Comparison of Heparin- and Dermatan Sulfate-mediated Catalysis of Thrombin Inactivation by Heparin Cofactor II

Abstract

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 substituted basic residues in the glycosaminoglycanbinding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant , demonstrated a ϳ130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates ␥-thrombin at a 65-fold slower rate than ␣-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 thrombin inactivation ϳ100-fold. We also examined the contribution of glycosaminoglycan-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 does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.Heparin cofactor II (HCII), 1 a serpin found in human plasma at a concentration of 1.2 M, selectively inactivates thrombin in a reaction that is accelerated Ͼ1000-fold by glycosaminoglycans (GAGs) such as heparin, dermatan sulfate, and heparan sulfate (1). A second serpin, antithrombin (AT), also inactivates thrombin but differs from HCII in four important ways. First, whereas HCII only inactivates thrombin, AT inactivates other coagulation enzymes including factors Xa and IXa (2). Second, the high affinity interaction of heparin with AT is mediated by a unique pentasaccharide sequence found only in a subpopulation of heparin molecules (3-5). In contrast, heparin does not possess a high affinity sequence for HCII (6). Furthermore, dermatan sulfate (DS), a GAG found in the extracellular matrix of connective tissue (7, 8), activates HCII, but has no effect on AT (1). Third, the uncatalyzed rate of thrombin inactivation by AT is about 10-fold faster than that for HCII, probably reflecting differences in the amino acid residue at their P-1 position, with AT containing an Arg residue and HCII a Leu (9). Fourth, HCII possesses a unique 75-amino acid domain at its amino terminus that binds to thrombin exosite I, an interaction analogous to the binding of the carboxyl terminus of hirudin to exosite I (1).Although the uncatalyzed rate of thrombin inactivation by HCII is slower than that for AT, in the presence of heparin or DS, HCII inactivates thrombin at a rate similar to that at which AT inactivates thrombin when heparin is present (1, 10). The current model to explain GAG-mediated catalysis of HCII inactivation of thrombin suggests ...

Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

“…3 (model 1). This model suggests that the acidic tail in native HCII would interfere with thrombin docking and is consistent with the higher basal rate of thrombin inhibition for the tail-deleted HCII variant (43). An attractive alternative to the positioning of the tail illustrated in the Fig.…”

Molecular and functional characterization of a natural homozygous Arg67His mutation in the prothrombin gene of a patient with a severe procoagulant defect contrasting with a mild hemorrhagic phenotype

“…The severely defective interaction of the mutant thrombin with HCII is in agreement with the involvement of the N-terminus of HCII in the ligation of thrombin FRS. 12,33 The HCII inhibitory activity toward thrombin is strongly enhanced by the presence of glycosaminoglycans (GAGs), such as dermatan sulfate, which allosterically "activates" HCII. A specific dermatan sulfate hexasaccharide sequence composed of repeats of iduronic acid 2-sulfate and N-acetylgalactosamine 4-sulfate has been shown to bind to a cationic domain of HCII.…”

“…34 This binding disrupts the intramolecular docking, allowing the free N-terminal acidic domain of the inhibitor to bind to the FRS of thrombin. 33 Given the selectivity with which dermatan sulfate activates HCII among all serpins interacting with GAGs, and the presence of dermatan sulfate in the extracellular matrix of a wide variety of tissues, HCII has been proposed to inhibit "extravascular" thrombin activity. This inhibitory mechanism has been proposed to oppose the proatherogenic activity of thrombin.…”

Molecular Mapping of the Thrombin-Heparin Cofactor II Complex

“…We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln 24 ) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCIIglycosaminoglycan inhibition reactions.…”

“…HCII appears to use a novel allosteric process in which glycosaminoglycan binding to the D-helix region allows a more permissive acidic domain to act as a "tethered ligand" for binding to exosite-1 of thrombin (5, 9, 16, 24, 30, 38, 51, 52, 61, 62). There have been two similar mechanisms proposed, with the only difference being the role of glycosaminoglycan acting as a secondary bridge between HCII and thrombin (28,30,38,51,62) versus a purely allosteric model without the need for ternary complex formation between HCII-glycosaminoglycanthrombin (5,6,(23)(24)(25)(26)52).…”