Effects of Prothrombin on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Abstract

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg 306 and Arg 506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg 506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg 306 and Arg 506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 M prothrombin, whereas half-maximal inhibition was obtained at 0.7 M prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg 306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.

Novel APC‐cleavage sites in FVa provide insights into mechanisms of action of APC and its cofactor protein S

“…For the FVa variants cleaved at only one site, and for 306Q/505Q/506Q/679Q-FVa that was cleaved at four equivalent and closely spaced sites, equation 1 was used [16,29].…”

“…1B), or non-purified FVa (Fig. 2), was subjected to APC-mediated degradation, and analyzed by Western blotting using AH5146 (epitope between positions 307 and 506), as described [29]. A Fuji LAS 3000IR CDD camera was used and the signals were quantified with the Image-Gauge (Fujifilm, Tokyo, Japan) program.…”

“…PS/PE (10/90) vesicles (PTase) were prepared fresh [29]. PS/PE/ PC (10/20/70) vesicles, prepared by extrusion, were used for 2 days [30].…”

“…Thereafter, phospholipids (25 lM) ± protein S (100 nM) were added. The reactions were started with APC (0-100 nM) (all concentrations being final), stopped at intervals by 1/25 dilution usingice-coldHNBSACaandtheFVaactivitymeasured(dilution plus low temperature completely stopped the degradation) [29].…”

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

“…The binding regions on FVa for prothrombin have been less well described. However, the fact that prothrombin inhibits APC-catalyzed FVa inactivation in the absence of FXa (33)(34) suggests that APC and prothrombin also share binding sites on FVa. The protective effect of prothrombin in the presence of OG-FXa can be explained by formation of a nonproductive ternary OG-FXa⅐FVa⅐prothrombin complex.…”