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

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 (fV^D659-663) and a mutant molecule in which all five residues were mutated to lysine (fV^5K, charge reversal). The recombinant molecules along with wild type fV (fV^WT) 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 fVa^WT. Kinetic analyses studying prothrombinase assembled with the mutant molecules demonstrated diminished k_cat values, while the affinity of all mutant molecules for factor plasma-derived fXa was similar to fVa^WT. 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 fVa^D659-663 or fVa^5K 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 fVa^WT or fVa^D659-663. The data demonstrate a delay for cleavage of FPR-meizothrombin at Arg²⁷¹ by prothrombinase assembled with fVa^D659-663 as compared to the same reaction catalyzed by prothrombinase assembled with fVa^WT. Quantitative scanning densitometry of fragment 1•2-A demonstrated a ∼4-fold delay in cleavage of FPR-meizothrombin at Arg²⁷¹ by prothrombinase assembled with fVa^D659-663, compared to cleavage at Arg²⁷¹ by prothrombinase assembled with fVa^WT. Direct comparison between the rates of cleavage of FPR-meizothrombin by membrane-bound fXa alone or by prothrombinase assembled with fVa_RVV^D659-663 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.