Cleavage of Factor Va Heavy Chain at Arg⁶⁴³ by Thrombin Partially Inactivates the Cofactor.

Prothrombinase, the enzyme complex required to activate prothrombin, is composed of the serine protease factor Xa and the cofactor factor Va, associated in 1:1 stoichiometry on a phospholipid surface in the presence of Ca²⁺. Incorporation of factor Va in prothrombinase is required for any meaningful rate of thrombin generation and the arrest of hemorrhage. Factor Va inactivation down-regulates thrombin production resulting in the termination of the hemostatic response. The principal enzyme involved in this down-regulation is activated protein C (APC). Factor Va is formed following enzymatic cleavage of the single chain procofactor, factor V (M_r 330,000) by thrombin. Thrombin cleaves and activates the procofactor sequentially at Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵. The active cofactor, factor Va, is composed of heavy (HC¹⁰⁵, M_r 105,000) and light (M_r 74,000) chains non-covalently associated in the presence of divalent ions. Previous studies of factor Va inactivation on human umbilical vein endothelial cells (HUVEC) have shown that thrombin cleaves the heavy chain at the COOH-terminus to produce a M_r 97,000 fragment containing the NH₂-terminal portion of the heavy chain and a M_r 8,000 peptide representing the COOH-terminus of the molecule which remains attached to the heavy chain by a disulfide bond. The thrombin cleavage appeared to occur between residues 586 and 654. This region contains a consensus sequence for cleavage by thrombin located between residues 640–643 (S-P-R). To evaluate the functional importance of thrombin cleavage at Arg⁶⁴³ for factor Va inactivation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations R⁶⁴³→Q (factor V^R643Q) and R⁶⁴³→A (factor V^R643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with thrombin. Under similar experimental conditions, cleavage of HC₁₀₅ and appearance of the M_r 97,000 heavy chain fragment in the wild type molecule correlated with partial loss of cofactor activity, while following incubation of factor V^R643Q and factor V^R643A with thrombin no cleavage of HC₁₀₅ at Arg⁶⁴³ was observed and no presence of the M_r 97,000 heavy chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant factor Va molecules following extended incubation with thrombin. The endothelial cell surface has been presumed to be the site of PC activation and factor Va inactivation in vivo. The relative phospholipid composition of endothelial membranes has been suggested to be consistent with their ability to support factor Va inactivation in a manner analogous to the commonly used phospholipid system composed of 25% phosphatidylserine and 75% phosphatidylcholine. In the experiments conducted on the HUVEC surface incubation of 20 nM plasma factor V with 0.1 nM thrombin resulted in almost complete cleavage of HC₁₀₅ over a 60 minute thrombin treatment. In the experiments presented herein much higher concentrations of thrombin were necessary to obtain a similar effect. The combined data suggest the presence of a cofactor for thrombin on the surface of endothelial cells that would facilitate cleavage of factor Va heavy chain at Arg⁶⁴³. Collectively, the data demonstrate that cleavage of HC₁₀₅ at Arg⁶⁴³ by thrombin results in a partially inactive cofactor molecule and provide for an APC-independent anticoagulant effect of thrombin.