Mapping the Location of Autoinhibitory B-Domain Motifs within Factor V: New Insights Into How Factor V Activation Unfolds

Abstract 495 Blood coagulation factor V (FV) circulates as a procofactor with little or no procoagulant activity. Proteolytic removal of a large central B-domain converts FV (domain organization of A1-A2-B-A3-C1-C2) to an active cofactor for membrane-bound FXa to form prothrombinase. Recently we found that discrete, evolutionarily conserved sequences within the B-domain serve an autoinhibitory function and are necessary to maintain FV as an inactive procofactor. The autoinhibitory region within the B-domain (termed the procofactor regulatory region or PRR) is sequence specific and remarkably short (∼100 amino acids out of 836 from B-domain) consisting of basic (963–1008; basic region or BR) and acidic (1493–1537; acidic region or AR) sequences. Dismantling this region appears to be the driving force to unveil a high affinity binding site(s) for FXa and thus underpins the procofactor to cofactor transition. While the BR and AR seem to work together to provide “on-site” repression of cofactor activity, there is no structural information about the FV B-domain that could provide clues into how this critical region functions. Here we use B-domain fragments tethered to an artificial protease in order to map the position of the PRR relative to the rest of the FV molecule. A BR peptide with an introduced free Cys at position 990 was stoichiometrically modified with FeBABE (Fe-p-bromoacetamidobenzyl-EDTA). FeBABE is a protein cutting reagent with a sulfhydryl-reactive moiety for attachment to Cys and an EDTA-chelated iron atom which, when triggered with ascorbic acid and peroxide, generates hydroxyl radicals that cut peptide bonds in a sequence-independent fashion. When a protein labeled with FeBABE binds its target, the Fe-BABE moiety facilitates cutting if it is orientated correctly and near ( Disclosures: Camire: Pfizer: Patents & Royalties, Research Funding.