Abstract
Heparin activates the serpin, antithrombin, to inhibit its target blood-clotting proteases by generating new protease interaction exosites. To resolve the effects of these exosites on the initial Michaelis docking step and the subsequent acylation and conformational change steps of antithrombin-protease reactions, we compared the reactions of catalytically inactive S195A and active proteases with site-specific fluorophore-labeled antithrombins that allow monitoring of these reaction steps. Heparin bound to N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled antithrombins and accelerated the reactions of the labeled inhibitor with thrombin and factor Xa similar to wild type. Equilibrium binding of NBD-labeled antithrombins to S195A proteases showed that exosites generated by conformationally activating antithrombin with a heparin pentasaccharide enhanced the affinity of the serpin for S195A factor Xa minimally 100-fold. Moreover, additional bridging exosites provided by a hexadecasaccharide heparin activator enhanced antithrombin affinity for both S195A factor Xa and thrombin at least 1000-fold. Rapid kinetic studies showed that these exosite-mediated enhancements in Michaelis complex affinity resulted from increases in k(on) and decreases in k(off) and caused antithrombin-protease reactions to become diffusion-controlled. Competitive binding and kinetic studies with exosite mutant antithrombins showed that Tyr-253 was a critical mediator of exosite interactions with S195A factor Xa; that Glu-255, Glu-237, and Arg-399 made more modest contributions to these interactions; and that exosite interactions reduced k(off) for the Michaelis complex interaction. Together these results show that exosites generated by heparin activation of antithrombin function both to promote the formation of an initial antithrombin-protease Michaelis complex and to favor the subsequent acylation of this complex.
Highlights
The serpin family protein protease inhibitor, antithrombin, together with its glycosaminoglycan activator, heparin, are the principal anticoagulant regulators of blood coagulation cascade proteases [1, 2]
In the case of antithrombin, the exosites that stabilize the Michaelis complex are generated on antithrombin and provided by a bridging heparin when heparin binds and conformationally activates the serpin
The exosites on heparin-activated antithrombin engage complementary exosites on thrombin and factor Xa in the Michaelis complex [10, 11, 21,22,23, 28, 29, 48]. The specificity of these exosite interactions is supported by the observation that heparin only marginally promotes the Michaelis complex interaction or accelerates antithrombin inhibition of the related protease, trypsin [47].The increased stabilization of the Michaelis complex by exosites was shown to have a negative effect on the subsequent serpin conformational change that leads to trapping of the acyl-intermediate complex in the case of the plasminogen activator inhibitor-1 reaction with tissue-type plasminogen activator [38]
Summary
Proteins—Antithrombin was purified from human plasma as described previously [30]. Wild-type and variant recombinant antithrombins with an N135Q mutation to eliminate glycosylation heterogeneity at the Asn-135 glycosylation site [31] were expressed in baby hamster kidney cells or in baculovirus-infected insect cells and purified as in previous studies [18, 24, 25]. Factor Xa was purchased from Enzyme Research Laboratories (South Bend, IN), and thrombin was obtained from purified prothrombin by activation of the zymogen followed by purification of the active enzyme as described previously [33, 34]. S195A thrombin and ␥-carboxyglutamic acid-domainless S195A factor Xa were recombinant enzymes expressed in mammalian cells and purified as described previously [35, 36].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
