The blood-clotting protein prothrombin can be converted to thrombin in free solution by the proteolytic enzyme, activated factor X. When prothrombin is bound to the surface of phospholipid vesicles, the rate of thrombin generation is increased more than 30-fold over the rate of unbound prothrombin. If the prothrombin activation process is terminated after a time interval in which less than 10% of the expected thrombin has been produced, two major products are found in the activation mixture. These products have been termed intermediate 1, a precursor of thrombin, and fragment 1. The conversion of prothrombin to thrombin in the blood coagulation process is markedly accelerated in the presence of phospholipid vesicles (1-3). Both prothrombin (3, 4) and activated factor X (the enzyme catalyzing the proteolyses involved in thrombin formation) (5) bind to phospholipid particles. This binding requires divalent cations and is reversible by removal of the divalent cations. In contrast to prothrombin, the final active product of the activation process, thrombin, does not bind to phospholipid surfaces (3,4).Investigations into the prothrombin activation mechanism have demonstrated the existence of at least two intermediates between prothrombin and thrombin and two activation fragments (6-10). As the relationship of some of the products described in earlier investigations to those seen by sodium dodecyl sulfate-acrylamide gel electrophoresis (8-10) Intermediate 1 and fragment 1 can be formed by the action of thrombin on prothrombin in both the presence and absence of factor Xa; however, factor Xa is required to form thrombin from prothrombin or either of the intermediates. Interaction between phospholipid vesicles and the activation products from partial activation of prothrombin with factor Xa were investigated in this study.
MATERIALS AND METHODSOleic acid (>99% purity), phospholipase D, bovine-serum albumin, Russell's viper (Vipera russellii) venom, and Tris * HCl were obtained from Sigma Chemical Co. DEAESephadex was a product of Pharmacia, and Bio-Gel A 0.5 m was obtained from Bio Rad Laboratories. Dialysis tubing was a product of Union Carbide Corp., and silica gel GF-254 was a product of E. Merck Darmstadt. Preparative thin-layer chromatography (TLC) plates were obtained from Analtech Inc. All common laboratory chemicals were of analytical reagent grade and were obtained from major domestic supply houses. Bovine brain "cephalin" was prepared by the procedure of Folch (11).Bovine factor X was purified (12)
1344Abbreviations: factor Xa, factor X that has been activated by the factor X activating enzyme of Russell's viper venom; TLC, thin-layer chromatography. * To whom requests for reprints should be sent.