Down regulation of prothrombinase by activated protein C during prothrombin activation

Abstract

SUMMARY Activated protein C (aPC) proteolytically inactivates factor Va (FVa) and thereby down regulates prothrombinase. Although FVa inactivation by aPC has been studied extensively, the inactivation of prothrombinase during prothrombin activation has not. Therefore, prothrombin activation initiated both without and with aPC (5.0, 7.5 or 10.0nM) was monitored over time by fluorescence. The experiments were performed with 0.075nM FVa and 1.0nM FXa, and with these concentrations reversed. The time courses of the residual prothrombinase activity with aPC, determined from the slopes of fluorescence over time, were pseudo first order with both limiting and excess FVa. With FVa limiting or in excess, the second rate constants for inactivation of prothrombinase were 1.98±0.09 × 105 M−1s−1 and 2.54±0.13 × 105 M−1s−1, respectively. The former value is 101-fold smaller than that for FVa inactivation by aPC alone. Since with limiting FVa the second order rate constants for prothrombinsase inactivation and FVa inactivation are equal, FVa is protected 101-fold, presumably by both FXa and prothrombin. In contrast, with excess FVa, the calculated rate constant for FVa inactivation exceeds that for prothrombinase inactivation 17.3-fold, which reflects a loss of protection by FXa. Since the protective effects of the two proteins are theoretically multiplicative, FXa protected 17.3-fold and prothrombin protected 5.8-fold. With 150nM protein S and limiting FVa, prothrombinase inactivation was two-fold faster, yet it was still protected 91-fold. These studies show that FVa is down-regulated by aPC during prothrombin activation, but FXa and prothrombin together are highly protective, both with and without protein S.

Identification and characterization of a factor Va-binding site on human prothrombin fragment 2

“…While the difference in initial rates of prothrombin activation by prothrombinase between wild-type and PT6 were minimal (Table 1), there was a large difference in total thrombin generation at completion (Figs 4 and 5), comparable with the effect observed in the peptide inhibition studies. A similar observation has been reported previously by our group 42 , whereby decreases in total thrombin generation are not accompanied by proportional decreases in the initial rates of prothrombin activation. Effects on the initial rates of thrombin generation, as well as total thrombin generation were accentuated only when prothrombin was activated in the presence of FVa, indicating FVa dependence of inhibition.…”

Modeling of human factor Va inactivation by activated protein C

“…It has been widely reported that FXa shields FVa from degradation by APC through its reversible association in the prothrombinase (FXaFVa) complex [ 34 - 38 , 44 ]. The mechanistic basis of this protection has been conceptualized as a mutually exclusive competition for association with membrane bound FVa, so that when FVa is associated with FXa it is not a substrate for APC [ 35 ], at least at the Arg 506 site [ 37 ].…”

“…We did so in stepwise fashion, analyzing the time course of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation [ 22 , 31 - 33 ], in the presence of FXa [ 14 , 34 - 38 ], and in the presence of prothrombin [ 39 , 40 , 58 ]. Model predictions were assessed against empirical time course data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with experiments done multiple times, with plasma proteins derived from multiple preparations.…”

“…Numerous studies examining how components like FXa, prothrombin and protein S modulate APC inactivation of FVa have been reported [ 14 , 34 - 40 ]. These studies routinely employ approaches like progress curve analysis based on curve fitting to compare initial rates, or natural and recombinant cleavage site mutants to detail mechanistic features.…”