Abstract
Proexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659-698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa709 (des710-1545), FVa699 (des700-1545), FVa(692 (des693-1545), FVa678 (des679-1545), and FVa658 (des659-1545). We found that FVa709, FVa699, FVa692, and FVa678 exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the Km and kcat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa709, FVa699, FVa692, and FVa678 bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa658 had an approximately 1% clotting activity and reduced affinity for FXa membranes (approximately 20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa(658) exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase.
Highlights
Blood coagulation factor Va (FVa)2 is a heterodimeric protein composed of a heavy chain and a light chain which arises from limited proteolysis of the pro-cofactor factor V (FV) [1]
Based on these findings it is reasonable to hypothesize that the corresponding binding site on FVa for proexosite I is found within the hirudin-like C-terminal region of the FVa heavy chain (659 – 698; Fig. 1)
Construction of rFVa Variants—To directly assess the contribution of hirudin-like FVa sequences (659 – 698) to cofactor function, the entire B-domain and specific heavy chain sequences were removed, and a PACE-furin cleavage site was introduced between these deleted sequences and the light chain generating rFVa658, rFVa678, rFVa692, rFVa699, and rFVa709
Summary
Materials—Bovine serum albumin (BSA) and 5,5Ј-dithiobis(2-nitrobenzoic acid) were purchased from Sigma. L-␣-Phosphatidylserine (brain, sodium salt) and L-␣-phosphatidylcholine (egg yolk) were purchased from Avanti Polar. Small unilamellar phospholipid vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine (PCPS) were prepared as described previously [36]. Prethrombin-1, prethrombin-2, and IIa were prepared from human prothrombin and purified using established procedures [40]. A recombinant B-domainless form of FV (rFV-DT) used to prepare rFVa was expressed and purified as previously described [41]. Human plasma derived FV (PD-FV) and rFV-DT were proteolytically processed with IIa to generate PD-FVa and rFVa, and both were subsequently purified on a Poros HS/20 (0.46 ϫ 10 cm) column as described [41, 42]. Protein concentrations were determined using the following molecular weights and extinction coefficients N-terminal sequence analysis was performed in the laboratory of Dr
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