Thrombin-Catalyzed Activation of Recombinant Human Factor V

Abstract

Proteolytic activation of human factor V by thrombin results from the cleavage of three peptide bonds at Arg709, Arg1018, and Arg1545. In order to define the functional importance of these sites, mutants with isoleucine substitutions blocking thrombin cleavage at one, two, or all three activation sites were expressed in COS-7 cells. The wild type protein is activated approximately 10-fold by thrombin or Russell's viper venom (RVV-V). Thrombin cleavage at Arg709 alone did not result in an increase in procoagulant activity. Cleavage at both Arg709 and Arg1018 resulted in an approximately 3.4-fold increase in activity. Cleavage at these sites was required for rapid cleavage by thrombin at Arg1545, however, which resulted in maximal activation of the factor V molecule. In contrast, isolated cleavage at Arg1545 by RVV-V was sufficient for efficient and complete activation of factor V. The effect of isoleucine substitutions at one or both thrombin cleavage sites in a B-domain deletion mutant lacking amino acids 811-1491 was also investigated. The specific activity of all four mutants was approximately 30% compared to thrombin activated factor V, indicating that these isoleucine substitutions do not drastically alter the structure of the protein and that cleavage at these sites is not required for the expression of partial procoagulant activity.

Structural Requirements for Expression of Factor Va Activity

“…No increase in cofactor activity was found when factor V Xa was treated with ␣-thrombin (factor V Xa/IIa ), and no differences in activities were observed between V Xa and factor V IIa . In contrast, using a clotting assay (5) or an assay that measures ␣-thrombin formation and employs limiting factor Xa concentrations (67), it was reported that cleavage of factor V at Arg 1545 and generation of the light chain alone was sufficient for optimum factor Xa activity within prothrombinase. The data presented here suggest that at saturating concentrations of factor Xa, cleavage at Arg 709 is sufficient for optimum factor Va cofactor expression, whereas under conditions of limiting factor Xa, cleavages at both Arg 1545 and Arg 709 are required to promote maximum cofactor activity.…”

“…Popular consensus is that the RVV-V protease activates factor V to produce a species with only a light chain, equivalent (by electrophoretic migration) and with similar clotting activity to factor V IIa (12,66,67). We have reported that APC-inactivated membranebound factor V (cleaved at Arg 306 , Arg 506 , Arg 679 , and Lys 994 ) is further cleaved by the RVV-V activator at Arg 1018 and Arg 1545 (65).…”

“…Activation of Factor V by Russell's Viper Venom Factor V Activator and the Importance of the Light Chain-In contrast to the NN protease, RVV-V activator is an efficient activator of factor V. Although several laboratories have used the RVV-V activator to activate factor V (12,66,67) no explicit description of the activation/cleavage sites has been reported. Popular consensus is that the RVV-V protease activates factor V to produce a species with only a light chain, equivalent (by electrophoretic migration) and with similar clotting activity to factor V IIa (12,66,67).…”

A Bipartite Autoinhibitory Region within the B-domain Suppresses Function in Factor V

“…Broadly, these studies found that variable amounts of cofactor activity are observed depending on which region of the B-domain is cleaved and which assay is used to evaluate activity. A clear consensus is that maximal activity correlates with cleavage at Arg 1545 (7,13,(15)(16)(17)(18)(19)(20)(21)(22). Although these studies have advanced the field, such correlative studies have not provided mechanistic insight into how FV activation unfolds.…”

“…A longstanding assumption is that the B-domain, due to its size/high carbohydrate content, nonspecifically prevents activity of the procofactor. Initial evidence for this came from the Kane laboratory (17,24), who generated a FV derivative with a shortened B-domain (FVdes 811-1491 or FV-810). Using a transient expression system, they found that this FV derivative had partial but seemingly constitutive activity (17).…”