Abstract
Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg271 followed by cleavage at Arg320. Factor Va, along with phospholipid and Ca2+, enhances the rate of the process by 300,000-fold, reverses the order of cleavages, and directs the process through the meizothrombin pathway, characterized by initial cleavage at Arg320. Previous work indicated reduced rates of prothrombin activation with recombinant mutant factor Va defective in factor Xa binding (E323F/Y324F and E330M/V331I, designated factor VaFF/MI). The present studies were undertaken to determine whether loss of activity can be attributed to selective loss of efficiency at one or both of the two prothrombin-activating cleavage sites. Kinetic constants for the overall activation of prothrombin by prothrombinase assembled with saturating concentrations of recombinant mutant factor Va were calculated, prothrombin activation was assessed by SDS-PAGE, and rate constants for both cleavages were analyzed from the time course of the concentration of meizothrombin. Prothrombinase assembled with factor VaFF/MI had decreased k(cat) for prothrombin activation with Km remaining unaffected. Prothrombinase assembled with saturating concentrations of factor VaFF/MI showed significantly lower rate for cleavage of plasma-derived prothrombin at Arg320 than prothrombinase assembled with saturating concentrations of wild type factor Va. These results were corroborated by analysis of cleavage of recombinant prothrombin mutants rMz-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A), which can be cleaved only at Arg320 or Arg271, respectively. Time courses of these mutants indicated that mutations in the factor Xa binding site of factor Va reduce rates for both bonds. These data indicate that the interaction of factor Xa with the heavy chain of factor Va strongly influences the catalytic activity of the enzyme resulting in increased rates for both prothrombin-activating cleavages.
Highlights
Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg271 followed by cleavage at Arg320
The prothrombinase complex catalyzes the activation of prothrombin following the second pathway (Arg320 followed by Arg271, Fig. 1, pathway II), resulting in a 300,000-fold increase in the rate of the overall reaction compared with the rate of prothrombin activation observed with factor Xa alone and represents the physiological relevant pathway for prothrombin activation [3, 12, 13]
Our findings suggest that the interaction of amino acids Glu323, Tyr324, Glu330, and Val331 from the heavy chain of factor Va with factor Xa is required for the positioning of the active site of the enzyme in a position necessary for optimal productive collisions between the enzyme and prothrombin, efficient initial cleavage at Arg320, and competent thrombin formation
Summary
Reagents, and Proteins—L-␣-Phosphatidylserine (PS) and L-␣-phosphatidylcholine (PC) were from Avanti Polar Lipids (Alabaster, AL). Human plasma factor V and factor Va were purified and concentrated using methodologies previously described employing the monoclonal antibody ␣hFV#1 coupled to Sepharose and heparin-Sepharose [30, 35]. The activity and the integrity of the molecules were verified before and after activation by thrombin by clotting assays using factor V-deficient plasma and by SDS-PAGE followed by Western blotting using well characterized monoclonal antibodies (provided by Dr Ken Mann, Department of Biochemistry, University of Vermont). The assay verifying the activity of the recombinant molecules was conducted as described by measuring thrombin formation by the change in the absorbance of a chromogenic substrate at 405 nm (Spectrozyme-TH) monitored with a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA) [31]. Once the value of the KDapp for the bimolecular interaction of each species of factor Va with factor Xa was determined, the amount of recombinant factor Va required to completely saturate factor Xa and provide similar amount of enzyme (prothrombinase) when using various mutant recombinant factor Va
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