The R306G and R506Q mutations in coagulation Factor V reveals additional cleavage sites for Activated Protein C in the R313-R321 region and at R505.

The procoagulant function of activated factor V (FVa) is inhibited by activated Protein C (APC) through proteolytic cleavages at R306, R506 and R679. Recombinant FVa mutated at all three APC-cleavage sites, FVa-GQA, was still inactivated by APC through at least two cleavages in the heavy chain of FVa; relatively rapid cleavage at R(x1) close to residue 506 and slower cleavage at R(x2) nearby residue 306. We investigated the exact location of these two cleavages, by substitution of arginines by glutamine within the R(x1)-region (R501, R505 or R510) and the R(x2)-region (R313, R316, R317 or R321). Immunoblot and kinetic analyses of the inactivation of activated R(x1)-mutants by APC revealed that using mutant FVa-GQA-505Q no R(x2)-R(x1) fragment was formed and that the inactivation reaction was first order with a rate constant of 1.0 x 10(4) M(-1) s(-1), similar to the rate constant of R(x2) cleavage (k(2)=1.3 x 10(4) M(-1) s(-1)). No single arginine could be pinpointed identified as R(x2). Individual replacement of arginine by glutamine at positions 313, 316, 317 or 321 in FV-GQA-505Q did not result in the disappearance of R(x2) as judged from kinetic and immunoblot analyses. However, replacement of all four arginines by glutamine completely prevented formation of the R(x2)-R(709) fragment. We conclude that substitution of arginine 506 by glutamine as in FV-Leiden, leads to the detection of a novel cleavage site at arginine 505 (R(x1)). Substitution of arginine 306 by glycine, like in FV-Cambridge, reveals several alternative cleavage sites near arginine 306, which together constitute a secondary cleavage site.