Defective regulation of platelet activation/aggregation is a predominant cause for arterial thrombosis, the major complication of atherosclerosis triggering myocardial infarction and stroke. A central regulatory pathway conveying inhibition of platelet activation/aggregation is nitric oxide (NO)/cyclic GMP (cGMP) signaling by cGMP-dependent protein kinase I (cGKI). However, the regulatory cascade downstream of cGKI mediating platelet inhibition is still unclear. Here, we show that the inositol-1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) is abundantly expressed in platelets and assembled in a macrocomplex together with cGKIβ and the inositol-1,4,5-trisphosphate receptor type I (InsP 3RI). cGKI phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted deletion of the IRAG-InsP 3RI interaction in IRAG Δ12/Δ12 mutant mice leads to a loss of NO/cGMP-dependent inhibition of fibrinogen-receptor activation and platelet aggregation. Intracellular calcium transients were not affected by DEA/NO or cGMP in mutant platelets. Furthermore, intravital microscopy shows that NO fails to prevent arterial thrombosis of the injured carotid artery in IRAG Δ12/Δ12 mutants. These findings reveal that interaction between IRAG and InsP 3RI has a central role in NO/cGMP-dependent inhibition of platelet aggregation and in vivo thrombosis. © 2007 by The American Society of Hematology.