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 (A PC) in the presence and absence of phospholipid (25% phosphatidylserin e, 75% phosphatidylcholine, PCPS) were evaluated using sedimentation v elocity/equilibrium methods in the analytical ultracentrifuge and by i mmunoprecipitation using an antibody directed against the light chain of the factor Va molecule, The molecular weight and sedimentation coef ficient 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 Arg(505) and Arg(662), Complete cleavage of the factor Va heavy chain (with APC-PC PS) at Arg(505), Arg(662) and Arg(306) results in a drastic change in the molecular weight observed for the product, Two products are resolv ed with sedimentation coefficients of 3.3 and 6.3 S with estimated mol ecular weights of 48,000 and 114,000, respectively, Immunoprecipitatio n studies showed that the products of factor Va cleavage at Arg(505) a nd Arg(662) (A1A2(N).A2(C).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 cle aved at Arg(505), Arg(662), and Arg(306) the precipitated complex cons isted of the Al domain (residues 1-306) and the light chain (residues 1537-2183) of factor Va (A1.LC). The fragments corresponding to residu es 307-505 (A2(N)) and 506-662 (A2(C)) are found in the supernatant, T he combined mass of these two products (48,000) is similar to the esti mated 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 frag ment, These data lead to the conclusion that cleavages at Arg(306), Ar g(505), and Arg(662) Of the factor Va molecule resulted in the dissoci ation of the entire A2 domain as two noncovalently associated fragment s (A2(N).A2(C)). Enzyme kinetic and light scattering data suggest that the complete inactivation of the factor Va molecule involves not only cleavage at Arg(306) but also the dissociation of the A2 domain, Thes e data also suggest that the complete APC inactivation of the factor V a molecule is analogous to the spontaneous inactivation of factor VIII a, which occurs via the dissociation of the A2 domain.