and Arg306 results in a drastic change in the molecular weight observed for the product. Two products are resolved with sedimentation coefficients of 3.3 and 6.3 S with estimated molecular weights of 48,000 and 114,000, respectively. Immunoprecipitation studies showed that the products of factor Va cleavage at Arg505 and Arg662 (A1A2N.A2C.LC) are mostly noncovalently associated and consequently immunoprecipitated with an antibody directed against the light chain of the factor Va molecule. In contrast, for factor Va cleaved at Arg505, Arg662, and Arg306 the precipitated complex consisted of the A1 domain (residues 1-306) and the light chain (residues 1537-2183) of factor Va (A1.LC). The fragments corresponding to residues 307-505 (A2N) and 506-662 (A2C) are found in the supernatant. The combined mass of these two products (48,000) is similar to the estimated mass of the 3.3 S fragment estimated from sedimentation velocity/equilibrium studies; while the combined mass of the 1-306 + 1537-2183 products corresponds to 114,000, the estimated mass of the 6.3 S fragment. These data lead to the conclusion that cleavages at Arg306, Arg505, and Arg662 of the factor Va molecule resulted in the dissociation of the entire A2 domain as two noncovalently associated fragments (A2N.A2C). Enzyme kinetic and light scattering data suggest that the complete inactivation of the factor Va molecule involves not only cleavage at Arg306 but also the dissociation of the A2 domain. These data also suggest that the complete APC inactivation of the factor Va molecule is analogous to the spontaneous inactivation of factor VIIIa, which occurs via the dissociation of the A2 domain.