Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombininactivation by heparin cofactor II

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably , presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin, To explore this model of activation, we systematically substit uted basic residues in the glycosaminoglycan-binding domain of HCII with ne utral amino acids and measured the rates of thrombin inactivation by the mu tants. Mutant D, with changes at Arg(184), Lys(185), Arg(189), Arg(192), Ap g(193) demonstrated a similar to 130-fold increased rate of thrombin inacti vation that was unaffected by the presence of glycosaminoglycans. The incre ased rate reflects displacement of the amino terminus of mutant D because ( a) mutant D inactivates gamma-thrombin at a 65-fold slower rate than alpha- thrombin, (b) hirudin-(54-65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thro mbin inactivation similar to 100-fold, We also examined the contribution of glycosaminoglyean-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate d oes not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater con formational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino termi nus.