title: The contribution of bovine Factor V and Factor Va to the activity of prothrombinase.
abstract: The rates of prothrombin activation under initial conditions of invariant concentrations of prothrombin and Factor Xa were studied in the presence of various combinations of Ca2+, homogeneous bovine Factor V, Factor Va, phosphatidylcholine-phosphatidylserine vesicles, and activated bovine platelets. Reactions were monitored continuously through the enhanced fluorescence accompanying the interaction of newly formed thrombin with dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide. The complete prothrombinase (Factor Xa, Ca2+, phospholipid, and Factor Va) behaved as a "typical" enzyme and catalyzed the activation of prothrombin with an apparent Vmax of 2100 mol of thrombin/min/mol of Factor Va or Factor Xa, whichever was the rate-limiting component. Regardless of whether the enzymatic complex was composed of Factor Xa, Ca2+, and plasma Factor Va plus phospholipid vesicles, or activated platelets in the place of the latter components, similar specific activity values were observed. The combination of Factor Va, Ca2+, and phospholipid enhanced the rate of the Factor Xa-catalyzed activation of prothrombin by a factor of 278,000. Factor Va itself when added to Factor Xa, Ca2+, and phospholipid, enhanced the rate of prothrombin activation by a factor of 13,000. Unactivated Factor V appears to possess 0.27% of the procoagulant activity of thrombin-activated Factor Va. From the kinetics of prothrombinase activity, an interaction between Factor Xa and both Factor V and Factor Va was observed, with apparent 1:1 stoichiometries and dissociation constants of 7.3 x 10(-10) M for Factor Va and 2.7 x 10(-9) M for Factor V. The present data, combined with data on the equilibrium binding of prothrombinase components to phospholipid, indicate that the model prothrombinase described in this paper consists of a phospholipid-bound, stoichiometric complex of Factor Va and Factor Xa, with bound Factor Va serving as the "binding site" for Factor Xa, in concert with its proposed role in platelets.
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000502:physiology; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003619:Dansyl Compounds / Q000494:pharmacology; D004789:Enzyme Activation; D005165:Factor V / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D011485:Protein Binding; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D003619:Dansyl Compounds; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D005165:Factor V; D005170:Factor X; D013925:Thromboplastin; D001120:Arginine; D002118:Calcium