The X-Ray Structure of a Variant of Human Factor V Provides Structural Insights into the Procofactor Activation Paradox
Authors: Kumar Shekhar , Stayrook Steven , Camire Rodney M. , Krishnaswamy Sriram 
Published: October 2019

Coagulation factor V (FV) circulates as an inactive procofactor with a domain organization of A1-A2-B-A3-C1-C2. Factor Va (FVa), the active cofactor, is produced in steps essential for rapid thrombin formation, by the proteolytic excision of the B domain which resolves the molecule into a heterodimer (A1-A2/A3-C1-C2). Removal of the B domain imbues the resulting FVa with the ability to bind factor Xa (Xa) on a membrane surface to assemble prothrombinase and greatly enhance the rate of thrombin formation. A recombinant variant of human factor V (HFVDT) with a shortened B domain exhibits constitutive cofactor activity. Cofactor activity even without proteolysis arises from its lack of a conserved basic region (BR) located in the large B domain of FV. Exogenously added BR peptide binds tightly to HFVDT in a Ca2+-dependent fashion and restores procofactor-like properties. The BR is proposed to restrict Xa binding and cofactor function by interacting with an acidic region at the C terminus of the B domain (AR2) and likely also an acidic sequence at the C terminus of the A2 domain (AR1). These two sequences are ~800 residues apart in FV with no structural information to explain how AR1 and AR2 might cooperate to engage the BR in the central portion of the B domain to autoinhibit FV. The available structures of an inactivated form of bovine factor Va (BFVai), of a FV ortholog from Pseudonaja textilis (FVPtex) and a lower resolution structure of B domainless human factor VIII (HFVIII) shed no light on this problem. We obtained diffraction quality crystals of HFVDT complexed with a single chain antibody (scFvE10) directed to FVa. Crystals were not obtained in the absence of scFvE10. The crystals diffracted to a resolution of 2.8 Å and the structure was solved by molecular replacement. The refined structure shows high similarity to BFVai, FVPtex and HFVIII. Insufficient electron density precluded the placement of scFvE10 in the modeled structure. The three homologous A domains in HFVDT adopt a typical cupredoxin-fold with the A domains arranged in a pseudo-three-fold axis of symmetry. The two C domains are cylindrical and oriented side-by-side to form the base of the A domain rosette. These features are equivalent to those seen in structures of BFVai, HFVIII and FVPtex. Two bound calcium ions are evident, one in A1 and the other in the A3 domain. The most important feature newly revealed in the structure of HFVDT is the close spatial proximity of AR1 and AR2 at the outer edge of the A domain rosette at the 3 o'clock position in the standard orientation. These acidic regions form adjacently positioned surfaces in spite of being bisected by the long primary sequence of the intervening B domain. Our observations provide the first structural evidence that the two distinct acidic regions come together in space to provide an extended surface. This provides a plausible explanation for how the BR in the middle of the B domain may bind to both AR1 and AR2 to restrict cofactor function in FV. The need for this extended but bipartite acidic surface, to which the BR may bind, also provides a plausible explanation for how proteolytic cleavage at position 1545 at the C terminus of AR2 destabilizes BR binding and results in cofactor formation. The Ca2+-stabilized loop in the A3 domain abuts the bisegmental acidic cluster potentially explaining why BR binding to HFVDT is strongly dependent on Ca2+. In the structure of FVPtex bound to snake venom factor X, AR1 extends away from the body of the cofactor to make intimate contacts with factor X. If this is mirrored in human prothrombinase, then our findings provide a structure-based model to phrase the long-standing procofactor activation paradox. BR binding to the AR1/AR2 extended surface ties up surfaces necessary for Xa binding and restricts cofactor activity. Proteolytic processing of the B domain and probably most importantly following AR2 destabilizes the BR/AR1/AR2 complex to free up surfaces including AR1 necessary to support Xa binding. This model reconciles the biochemical evidence with structural findings to provide new insights into the role played by the BR/AR1/AR2 complex in restricting Xa binding and cofactor function in FV. It provides a platform to further explore mechanistic details of FV and FVa function and for the development of novel strategies to modulate their functions to regulate thrombin formation for therapeutic gain.

Disclosures

Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Spark Therapeutics: Other: Scientific advisory board.