Differentiation of the Two Forms of GPIb Functioning as Receptors for α-Thrombin and von Willebrand Factor:  Ca²⁺ Responses of Protease-Treated Human Platelets Activated with α-Thrombin and the Tethered Ligand Peptide

Abstract

Previous results have shown that both GPIb and the seven transmembrane domain receptor (STDR) are required for optimal thrombin-induced platelet activation (Greco et al., 1996). Limited degradation (approximately 10%) of GPIb and the STDR by elastase reduced the Ca2+ response to 0.5 nM alpha-thrombin by only 10% whereas Serratia marcescens metalloprotease reduced the Ca2+ response by 80% and fully abrogated high-affinity thrombin binding and aggregation. vWF/ristocetin-induced agglutination was only slightly reduced (20%) while Ca2+ and aggregation response to higher thrombin concentrations were retained. At increasing elastase and Serratia protease concentrations, degradation of the STDR proceeded from the amino-terminal domain, but Ca2+ responses to the tethered ligand peptide SFLLRNPNDKYEPF were not affected by either protease. These results show that both putative thrombin receptors are susceptible to protease degradation and suggest that Serratia protease is able to differentiate the GPIb-mediated events associated with thrombin activation from those associated with ristocetin-induced agglutination.

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

“…In addition, GpIb also binds to thrombin with high affinity (1). The inhibition of this interaction causes a reduced thrombin-induced platelet activation through an unclear mechanism (3,4).…”

“…At each AT concentration, the reaction was quenched at various intervals by addition of 2 ml of 100 M Phe-PipArg-pNA in 50 mM Tris-HCl, 0.1 M NaCl, 0.1% PEG 6000, pH 8.00. The k obs value was then calculated using Equation 4.…”

The Platelet High Affinity Binding Site for Thrombin Mimics Hirudin, Modulates Thrombin-induced Platelet Activation, and Is Distinct from the Glycoprotein Ib-IX-V Complex

“…There is no enhanced platelet activation response, suggesting that this Ab does not recognize the high affinity binding site for thrombin which we hypothesize also contains a hirudin-like peptide. Based on studies of thrombin interactions with platelets and GP Ib using anti-GP Ib␣ antibodies, several investigators propose that GP Ib is a high affinity binding site for thrombin on platelets (25,30,31). An alternative explanation for these data includes the possibility that the anti-GP Ib Ab used in these experiments cross-reacts with hirudin-like regions found on other platelet membrane proteins including PAR-1 and the unique high affinity binding site identified in this study.…”

Thrombin and cardiopulmonary bypass – A paradigm for evaluation of the regulation of hemostasis

“…In addition, kinetic studies of ligand-receptor binding and binding of specific antibodies to platelet receptors suggest that the platelet glycoprotein receptor for von Willebrand factor (GP Ib) is an important component of thrombin activation of human platelets [47, 82,216]. Results using ligand binding studies suggest that GP Ib and PAR receptors are required to ensure the optimal rate and extent of thrombin induced human platelet activation over the physiologic range of concentrations of thrombin [82].…”