IRAG is involved in NO/cGMP-mediated inhibition of platelet function

Abstract

The inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) is highly expressed in platelets. It is assembled in a macrocomplex together with cGMP-dependent protein kinase Iβ (cGKIβ) and the inositol 1,4,5-trisphosphate receptor type I (IP 3 RI). In response to cGKI activation, IRAG is phosphorylated in vivo. By mass spectrometric analysis of purified in vivo phosphorylated IRAG from cGMP-stimulated human platelets, Ser664 and Ser677 were identified as phosphorylated amino acids. Generation of phosphospecific antibodies confirmed these in vivo phosphorylation sites. To examine the role of IRAG in platelet function, we analysed the aggregation of platelets from an IRAG mouse mutant (IRAG ∆12/∆12 ). In this mutant, exon 12 of the IRAG gene was deleted and thereby the IRAG-IP 3 RI interaction was abolished [1]. Interestingly, collagen-induced aggregation of IRAG ∆12/∆12 platelets was not inhibited by nitric oxide (NO) and the cGMP analogue 8-pCPT-cGMP in contrast to wild type platelets. The shape change was not affected, neither in mutant nor in wild type platelets. However, iloprost and the cAMP-analogue cBIMPS still inhibited aggregation and shape change in IRAG ∆12/∆12 and wild type platelets. Additionally, we analysed fibrinogen binding to thrombin-stimulated platelets. In wild type platelets, pre-treatment with NO and cGMP decreased fibrinogen binding by about 50%. In contrast, in IRAG ∆12/ ∆12 platelets the response to NO and cGMP was nearly absent. Preincubation with iloprost and cBIMPS clearly reduced agonist-induced fibrinogen binding to both, IRAG ∆12/∆12 and wild type platelets. These results suggest that signalling through IRAG/IP 3 RI is essential for NO/ cGMP-dependent inhibition of platelet aggregation and activation of the fibrinogen receptor