Glycoprotein Ib-mediated platelet activation: a signaling pathway evoked by thrombin

Abstract number: P0723

Adam F., Guillin M. C., Jandrot-Perrus M.

INSERM E9907, France

Platelet activation by thrombin plays a major role in hemostasis and thrombosis. Thrombin activates platelets by cleaving G-protein-coupled protease activated receptors (PARs). On the other hand, the platelet membrane glycoprotein (GP)Ib acts as a thrombin-binding site and promotes platelet activation by low thrombin concentrations. The hypothesis that thrombin triggers GPIb-coupled signals undetectable by conventional methods because of their low intensity was investigated. We developed a model based on the immobilization of thrombin to study events emphasized by agonist immobilization and independent of PARs by using active site-blocked thrombin. The firm attachment of platelets to immobilized inactive thrombin was observed and reached 6.8 ± 0.4 and 14.9 ± 0.6% in 30 and 60 min, respectively. Fluorescence and confocal microscopy using FITC-phalloidin showed morphologic changes (spreading, lamellipodia, filopodia, stress fibers) and platelet aggregates. Binding was GPIb-dependent because inhibited by an anti-GPIb MoAb (SZ2) or by an excess of glycocalicin, the extracellular domain of GPIba, and reduced by more than 90% with Bernard–Soulier platelets. Dense granule content was slowly secreted (6.4 ± 0.9% in 60 min) upon platelet incubation with immobilized inactive thrombin compared to immobilized albumin (0.3 ± 0.1%). In the presence of a; IIbb3 antagonists, formation of aggregates was inhibited, but platelets still bound to thrombin were spread. Platelet firm binding was reduced by antagonists of the P2Y1 and P2Y12 receptors. These results indicate that GPIb interaction with thrombin activates signals leading to ADP secretion in turn involved in a; IIbb3 activation and platelet aggregation. Activation of protein tyrosine kinases was observed upon platelet binding to thrombin. Antagonists of PI3-kinases and protein kinase C (PKC) inhibited platelet–platelet interactions and tyrosine phosphorylations. This study provide evidences that thrombin induced GPIb-dependent PAR-independent activation pathway.