The Regulatory Function of Amino Acid Region 659–663 of Factor Va on Prothrombinase during Thrombin Formation.

Following vascular injury, the process of hemostasis facilitates the generation of thrombin, which in turn allows the formation of a fibrin clot. Without the proper regulation of this process, serious life threatening conditions, such as DVT (deep vein thrombosis), can occur. The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. The incorporation of factor Va (fVa) into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of factor Xa (fXa) for thrombin generation. Factor Va is composed of heavy and light chains. The light chain of the cofactor contains the binding sites of the cofactor to the membrane surface while the heavy chain contains binding sites for the other components of prothrombinase. Portions of the fVa heavy chain have been found to act as fXa binding sites. It has been demonstrated that the COOH-terminal region of factor Va contains cluster of acidic amino acids that are crucial for its cofactor activity. More specifically, amino acid region 695–698 from fVa heavy chain regulates the rate of cleavage of prothrombin at Arg²⁷¹ by prothrombinase. The COOH-terminal portion of the heavy chain also contains another cluster of acidic amino acids (encompassing residues 659–663). Site-directed mutagenesis was performed to generate a factor V (fV) molecule with region Asp⁶⁵⁹-Asp⁶⁶³ (fV⁶⁶³) deleted. We have also constructed mutant molecules with regions Lys⁶⁸⁰-Arg⁷⁰⁹ and Asp⁶⁵⁹-Asp⁶⁶³ (fV^663+709)) deleted from the COOH-terminal region of the heavy chain. Finally, a mutant molecule containing point mutations in region Asp⁶⁵⁹-Asp⁶⁶³ where the five amino acids in this sequence are mutated to all lysines (fV^5K), was also constructed. These recombinant molecules along with wild type fV (fV^WT) were transiently expressed in COS7L cells and assessed for their capability to promote prothrombin activation following activation by thrombin. Prothrombin activation by prothrombinase assembled with the mutant molecules was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with the recombinant mutant molecules was slower. Two-stage clotting assays revealed that FV^663+709, fVa^5K, and fVa⁶⁶³ all had reduced clotting activities compared to fVa^WT and plasma-derived fVa. Kinetic analyses demonstrated that K^d values for fXa of all the mutants were similar to fVa^WT. However, k^cat values for the various molecules varied. The k^cat values for prothrombinase assembled with fVa^5K, and fVa⁶⁶³ were 10-fold reduced when compared to the values obtained with prothrombinase assembled with fVa^WT, while prothrombinase assembled with fVa^663+709 had a k^cat value that was sligtly lower than that of fVa^WT. Our data suggest that amino acid region 659–663 from fV plays a crucial role for fVa cofactor acivity and overall the data demonstrate that acidic amino acids from the COOH-terminus of the factor Va heavy chain play a preeminent role in proper prothrombinase complex assembly and function, resulting in competent thrombin formation. These data assign an important regulatory role of the acidic COOH-terminal region of fVa to the activity of factor Xa within prothrombinase. Finally, our data aid in further studies that may lead to the development of small synthetic molecules that could be used as anticoagulants in individuals with thrombotic tendencies.