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 Arg⁷⁰⁹, Arg¹⁰¹⁸ followed by Arg¹⁵⁴⁵ 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 Arg¹⁵⁴⁵. 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 Arg⁷⁰⁹, to generate the heavy chain (105 kDa) followed by cleavage at Arg¹⁰¹⁸ and Arg¹⁵⁴⁵ to produce the light chain (74 kDa). In contrast to these results, elimination of the Arg¹⁰¹⁸ cleavage site via mutagenesis (FV-1033^R1018Q) or deletion (FV-1015; 1016–1491 deleted) dramatically delayed cleavage at Arg¹⁵⁴⁵ while proteolysis at Arg⁷⁰⁹ was unaltered compared to FV-1033. Control experiments with RVV-V revealed that FV-1033^R1018Q and FV-1015 were rapidly cleaved at Arg¹⁵⁴⁵ 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; Arg¹⁰¹⁸ not present) surprisingly restored rapid cleavage at Arg¹⁵⁴⁵. These data suggest that sequences N-terminal to the 1018 site (902-1017) are involved in regulating thrombin cleavage at Arg¹⁵⁴⁵ and that the purpose of cleavage at Arg¹⁰¹⁸ 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 Arg¹⁵⁴⁵ whether or not Arg¹⁰¹⁸ was present. Furthermore, we were able to demonstrate that residues 964–1007 are the principal determinants in restricting thrombin cleavage at Arg¹⁵⁴⁵. Functional measurements, including clotting assays, thrombin generation assays, and purified component systems revealed that FV derivatives which exhibited delayed cleavage at Arg¹⁵⁴⁵ 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 Arg¹⁰¹⁸. These sequences, which conformationally restrict the ability of thrombin to access Arg¹⁵⁴⁵, affect the rate and order of bond cleavage. Thus proteolysis at Arg¹⁰¹⁸ is required to alleviate these constraints allowing for accelerated cleavage at Arg¹⁵⁴⁵ by thrombin. Furthermore, initial cleavage at Arg⁷⁰⁹ 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.