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

Abstract

The procoagulant function of activated factorV (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 1.0x10(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. (C) 2009 Elsevier Ltd. All rights reserved.