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

Abstract

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 GpIbα, we examined the effect of GpIbα1-282, a GpIbα fragment released by the cobra venom mocarhagin on the heparin- catalyzed rate of thrombin inhibition by antithrombin III (AT). GpIbα1-282 inhibited the reaction in a dose-dependent and competitive fashion. In contrast, the GpIbα1-271 fragment, produced by exposing GpIbα1-282 to carboxypeptidase Y, had no effect on thrombin inhibition by the heparin-AT complex. Measurements of the apparent equilibrium constant of the GpIbα1-282 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 (Γ = -4.5), similar to that pertaining to the heparin binding to thrombin. The importance of thrombin HBS in its interaction with GpIbα 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 GPIbα1-282. Furthermore, the proteolytic derivative γ(T)-thrombin, which lacks the fibrinogen recognition site, binds to GpIbα via its intact HBS in a reaction that is inhibited by HD22. Neither α- nor γ(T)-thrombin bound to GpIbα1-271, suggesting that the Asp272-Glu282 region of GpIbα may act as a 'heparin-like' ligand for the thrombin HBS, 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 GpIbα involving the Asp272-Glu282 segment, protecting the enzyme from the inactivation by the heparin-AT system.