Abstract
The fragment 2 domain (F2) of prothrombin and its interaction with factor (F) Va is known to contribute significantly to prothrombinase-catalyzed activation of prothrombin. The extent to which the F2-FVa interaction affects the overall thrombin generation, however, is uncertain. To study this interaction, nuclear magnetic resonance spectroscopy of recombinant F2 was used to identify seven residues within F2 that are significantly responsive to FVa binding. The functional role of this region in interacting with FVa during prothrombin activation was verified by the FVa-dependent inhibition of thrombin generation using peptides that mimic the same region of F2. Because six of the seven residues were within a 9-residue span, these were mutated to generate a prothrombin derivative (PT6). These mutations led to a decreased affinity for FVa as determined by surface plasmon resonance. When thrombin generation by an array of FXa containing prothrombinase components was monitored, a 54% decrease in thrombin generation was observed with PT6 compared with the wild-type, only when FVa was present. The functional significance of the specific low-affinity binding between F2 and FVa is discussed within the context of a dynamic model of molecular interactions between prothrombin and FVa engaging multiple contact sites.
Highlights
The immediate need to minimize blood loss upon vascular injury is met with the formation of a stable fibrin clot that is triggered by the rapid activation of the blood coagulation cascade
Proton and 15N/1H HSQC nuclear magnetic resonance (NMR) spectra of recombinant fragment 2 domain (F2) exhibited progressive changes with increasing concentrations of FVa-HC from 10 to 30 μM (Fig. 2), indicating a binding affinity (Kd) in the low to middle μM range. 15N/1H HSQC spectra at the highest (30 μM) FVa-HC concentration collected for the 15N-labeled F2 showed that a majority of the 15N/1H cross-peaks for F2 were not affected by the presence of the FVa-HC (Fig. 2)
The functional role of F2 in the prothrombin-FVa interaction has mostly been studied by the use of prothrombin variants with or without the F2 domain, and quantifying its activation by FXa/FVa
Summary
Identifying F2 residues involved in binding FVa-HC. Recombinant F2 domain of prothrombin and purified F2 from human plasma prothrombin (Fig. 1) exhibited essentially the same functional activities for enhancing the activation of prethrombin-2 by prothrombinase. With full prothrombinase (i.e. FXa/FVa/PCPS/Ca2+), a statistically significant difference in the initial rates of wild-type prothrombin (66.6 ± 9.6 s−1) or PT6 (55.1 ± 8.7 s−1) activation was observed (p < 0.05). Total thrombin generated was 42% lower for PT6 than wild-type prothrombin (Fig. 5) Taken together, these data suggest that the differences in activation between wild-type and PT6 prothrombin are apparent only in the presence of FVa. Effects of selected residue mutations on the binding of prothrombin to FVa. The binding of wild-type prothrombin or PT6 to FVa was monitored in real-time and quantified using surface plasmon resonance (SPR). These data demonstrate that binding of prothrombin to FVa clearly involves the six residues identified within the F2 domain of prothrombin
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