Molecular Mechanism Underlying the Need for Three Cleavage Sites within the B-Domain to Activate Factor V by Thrombin

Abstract 2219 Blood coagulation factor V (FV) is a multi-domain protein (A1-A2-B-A3-C1-C2) with little or no procoagulant activity and circulates in blood as a procofactor. Recent data indicate that specific sequences within the B-domain (963-1008) play a predominant role in keeping FV inactive. Removing these sequences via deletion or mutagenesis drives the activation of FV. In normal physiological situations, proteolytic processing of FV by thrombin within the B-domain (710-1545) ultimately removes these inhibitory sequences. Thrombin cleaves FV at three sites in a kinetically preferred order at Arg 709 , Arg 1018 followed by Arg 1545 to ultimately generate the active cofactor species, FVa consisting of a heavy and light chain. The need for three thrombin cleavage sites to generate an active cofactor species is not entirely obvious considering that RVV-V, a snake venom protease, activates FV following a single cleavage at Arg 1545 . Here we uncover new structure/function insights into how specific B-domain sequences restrict thrombin recognition of FV thereby forcing the need for three cleavage sites. To investigate this, we generated a panel of recombinant FV derivatives in BHK cells with different regions of the B-domain eliminated and combined functional measurements with SDS-PAGE to examine FV activation. As a control, we generated FV-1033 (residues 1034–1491 deleted) which is functionally equivalent to full-length FV and retains all three thrombin cleavage sites. Like FV, incubation of FV-1033 with thrombin resulted in rapid cleavage at Arg 709 , to generate the heavy chain (105 kDa) followed by cleavage at Arg 1018 and Arg 1545 to produce the light chain (74 kDa). In contrast to these results, elimination of the Arg 1018 cleavage site via mutagenesis (FV-1033 R1018Q ) or deletion (FV-1015; 1016–1491 deleted) dramatically delayed cleavage at Arg 1545 while proteolysis at Arg 709 was unaltered compared to FV-1033. Control experiments with RVV-V revealed that FV-1033 R1018Q and FV-1015 were rapidly cleaved at Arg 1545 similar to FV-1033. These data are consistent with prior mutagenesis studies with full length FV and collectively suggest that the middle cleavage site somehow potentiates cleavage at 1545. However, further elimination of the B-domain using FV-902 (903-1491 deleted; Arg 1018 not present) surprisingly restored rapid cleavage at Arg 1545 . These data suggest that sequences N-terminal to the 1018 site (902-1017) are involved in regulating thrombin cleavage at Arg 1545 and that the purpose of cleavage at Arg 1018 is to remove these constraints. In support of this, FV derivatives in which these sequences are exchanged with non-homologous regions of the FVIII B-domain were rapidly cleaved at Arg 1545 whether or not Arg 1018 was present. Furthermore, we were able to demonstrate that residues 964–1007 are the principal determinants in restricting thrombin cleavage at Arg 1545 . Functional measurements, including clotting assays, thrombin generation assays, and purified component systems revealed that FV derivatives which exhibited delayed cleavage at Arg 1545 all have a markedly reduced ability to generate thrombin compared to FV-1033 or FV. Collectively these data demonstrate that FV cleavage site preferences are not only influenced by the geometric constraints imposed on thrombin but are also influenced in a dramatic way by specific B-domain sequences adjacent to Arg 1018 . These sequences, which conformationally restrict the ability of thrombin to access Arg 1545 , affect the rate and order of bond cleavage. Thus proteolysis at Arg 1018 is required to alleviate these constraints allowing for accelerated cleavage at Arg 1545 by thrombin. Furthermore, initial cleavage at Arg 709 appears unaltered by any region of the B-domain as each of FV derivatives studied show equivalent processing at this site. These data provide new structure/function insights into the relationship between the removal of B-domain constraints and accessibility of thrombin cleavage sites which lead to the activation of FV. Disclosures: Camire: Pfizer: Patents & Royalties, Research Funding.