Amino Acid Region 1000–1008 of Coagulation Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Abstract 2221 The intricate process of hemostasis is a highly regulated mechanism which implements the conversion of prothrombin to thrombin and the crucial formation of a fibrin clot. The direct progression of hemostasis is pivotal to the prevention of various clotting disorders associated to hypercoagulation and excess bleeding. Upon vascular injury, the proteolytic conversion of prothrombin to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of divalent metal ions. Coagulation factor V (fV) is synthesized as a multi-domain (A1-A2-B-A3-C1-C2) quiescent procofactor with nominal procoagulant activity. Following the three sequential catalytic cleavages by a-thrombin at Arg709, Arg1018 and Arg1545 amino acids 710–1545 of the B-domain are liberated to generate the noncovalently associated light and heavy chains of fVa. The cleavage at Arg1545 is crucial for full procoagulant activity. The heterodimer of fVa is composed of a heavy chain associated with the 2 A domains (residues 1–303 and 317–656) and a light chain composed of one A domain (1546-1877) and two C domains (residues 1878–2036 and 2037–2196). Since single chain fV does not bind fXa, the proper removal of the B-domain is vital to generate procoagulant activity. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Appropriate binding of fVa to fXa during prothrombinase function is essential to the proper activation of the substrate, prothrombin. Previous studies have determined the heavy and light chains of fVa to have fXa interactive sites. A highly basic region of amino acids in the B-domain suggests a potential sheathing of either the heavy or light chain fXa interface sites. To verify this hypothesis we investigated the role of amino acid region 1000–1008 that contains seven basic amino acid residues. To ascertain the role of this region we have constructed a recombinant mutant fV molecule with all activation cleavage sites (R709/R1018/R1545) mutated to glutamine (fV*T3Q), a mutant fV molecule with region 1000–1008 deleted (fVΔ1000-1008), and a mutant fV molecule containing the same deletion with all activation cleavage sites changed to glutamine (fVΔ1000-1008/*T3Q). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for their capability to bind fXa within prothrombinase prior (fV) and after incubation with thrombin (fVa). The data showed that fV*T3Q and fVa*T3Q were unable to interact with fXa. In contrast, the Kd values for fVΔ1000-1008 (0.9 nM), fVaΔ1000-1008 (0.4 nM), fVΔ1000-1008*T3Q (0.7 nM) and fVaΔ1000-1008*T3Q (0.5 nM), were similar to the affinity of fVaWT for fXa (0.22 nM). Two-stage clotting assays revealed that while fVa*T3Q was practically devoid of clotting activity, the mutant molecules fVaΔ1000-1008, and fVaD1000-1008*T3Q had clotting activities comparable to fVaWT. Thus, unactivated fVΔ1000-1008*T3Q has an affinity for fXa that is similar to the affinity of fVaWT for the enzyme. In addition, fVΔ1000-1008*T3Q that cannot be cleaved and activated by thrombin or activated during the course of the clotting assay, has similar clotting activity as fVaWT (∼3110 U/mg). The data presented in this study provide an important insight into one of the possible roles of the B domain of factor V, explicitly the fXa interactive sites on fVa are covered/inhibited by amino acids 1000–1008 of the fV B-domain. These data strongly suggest that amino acid region 1000–1008 of fV contains a regulatory sequence protecting the organisms from spontaneous binding of the procofactor to fXa and unnecessary prothrombinase complex formation which will result in catastrophic physiological consequences. Disclosures: No relevant conflicts of interest to declare.