Enzymic and nonenzymic properties of human beta-thrombin.
Autolysis or tryptic hydrolysis converts human alpha-thrombin to its beta-derivative and subsequently to gamma-thrombin. Human beta-thrombin was obtained by tryptic digestion of alpha-thrombin and isolated by BioRex chromatography. The kinetic parameters for human alpha- and beta-thrombins with H-D-phenylalanyl-L-pipecolyl-L-arginine-para-nitroanilide were similar, as well as the rate of inactivation by tosyl-lysine chloromethyl ketone. By contrast, the rate of inactivation by diisopropyl fluorophosphate was reduced by half, and the inhibition constant for benzamidine was increased 2.5-fold. Moreover, the beta cleavages induced a drastic reduction in reactivity toward protein C, affinity for thrombomodulin, and fibrinogen clotting activity. Unlike alpha-thrombin, beta-thrombin was not protected from inhibition by diisopropyl fluorophosphate in the presence of fibrinogen and failed to bind to fibrin-Sepharose. Our results indicate that the beta cleavages induce multiple defects in the functions of human thrombin. Although the three catalytic residues remain in an active configuration, subtle changes are induced in the microenvironment of the active serine. However, the drastic reduction of fibrinogen clotting activity should rather be ascribed to major alterations observed in both the fibrinopeptide groove and the fibrin recognition site. These observations provide further evidence for a double-site mechanism in the interaction of fibrinogen with thrombin.
Bezeaud
A
A
Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
Guillin
M C
MC
D001777:Blood Coagulation; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011486:Protein C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D018179:Receptors, Thrombin; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin