MECHANISM OF FACTOR IXA INHIBITION BY ANTITHROMBIN IN THE PRESENCE OFUNFRACTIONATED AND LOW-MOLECULAR-WEIGHT HEPARINS AND FUCOIDAN

Heparin exerts its anticoagulant activity by catalysing the inhibition of coagulation proteases by antithrombin (AT). Its main target is thr ombin but it also catalyses the inhibition of the other serine-proteas es of the coagulation cascade, such as factor IXa (fIXa). The aim of t his study was to compare the catalysis of inhibition of blood fIXa by antithrombin in the presence of several sulfated polysaccharides with anticoagulant activity, i.e. heparin, three widely used in therapeutic s low molecular weight heparins (LMWH) and fucoidan. Plots of the seco nd-order rate constants of the fIXa-antithrombin reaction vs. the conc entration of added heparin and LMWH are bell-shaped and fit the kineti c model established for thrombin-antithrombin reaction by Jordan R., B eeler D., Rosenberg R. (1979) J. Biol. Chem., 254, 2902-2913. In the a scending branch, the catalyst (C) binds quickly to the inhibitor (I) t o form a catalyst-inhibitor (CI) complex which is more reactive toward s the enzyme (E) than the free inhibitor, leading to the formation of an inactive enzyme-inhibitor complex (EI) and the release of free cata lyst, in a rate-limiting second step. After a maximum corresponding to an optimal catalyst concentration, the decrease in the reaction rate was in keeping with the formation of a catalyst-enzyme (CE) complex, w hose inactivation by the CI complex was slower than that of the free e nzyme. Maximum second-order rate constants for the inhibition of fIXa by AT were 105, 6.8, 12.24 and 22 mu M-1 min(-1) with heparin, Enoxapa rin, Fraxiparin and Fragmin, respectively, leading to 3500-, 225-, 405 - and 728-fold increases in the inhibition rate in the absence of poly saccharide, respectively. Fucoidan yielded 23-fold increase in the fIX a-antithrombin interaction rate. The kinetic profiles obtained with th is polysaccharide exhibited ascending branch which correlated well wit h the kinetic model based on the formation of binary complexes (CI or CE). Fucoidan was covalently conjugated with a fluorescent probe (DTAF ) and used in conjunction with fluorescence anisotropy to follow its b inding to antithrombin, heparin cofactor II (HCII), thrombin and fIXa. The binding of fucoidan to these proteins occurred with low affinitie s when compared to heparin and LMWH. Fucoidan had higher affinity for the inhibitor HCII compared to antithrombin and enzymes. These data su ggest that binding of heparins and fucoidan to the inhibitor (CI) is r equired for the polysaccharide-dependent enhancement in the rate of ne utralization of the enzyme by the inhibitor. (C) 1998 Elsevier Science B.V. All rights reserved.