Cleavage at both Arg306 and Arg506 is required and sufficient for timely and efficient inactivation of factor Va by activated protein C

Abstract

Background Activated protein C (APC) inactivates membrane-bound factor Va following cleavages of the heavy chain at Arg306, Arg506, and Arg679. Objectives To examine which cleavage is most important for inactivation, recombinant factor V molecules were constructed as follows: factor V306Q (R306→Q), factor V506Q (R506→Q), and factor V306Q/506Q (R306→Q and R506→Q). Methods The recombinant molecules were expressed in mammalian cells, purified, and assayed prior and after incubation with APC and lipids for 30 min (factor Vai) in clotting assays and in an assay using purified reagents and saturating concentrations of factor Va. Results Clotting assays demonstrated that factor VaiWT, factor Vai306Q and factor Vai506Q were devoid of activity, while factor Vai306Q/506Q maintained ~70% activity following a 30 minute incubation with APC. Prothrombinase assembled with all mutant cofactor molecules before and after treatment with APC had Km values similar to values found with prothrombinase assembled with factor VaWT. Prothrombinase assembled with factor VaiWT demonstrated 20-fold reduction in kcat, while prothrombinase assembled with factor Vai506Q had a 2-fold reduction in kcat as compared to prothrombinase assembled with factor VaWT. In contrast, factor Vai306Q and factor Vai306Q/506Q didn’t show any loss in kcat under similar experimental conditions. Conclusion Our data demonstrate that: 1) the activity of an APC-treated factor Va molecule bearing a single mutation at Arg306 or Arg506 depends on the assay used; and 2) regardless of the assay employed, in the absence of the APC-cleavage sites at Arg306 and Arg506, the active cofactor is unable to be significantly inactivated by APC in the presence of a membrane surface.

Modeling of human factor Va inactivation by activated protein C

“…The more rapid clearance of FVa 1-506 observed empirically suggests two possible problems with the model construct: 1) the binding affinity between FVa i 506 and FXa is weaker than the model value of 1 nM based on studies with bovine FVa [ 32 ] or 2) APC can cleave at Arg 306 when the FVa i 506 species is bound to FXa. Previous studies with recombinant human proteins have reported K D values ranging from ~3.9 nM [ 31 ] to ~1.35 nM [ 51 ]. To evaluate the likelihood that a weaker binding affinity was the culprit, mathematical simulations were conducted where the K D for the FXaFVa i 506 complex was varied; to fit the empirical data the K D for this complex would have to be greater than 10 nM (data not shown) and as such this adjustment was discarded as a viable option.…”

“…To incorporate the formation of the ternary prothrombinase substrate complex, four additional reactions are required (Table 3 , Eqns 19–22). Based on studies indicating that prothrombinase complexes formed with partially proteolyzed FVa species have a K M similar to the complex formed with intact FVa [ 31 , 51 ], we set the K M of prothrombin for any prothrombinase complex to be the same and consistent with previous modeling studies [ 46 , 47 ]. We included two additional reactions (Table 3 , Eqns 23–24) to allow for the dissociation of the ternary complex formed with some of the partially proteolyzed FVa species.…”

Quantitative measurement of APC-Resistant factor V clotting activity (FV-Leiden) and potential graduation of the associated thrombo-embolic risk

“…Many mutations have been identified on FV, some of them without any impact on its activities but associated to FV variations in blood concentration 32 , and others with a decreased APC dependent inhibition 21,[33][34][35][36] . The most frequent mutation, which has a unique origin, is the R506Q one, and it is located on one of the APC cleaving sites on FV, at position 506 12,18,19 . FV-L is resistant to the APC-PS cleavage, especially in its activated form, and when bound to phospholipids.…”

“…One year later, Bertina's group in Leiden (NL) showed that this defect is the consequence of a FV mutation at position 506, where arginine is replaced by glutamine (noted R506Q), and the mutated factor was named Factor V Leiden (FV-L) 12 . This FV mutation is located on one of the FV cleaving sites by APC 6,[16][17][18][19] . Thrombin activated FV-L has then an increased activity survival time in plasmas from patients with this R506Q mutation, and this can propagate activation of coagulation pathways.…”

APC Resistance

“…As mentioned above, the main proteolytic process responsible for FVa/FVIIIa regulation is limited proteolysis by the serine protease activated protein C (APC). The inactivation process occurs much in analogy to the activation described before: APC targets its substrates at multiple but specific cleavage sites, provided that both the substrate and the enzyme are bound to a membrane surface (Kalafatis et al, 1994;Nicolaes et al, 1995, Egan et al, 1997, Barhoover & Kalafatis, 2011. In the absence of a lipid surface, reactions occur too slowly to be physiologically relevant (Bakker et al, 1992;Nicolaes et al, 1995).…”