A Signaling Mechanism By Which Platelet Glycoprotein Ib-IX Promotes Thrombin-Induced Platelet Activation

Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. We reconstituted GPIb-IX (GPIb) /Protease-activated receptor (PAR) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression, and this effect of GPIb appears to require GPIb signaling, as deletion of the cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown in human platelets, in which pretreatment with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of the GPIbα, inhibited thrombin-induced platelet activation. Furthermore, 14-3-3 binding site deletion in GPIba or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1, both of which have been shown to mediate von Willebrand factor-induced GPIb signaling, and the role of GPIb in promoting thrombin signaling was abolished with a Rac-inhibitor, NSC23766 or in Rac1^-/- platelets. Importantly, LIMK1^-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation.