Abstract 993: NO-Dependent Inhibition of Platelet Adhesion/Aggregation is Mediated by IRAG Phosphorylation in vivo
Background: Defective regulation of platelet adhesion/aggregation within the arterial vasculature is crucial for the pathogenesis of arterial thrombosis, the major complication of atherosclerosis triggering myocardial infarction and stroke. Nitric oxide (NO)/cyclic GMP (cGMP) signaling via cGMP-dependent protein kinase I (cGKI) is the central regulatory pathway conveying inhibition of platelet activation/aggregation. However, the signaling cascade downstream of cGKI mediating platelet inhibition is still unclear.
Methods and Results: We assessed the role of the inositol-1.4.5-trisphosphate receptor-associated cGMP kinase substrate IRAG for the regulation platelet function. We show that IRAG is abundantly expressed in platelets and assembled in a macrocomplex together with cGKIβ and the inositol-1.4.5-trisphosphate receptor type I (InsP3RI). cGKI phosphorylates platelet IRAG at Ser664 and Ser677 in vivo. Next, platelet function was investigated using IRAGΔ12/Δ12 mutant mice, in which the IRAG-InsP3RI interaction is completely disrupted. Mutant platelets were unresponsive to NO/cGMP-dependent inhibition of fibrinogen-receptor activation and platelet aggregation. Furthermore, intravital microscopy revealed that NO failed to prevent arterial thrombosis of the injured carotid artery in IRAGΔ12/Δ12 mutants, indicating that IRAG is essentially involved in NO/cGMP-dependent regulation of platelet function.
Conclusion: These findings reveal for the first time in vivo that interaction between IRAG and InsP3RI has a central role in NO/cGMP-dependent inhibition of platelet activation and aggregation. We conclude that the physiological down-regulation of platelet activation by intact IRAG signaling is an essential endogenous anti-platelet strategy in vivo.