The Asp272-Glu282 region of platelet glycoprotein Ibα interacts with the heparin-binding site of α-thrombin and protects the enzyme from the heparin-catalyzed inhibition by antithrombin III

Platelet glycoprotein Ib (GpIb) mediates interaction with both von Willebrand factor and thrombin. Thrombin binds to GpIb via its heparin-binding site (HBS) (De Candia, E., De Cristofaro, R., De Marco, L., Mazzucato, M., Picozzi, M., and Landolfi, R. (1997) Thromb. Haemostasis 77,735-740; De Cristofaro, R., De Candia, E., Croce, G., Morosetti, R., and Landolfi, R, (1998) Biochem. J. 332, 643-650). To identify the thrombin-binding domain on GpIb alpha, we examined the effect of GpIb alpha(1-282), a GpIb alpha fragment released by the cobra venom mocarhagin on the heparin-catalyzed rate of thrombin inhibition by antithrombin III (AT). GpIb alpha(1-282) inhibited the reaction in a dose-dependent and competitive fashion. In contrast, the GpIb alpha(1-271) fragment, produced by exposing QpI alpha(1-282) to carboxypeptidase Y, had no effect on thrombin inhibition by the heparin-AT complex. Measurements of the apparent equilibrium constant of the GpIb alpha(1-282) binding to thrombin as a function of different salts (NaCl and tetramethyl-ammonium chloride) concentration (0.1-0.2 M) indicated a large salt dependence (Gamma(+/-) = -4.5), similar to that pertaining to the heparin binding to thrombin. The importance of thrombin HBS in its interaction with GpIb alpha was confirmed using DNA aptamers, which specifically bind to either HBS (HD22) or the fibrinogen recognition site of thrombin (HD1). HD22, but not HD1, inhibited thrombin binding to GpIb alpha(1-282). Furthermore, the proteolytic derivative gamma(T)-thrombin, which lacks the fibrinogen recognition site, binds to GpIb alpha via its intact HBS in a reaction that is inhibited by HD22. Neither alpha- nor gamma(T)-thrombin bound to GpIb alpha(1-271), suggesting that the Asp(272)-Glu(282) region of GpIb alpha may act as a "heparin-like" ligand for the thrombin DBS, thereby inhibiting heparin binding to thrombin. It was also demonstrated that intact platelets may dose-dependently inhibit the heparin-catalyzed thrombin inhibition by AT at enzyme concentrations <5 nM. Altogether, these findings show that thrombin HBS binds to the region of GpIb alpha involving the Asp(272)-GlU(282) segment, protecting the enzyme from the inactivation by the heparin-AT system.