The preferred pathway of glycosaminoglycan-accelerated inactivation of thrombin by heparin cofactor II

Thrombin ( T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin ( H). Equilibrium binding and thrombin inactivation kinetics at pH 7.8 and ionic strength ( I) 0.125 M demonstrated that DS and heparin bound much tighter to thrombin (K-T(DS) 1 - 5.8 muM; K-T(H) 0.02 - 0.2 muM) than to HCII (K-HCII(DS) 236 - 291 muM; K-HCII(H) 25 - 35 muM), favoring formation of T . GAG over HCII . GAG complexes as intermediates for T GAG . HCII complex assembly. At [GAG] << KHCII( GAG) the GAG and HCII concentration dependences of the first-order inactivation rate constants (k(app)) were hyperbolic, reflecting saturation of T . GAG complex and formation of the T . GAG . HCII complex from T . GAG and free HCII, respectively. At [GAG] >> K-HCII(GAG), HCII . GAG complex formation caused a decrease in k(app). The bell-shaped logarithmic GAG dependences fit an obligatory template mechanism in which free HCII binds GAG in the T . GAG complex. DS and heparin bound fluorescently labeled meizothrombin(des-fragment 1) (MzT(- F1)) with KMzT(-F1)(GAG) 10 and 20 muM, respectively, demonstrating a binding site outside of exosite II. Exosite II ligands did not attenuate the DS-accelerated thrombin inactivation markedly, but DS displaced thrombin from heparin-Sepharose, suggesting that DS and heparin share a restricted binding site in or nearby exosite II, in addition to binding outside exosite II. Both T . DS and MzT(- F1) . DS interactions were saturable at DS concentrations substantially below K-HCII( DS), consistent with DS bridging T . DS and free HCII. The results suggest that GAG template action facilitates ternary complex formation and accommodates HCII binding to GAG and thrombin exosite I in the ternary complex.