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- IntroductionPlatelet activation and aggregation at foci of vascular injury is essential for primary hemostasis, but it also initiates arterial thrombosis, the leading cause of myocardial infarction and stroke. 1 The gaseous molecule nitric oxide (NO) is an endogenous platelet antagonist and inhibits platelet activation and aggregate formation both in vitro and in vivo. [2][3][4] NO activates soluble guanylyl cyclases that initiate a subsequent rise in platelet cyclic GMP (cGMP). 2,3 Several mechanisms have been proposed by which NO/cGMP signaling abolishes platelet activation and aggregation, including inhibition of G-proteincoupled receptors and rearrangement of the cytoskeleton. 3,5 In addition, NO/cGMP prevents inositol-1,4,5-trisphosphate (InsP 3 )-mediated intracellular calcium release, 3,5 the critical step in the signal transduction pathway that leads to full platelet activation. 6 The cGMP-dependent protein kinase type I (cGKI) is strictly required for inhibition of platelet activation by NO/cGMP. 2,7 Although cGKI has been reported to inhibit intracellular Ca 2ϩ release in platelets, [8][9][10] the exact molecular targets downstream of cGKI involved in NO/cGMP-dependent inhibition of platelet activation have not been defined.In smooth muscle cells, we have identified the cGKI substrate IRAG 4, ] receptorassociated cGKI substrate protein), a 125-kDa protein which copurifies in a macrocomplex together with cGKI and the InsP 3 receptor type I (InsP 3 RI). 11 IRAG is essential for NO/cGMPdependent smooth muscle cell relaxation, because it negatively regulates InsP 3 -induced calcium release. 11,12 Because of the importance of cGKI signaling in platelets, we studied here the expression and cGKI-dependent phosphorylation of IRAG in platelets and the physiologic relevance of the IRAG-InsP 3 RI interaction for the regulation of platelet function. We provide first evidence that the IRAG-InsP 3 RI interaction mediates NO/cGMP-dependent inhibition of thrombin-induced increases in [Ca 2ϩ ] i in platelets and is the major determinant of NO/cGMPdependent prevention of platelet aggregation in vitro and arterial thrombosis in vivo. Materials and methods MaterialsWe used 8-pCPT-cGMP, Rp-8-Br-PET-cGMPS, Sp-5,6-DCl-cBIMPS (cBIMPS), 8-AET-cGMP-agarose, ethanolamine-agarose (Biolog, Bremen, Germany), forskolin (Calbiochem, Darmstadt, Germany), prostacyclin (Sigma, Deisenhofen, Germany), iloprost (Axxora, San Diego, CA), DEA/NO (Axxora), NO-spermine (Axxora), GEA-NO 3162 (Axxora), sodium nitroprusside (...

SummaryBurkholderia 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.

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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