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Platelet activation and aggregation at sites of vascular injury are essential for primary hemostasis, but are also major pathomechanisms underlying myocardial infarction and stroke. Changes in [Ca 2؉ ] i are a central step in platelet activation. In nonexcitable cells, receptormediated depletion of intracellular Ca 2؉ stores triggers Ca 2؉ entry through storeoperated calcium (SOC) channels. STIM1 has been identified as an endoplasmic reticulum (ER)-resident Ca 2؉ sensor that regulates store-operated calcium entry (SOCE) in immune cells and platelets, but the identity of the platelet SOC channel has remained elusive. Orai1 (CRACM1) is the recently discovered SOC (CRAC) channel in T cells and mast cells but its role in mammalian physiology is unknown. Here we report that Orai1 is strongly expressed in human and mouse platelets. To test its role in blood clotting, we generated Orai1-deficient mice and found that their platelets display severely defective SOCE, agonist-induced Ca 2؉ responses, and impaired activation and thrombus formation under flow in vitro. As a direct consequence, Orai1 deficiency in mice results in resistance to pulmonary thromboembolism, arterial thrombosis, and ischemic brain infarction, but only mild bleeding time prolongation. These results establish Orai1 as the long-sought platelet SOC channel and a crucial mediator of ischemic cardiovascular and cerebrovascular events. IntroductionAt sites of vascular injury the subendothelial extracellular matrix (ECM) is exposed to the flowing blood and triggers sudden platelet activation and the formation of a fibrin-containing thrombus. This process is essential to prevent excessive posttraumatic blood loss, but if it occurs at sites of atherosclerotic plaque rupture it can also lead to vessel occlusion and the development of myocardial infarction or ischemic stroke, which are among the leading causes of mortality and severe disability in industrialized countries. 1,2 Therefore, the inhibition of platelet activation has become an important strategy to prevent or treat such acute ischemic events. 3 Platelet activation can occur through different signaling pathways that culminate in the activation of phospholipase C (PLC) isoforms and production of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP 3 ). IP 3 binds its receptors on the membrane of the intracellular Ca 2ϩ stores and mediates Ca 2ϩ release into the cytosol. In platelets, the dense tubular system, referred to as sarcoplasmic reticulum (SR), is thought to be the major Ca 2ϩ store. The resulting decline in Ca 2ϩ store content in turn triggers a sustained influx of extracellular Ca 2ϩ by a mechanism known as store-operated Ca 2ϩ entry (SOCE). 4,5 Although SOCE has pharmacologically been well defined for more than a decade, not much was known about the underlying molecular machinery until recently, when stromal interaction molecule 1 (STIM1) was identified as a sarco/ endoplasmic (SR/ER)-store calcium sensor that controls SOCE in T cells and mast cells. [6][7][8][9] Shortly after that, ...

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Background and Purpose-Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury.

Aberrant Wnt-signaling caused by mutants of β-catenin, a key regulator of the canonical Wntsignaling pathway, is frequently detected in cancer. Only recently, it was suggested that in hepatocellular carcinoma (HCC) the expression of the target gene glutamine synthetase (GS) is a highly reliable marker for the identification of β-catenin mutations. In order to prove this hypothesis, 52 samples from human hepatocellular carcinomas were analysed for the activation of β-catenin and the expression of GS. In total, 45 samples stained positive for cytoplasmic/nuclear β-catenin. A strong correlation between expression of GS and activated β-catenin (100% of nuclear and 84% of cytosolic) was found. However, among 35 GS positive tumors that were analysed for β-catenin mutations no mutations were detected in 25 GS-positive carcinomas although 24 out of the 25 carcinomas exhibited at least abnormal expression of β-catenin. Since the mutational analysis identified 9 different point mutations of the β-catenin gene including the rare mutation H36P and the yet unknown mutation P44A it was asked whether these mutations may differently effect β-catenin target genes. Therefore, expression plasmids for different mutations were constructed and cotransfected with the TOP-flash luciferase reporter and a reporter carrying the GS-5'-enhancer. The experiments confirmed that there are differences between different β-catenin target sequences and different β-catenin mutations. In addition, the failure that the endogenous expression of GS in GS-negative cells was not induced by the transient transfection experiment indicated that the effect of β-catenin on the GS-5'-enhancer is only one aspect of gene activation induced by β-catenin.

Kinins are proinflammatory and vasoactive peptides that are released during tissue damage and may contribute to neuronal degeneration, inflammation, and edema formation after brain injury by acting on discrete bradykinin receptors, B1R and B2R. We studied the expression of B1R and B2R and the effect of their inhibition on lesion size, blood-brain barrier (BBB) disruption, and inflammatory processes after a focal cryolesion of the right parietal cortex in mice. B1R and B2R gene transcripts were significantly induced in the lesioned hemispheres of wild-type mice (P<0.05). The volume of the cortical lesions and neuronal damage at 24 h after injury in B1R(-/-) mice were significantly smaller than in wild-type controls (2.5+/-2.6 versus 11.5+/-3.9 mm(3), P<0.001). Treatment with the B1R antagonist R-715 1 h after lesion induction likewise reduced lesion volume in wild-type mice (2.6+/-1.4 versus 12.2+/-6.1 mm(3), P<0.001). This was accompanied by a remarkable reduction of BBB disruption and tissue inflammation. In contrast, genetic deletion or pharmacological inhibition of B2R had no significant impact on lesion formation or the development of brain edema. We conclude that B1R inhibition may offer a novel therapeutic strategy after acute brain injuries.

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Oxygen depletion (O2) and a decrease in pH are initial pathophysiological events in stroke development, but secondary mechanisms of ischemic cell death are incompletely understood. By patch-clamp recordings of brain slice preparations we show that TASK1 and TASK3 channels are inhibited by pH-reduction (42 ± 2%) and O2 deprivation (36 ± 5%) leading to membrane depolarization, increased input resistance and a switch in action potential generation under ischemic conditions. In vivo TASK blockade by anandamide significantly increased infarct volumes at 24h in mice undergoing 30 min of transient middle cerebral artery occlusion (tMCAO). Moreover, blockade of TASK channels accelerated stroke development. Supporting these findings TASK1−/− mice developed significantly larger infarct volumes after tMCAO accompanied by worse outcome in functional neurological tests compared to wild type mice. In conclusion, our data provide evidence for an important role of functional TASK channels in limiting tissue damage during cerebral ischemia.