The Role of Amino Acids 659-663 of Factor Va Heavy Chain During Prothrombin Activation by Prothrombinase.

Abstract Abstract 2131 Poster Board II-108 The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), and the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of Ca2+. FXa alone can activate prothrombin following sequential cleavages at Arg271 and Arg320 yielding the transient inactive intermediate prethrombin 2. However, the interaction of fVa with fXa on a membrane/cell surface in the presence of divalent metal ions and formation of the prothrombinase complex results in the reversal of the order of cleavages and a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. A first cleavage of prothrombin by prothrombinase at Arg320 produces the active intermediate meizothrombin, while the second cleavage at Arg271 produces thrombin. Thrombin and prothrombin contain two positively charged binding regions (anion binding exosite I, ABE-I and anion binding exosite II, ABE II), that are crucial for protein function. Initial cleavage of prothrombin at Arg320 by prothrombinase which is absolutely factor Va dependent, entirely exposes (pro)exosite I. FVa is required for the specific recognition of prothrombinase by (pro)exosite I of prothrombin. The COOH-terminal region of the heavy chain of fVa contains acidic amino acid clusters that are important for cofactor activity. We have investigated the role of amino acid region 659-663 that contains five consecutive acidic amino acid residues. To ascertain the function of this region, site-directed mutagenesis was performed. We have constructed a mutant molecule with this region deleted (fVD659-663) and a mutant molecule in which all five residues were mutated to lysine (fV5K, charge reversal). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to fVaWT. Kinetic analyses studying prothrombinase assembled with the mutant molecules demonstrated diminished kcat values, while the affinity of all mutant molecules for factor plasma-derived fXa was similar to fVaWT. Gel electrophoresis analyzing plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg320 and Arg271 by prothrombinase assembled with the mutant molecules. Using recombinant prothrombin molecules we determined that cleavage at Arg271 by prothrombinase assembled with either fVaD659-663 or fVa5K was severely impaired compared to cleavage at Arg320 by prothrombinase assembled with the same recombinant cofactor molecules, resulting in lingering of meizothrombin throughout the activation process. To ascertain the effect of the mutations of the fVa heavy chain on the cleavage at Arg271 alone following the transition that occurs after cleavage at Arg320, we compared the rate of cleavage of active-site blocked meizothrombin (FPR-meizothrombin) by prothrombinase assembled with either fVaWT or fVaD659-663. The data demonstrate a delay for cleavage of FPR-meizothrombin at Arg271 by prothrombinase assembled with fVaD659-663 as compared to the same reaction catalyzed by prothrombinase assembled with fVaWT. Quantitative scanning densitometry of fragment 1•2-A demonstrated a ∼4-fold delay in cleavage of FPR-meizothrombin at Arg271 by prothrombinase assembled with fVaD659-663, compared to cleavage at Arg271 by prothrombinase assembled with fVaWT. Direct comparison between the rates of cleavage of FPR-meizothrombin by membrane-bound fXa alone or by prothrombinase assembled with fVaRVVD659-663 do not show any significant differences. Thus, deletion of amino acid region 659-663 virtually eliminates the acceleration in the rate of cleavage at Arg271 of meizothrombin attributed to the interaction of fVa with fXa. These data demonstrate that amino acid sequence 659DDDED663 from the factor Va heavy chain, regulates meizothrombin concentration during activation of prothrombin by prothrombinase. Disclosures: No relevant conflicts of interest to declare.