IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation.

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 cGKIbeta and the inositol-1,4,5-trisphosphate receptor type I (InsP3RI). cGKI phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted deletion of the IRAG-InsP3RI interaction in IRAGDelta12/Delta12 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 IRAGDelta12/Delta12 mutants. These findings reveal that interaction between IRAG and InsP3RI has a central role in NO/cGMP-dependent inhibition of platelet aggregation and in vivo thrombosis.

Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system.

Burkholderia cepacia H111, which was isolated from a cystic fibrosis patient, effectively kills the nematode Caenorhabditis elegans. Depending on the medium used for growth of the bacterium two different killing modes were observed. On high-osmolarity medium the nematodes became paralysed and died within 24 h. Using filter assays we provide evidence that this killing mode involves the production of an extracellular toxin. On nematode growth medium killing occurs over the course of 2-3 days and involves the accumulation of bacteria in the intestinal lumen of C. elegans. We demonstrate that the cep quorum-sensing system of H111 is required for efficient killing of C. elegans under both killing conditions. Using the C. elegans phm-2 mutant that has a non-functional grinder evidence is provided that the cep system is required to enter the intestinal lumen but is dispensable for the colonization of the gut. Furthermore, we demonstrate that the type II secretion machinery is not essential for nematode killing.