Abstract 598: A Platelet Glycoprotein Ib-IX-specific 14-3-3/Rac1/LIMK1 Signaling Pathway Promotes Thrombin-Induced Platelet Activation

Abstract

Rationale: Thrombin-induced platelet activation requires protease-activated receptors. However, the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds to thrombin and is important for low dose thrombin-induced platelet activation. It is unclear how GPIb-IX promotes thrombin-induced platelet activation. Objective: To clearly elucidate the mechanism by which GPIb-IX promotes thrombin-induced platelet activation. Methods and Results: We reconstituted GPIb-IX (GPIb) /Protease-activated receptors (PARs) 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. This effect of GPIb appears to require GPIb signaling, as mutation of a cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-IX-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown by pretreating human platelets with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of GPIbα, which inhibited thrombin-induced platelet activation, but did not affect thrombin binding to platelets. Furthermore, 14-3-3 binding site deletion in GPIbα or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1. To determine the role of the Rac1/LIMK1 signaling pathway in mediating thrombin-induced GPIb signaling and platelet activation, we examined the effects of Rac1 knockout, LIMK1 knockout and Rac1-inhibitor on low dose thrombin-induced calcium response and platelet activation. Rac1 inhibitor, NSC23766, abolished the GPIb-dependent cell response in a reconstituted CHO cell model. Rac1 knockout platelets showed diminished platelet response to thrombin and were not different from wild type platelets in the presence of MPαC. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation. Conclusions: The stimulatory role of GPIb in thrombin-induced platelet activation requires a thrombin-induced GPIb-specific 14-3-3/Rac1/LIMK1 signaling pathway.