The Preferred Pathway of Glycosaminoglycan-accelerated Inactivation of Thrombin by Heparin Cofactor II

Abstract

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 inactivationThe blood clotting proteinase, ␣-thrombin (T), 1 possesses two electropositive sites, exosites I and II (1, 2), that play distinct roles in its covalent inactivation by the serine proteinase inhibitors (serpins), antithrombin (AT), and heparin cofactor II (HCII), in the absence and presence of rate-accelerating glycosaminoglycans (GAGs) (3-8 (9,10). Inactivation by AT is accelerated up to 50,000-fold by high molecular weight heparin, which acts as a template, by binding to thrombin exosite II and AT helices A and D (4, 11). Whereas heparin accelerates thrombin inactivation by both AT and HCII, dermatan sulfate (DS), and other polyanions (5, 12-17) selectively enhance HCII inhibitory action. The HCII mechanism was initially thought to utilize GAGs as templates between exosite II and HCII helix D (5, 12, 13), but later hypothesized to involve allosteric, GAG-induced interaction of an acidic N-terminal domain of HCII with exosite I (6, 7, 18 -21). A synthetic HCII-(54 -75) peptide was shown to compete with hirudin-(54 -65) for thrombin binding (18). Residues 52-75 in HCII contain two hirudin-like repeats, the first of which interacts directly with exosite I (21). The crystal structure of the HCII⅐S195A-thrombin Michaelis complex confirmed this exosite I interaction (22). Deletion of the first HCII acidic repeat (6,19), and mutations in exosite I residues Arg 67 and Arg 73 (20) caused large decreases in the rate constants for the heparin-and DS-catalyzed inactivation, further suggesting the importance of the direct HCII-exosite I interaction. Selective thrombin exosite II mutations did not affect the DS-catalyzed inactivation rate significantly (23,24). Blocking exosite II with HD22, a single-stranded DNA aptamer only had a modest effect on this inactivation rate, and DS with a chain length of 12-20 saccharide units was found to be as effective as higher M r DS in catalyzing thrombin inactivation (25). Whereas some studies report DS binding to thrombin (13,23,26), others postulate that DS does not interact with thrombin (27, 28). These findings, and the assumption that DS template action, like heparin, engages exosite II, led to the proposal of a mechanism in which direct interaction of the HCII N-terminal domain with exosite I is predominant, and GAG template action may be less important, or not even obligatory.In the mechanism of high affinity heparin-catalyzed throm-

Heparin Is a Major Activator of the Anticoagulant Serpin, Protein Z-dependent Protease Inhibitor

“…Heparin was found to accelerate the reactions of ZPI with factor Xa up to ϳ100-fold in a calcium-dependent manner and to accelerate the reaction with factor XIa up to ϳ20-fold independent of calcium to yield association rate constants of ϳ10 6 to 10 7 M Ϫ1 s Ϫ1 that are in the diffusion-limited, physiologically relevant range. The maximal rate enhancements occur at heparin concentrations comparable with those that mediate physiologically significant accelerations of other serpin reactions, such as those of heparin cofactor II (22,29), and at physiologic calcium concentrations. Prior studies did not consider heparin to be a significant effector of ZPI anticoagulant function based on findings of only modest ϳ2-3-fold heparin rate enhancements of ZPI reactions with factor Xa and factor XIa (7).…”

Sucrose Octasulfate Selectively Accelerates Thrombin Inactivation by Heparin Cofactor II

“…Inactivation by HCIIThrombin, MzT, and Mz(-F1) inactivation was measured by continuous competitive chromogenic substrate hydrolysis (9,37,38). K m and k cat for hydrolysis of S2238 and Chromozym TH were 1.5 M and 90 s Ϫ1 (39), and 11 M and 167 s Ϫ1 , respectively, determined from progress curve analysis of pNA formation at 405 nm (40).…”

“…In reactions utilizing template-forming GAGs, both the template and allosteric interactions contribute to the mechanism by binding of GAG to thrombin exosite II and interaction of thrombin-complexed GAG with the heparin binding site in HCII, thereby triggering interaction of the HCII NH 2 -terminal sequence with exosite I. The intermediate T⅐GAG⅐HCII complexes, stabilized by two interactions, are significantly tighter than the T⅐GAG⅐AT complexes (9).…”

Directing thrombin

“…55 Thrombin is inhibited by another SERPIN, heparin cofactor II, in a reaction accelerated up to 70 000-fold by heparin, heparan sulfate, and dermatan sulfate. 58,59 The mechanism is more complex than the heparin-accelerated inhibition of thrombin by antithrombin. 12 In addition to a template acceleration reaction mediated by exosite II, the polysaccharide releases a sequestered highly acidic N-terminal tail of heparin cofactor II, which is then able to bind to exosite I and position the thrombin for attempted cleavage of the Leu-Ser reactive center bond (Table 1).…”