The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.

Abstract

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.