Clevage at Both Arg³⁰⁶ and Arg⁵⁰⁶ Are Required for Timely and Efficient Inactivation of Factor Va by Activated Protein C: Influence of the Assay.

Abstract 2121

Poster Board II-98

The coagulation cascade culminates in the production of thrombin from prothrombin by the prothrombinase complex (factor Xa (fXa), factor Va (fVa), Ca²⁺, on a membrane surface). Once enough thrombin is produced to stop bleeding when there is an injury to the vasculature, down-regulation of the coagulation cascade begins. Thrombin is a procoagulant (promotes clotting by aiding in the formation of the fibrin plug) and also an anti-coagulant (initiates the down-regulation of the coagulation cascade). The anti-coagulant function of thrombin is to activate protein C (PC), to its active form, activated protein C (APC). APC down regulates the coagulation cascade by inactivating the protein cofactors, fVa and factor VIIIa (fVIIa), following proteolysis. APC inactivates factor Va following proteolytic cleavages at Arg³⁰⁶, Arg⁵⁰⁶, and Arg⁶⁷⁹. APC also inactivates fV following cleavages at Arg³⁰⁶, Arg⁵⁰⁶, Arg⁶⁷⁹ and Arg⁹⁹⁴. In order to examine the molecular mechanism by which APC down-regulates fV/Va in the presence of a membrane surface, recombinant fV molecules were constructed with R → Q mutation at the APC cleavage sites as follows: fV³⁰⁶ (R306Q), fV⁵⁰⁶ (R506Q), and fV^306/506 (R306Q and R506Q). We first analyzed the fV and fVa recombinant molecules for clotting activity. Clotting times were recorded before the addition of a membrane surface and APC and following 15 and 30 minutes of APC/membrane incubation. Following 15 minutes of APC inactivation in the presence of a membrane surface, fVa_i^WT and fV_i^WT had no clotting activity. This data demonstrate that the procofactor and activated cofactor behave similarly during inactivation. FV_i³⁰⁶ behave like the wild type molecule. Conversely, fV⁵⁰⁶ and fVa⁵⁰⁶ have comparable clotting times when exposed to APC and a membrane surface. These recombinant molecules retain partial clotting activity even following prolonged incubation with APC. These data demonstrate that cleavage at Arg³⁰⁶ has minimal consequences on clotting activity. FVa^306/506 is not susceptible to APC inactivation as evidenced by unchanging clotting times following extended incubation with APC and a membrane surface. We next assayed the recombinant molecules for their capability to promote thrombin formation in an assay using purified reagents and a chromogenic substrate. Prothrombinase assembled with factor Va_i⁵⁰⁶ or factor Va_i^306/506, were found to have K_m values close to prothrombinase assembled with factor Va^WT. These results suggest that cleavage at Arg³⁰⁶ does not have an effect on binding the substrate of prothrombinase. Prothrombinase assembled with factor Va_i^WT has a 40-fold reduction in the k_cat, while prothrombinase assembled with factor Va_i⁵⁰⁶had only a 2-fold reduction in the catalytic efficiency of prothrombin activation as compared to prothrombinase assembled with factor Va^WT. Prothrombinase assembled with factor Va_i³⁰⁶ had similar k_cat value as the prothrombinase assembled with fVa^WT. Our data demonstrate that 1) cleavage at Arg⁵⁰⁶ doesn't completely inactivate the cofactor, and 2) the activity of the corresponding cofactor molecule depends on the assay used. In a clotting assay, where there is limiting amounts of fVa, the effect of the mutations is more pronounced. In contrast, when cofactor activity is measure in an assay using saturating concentrations of fVa to determine thrombin formation, the effect of the Arg⁵⁰⁶ cleavage appears less important than cleavage at Arg³⁰⁶. In addition, our data also demonstrate that regardless of the assay, in the absence of the APC inactivation cleavage sites at Arg³⁰⁶ and Arg⁵⁰⁶, the procofactor and active cofactor molecules are unable to be proteolytically inactivated by APC in the presence of a membrane surface.