Human prothrombinase complex assembly and function on isolated peripheral blood cell populations.
A membrane-bound Ca2+-dependent complex of the cofactor Factor Va and the enzyme Factor Xa comprises the prothrombinase coagulation complex which catalyzes the proteolytic conversion of prothrombin to thrombin. Analyses of the kinetics of prothrombin activation permit calculation of the stoichiometry and binding parameters governing the functional interactions of Factor Va and Factor Xa with isolated thrombin-activated human platelets and isolated leukocyte subpopulations. Our kinetic approach indicates that Factor Xa binds to approximately 2700 +/- 1000 (n = 8) functional sites on the surface of thrombin-activated platelets with an apparent dissociation constant (Kd) equal to 1.18 +/- 0.53 X 10(-10) M and kcat equal to 19 +/- 7 mol of thrombin/s/mol of Factor Xa bound. The store of Factor V in normal platelets prevents an analogous determination of the functional Factor Va platelet binding sites. Factor Va and Factor Xa titrations performed using platelets from a Factor V antigen-deficient individual indicate that Factor Va and Factor Xa form a 1:1 stoichiometric complex on the surface of thrombin-activated platelets. Both binding isotherms are governed by the same apparent Kd (approximately equal to 10(-10) M) and expressed the same kcat/site (14-17 s-1. Factor Xa-platelet binding parameters are not altered by the use of different platelet agonists, the choice of anticoagulant, or platelet washing procedure. Kinetics of prothrombin activation indicate also that monocytes, lymphocytes, and neutrophils possess, respectively, 16,000, 45,000, and 8,000 Factor Va-Factor Xa receptor sites/cell, which are all governed by apparent KdS approximately equal to 10(-10) M. Enzymatic complexes bound to monocytes or neutrophils exhibit kcat values similar to the platelet-bound complex. Complexes bound to lymphocytes are only 25% as active.
D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D005165:Factor V / Q000378:metabolism*; D005166:Factor V Deficiency / Q000097:blood; D015943:Factor Va; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
C022475:prothrombinase complex; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D005170:Factor X; D013917:Thrombin; D015951:Factor Xa