Article

Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation

Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, Würzburg, Germany.
Blood (Impact Factor: 10.43). 11/2008; 113(9):2056-63. DOI: 10.1182/blood-2008-07-171611
Source: PubMed

ABSTRACT 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, receptor-mediated depletion of intracellular Ca(2+) stores triggers Ca(2+) entry through store-operated 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.

Download full-text

Full-text

Available from: Markus Bender, Jan 20, 2014
0 Followers
 · 
160 Views
  • Source
    • "At testing, mice were restrained in a plexiglass chamber during the measurement of bleed time. Methods for determining bleed time have been described previously (Braun et al., 2009). A 3-mm segment of the tail was removed using surgical scissors, and tail bleeding was monitored by gently blotting drops of blood leaving the tail with filter paper every 20 seconds. "
    [Show abstract] [Hide abstract]
    ABSTRACT: There are ongoing efforts to develop pain therapeutics with novel mechanisms of action that avoid common side effects associated with other analgesics. The anticonvulsant neuropeptide galanin is a potent regulator of neuronal excitability and has a well-established role in pain modulation, making it a potential target for novel therapies. Our previous efforts focused on improving blood-brain-barrier penetration and enhancing the metabolic stability of galanin analogs to protect against seizures. More recently, we designed peripherally-acting galanin analogs that reduce pain-related behaviors by acting in the periphery and exhibit preferential binding towards GAL2 over GAL1 galanin receptors. Here we report preclinical studies of a monodisperse oligoethylene glycol (dPEG)-containing galanin analog, NAX 409-9 (previously reported as Gal-R2-dPEG24 in Zhang et al., 2013), in rodent analgesic and safety models. Results obtained with NAX 409-9 in these tests were compared to the representative analgesics gabapentin, ibuprofen, acetylsalicylic acid, acetaminophen, and morphine. In mice that received intraplantar carrageenan, NAX 409-9 increased paw withdrawal latency with an ED50 of 6.6 mg/kg, i.p. NAX 409-9 also increased the paw withdrawal threshold to mechanical stimulation following partial sciatic nerve ligation in rats (2 mg/kg). Conversely, NAX 409-9 had no effect in the tail flick or hot plate assays (up to 24 mg/kg). Importantly, NAX 409-9 did not negatively affect gastrointestinal motility (4-20 mg/kg), respiratory rate (40-80 mg/kg), or bleed time (20 mg/kg). These studies illustrate that this non-brain-penetrating galanin analog reduces pain behaviors in several models and does not produce some of the dose-limiting toxicities associated with other analgesics.
    Journal of Pharmacology and Experimental Therapeutics 10/2014; 352(1). DOI:10.1124/jpet.114.219063 · 3.86 Impact Factor
  • Source
    • "Independent of the signaling pathway triggered by any of the physiological platelet agonists, a sustained and significant increase in intracellular calcium concentration [Ca 2+ ] i occurs. This increase consists of the release of compartmentalized calcium through the store-operated Ca 2+ entry (SOCE) (Alonso et al., 1991) and the entry of extracellular Ca 2+ at the plasma membrane through the stromal interaction molecule (STIM1), a sensor in the dense tubular system, and Orai1, the major store-operated Ca 2+ (SOC) channel in the plasma membrane or the Na + /Ca 2+ exchanger (Braun et al., 2009), contributing to hemostatic platelet responses. The Epithelial sodium channel/Degenerin (ENaC/Deg) family comprises cation-selective ion channels found widely expressed in animals from hydra and nematodes to vertebrates. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca2+ concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na+ channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and β-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na+ influx through this channel is fundamental for platelet collagen activation.
    European journal of cell biology 03/2014; DOI:10.1016/j.ejcb.2014.02.003 · 3.70 Impact Factor
  • Source
    • "Dysfunction of store-operated Ca 2+ channels causes some diseases, for example, the mutations of ORAI1 and STIM1 causing the deficiency of I CRAC in T cells that has been regarded as the aetiology of severe combined immune deficiency syndrome [15]. ORAI1 deficiency in mice results in resistance to pathological thrombus formation, which is an important new clue for preventing ischemic cardiovascular and cerebrovascular events [16]. In addition, the alteration of channel expression or channel activity is implicated in angiogenesis [17], smooth muscle cell proliferation and migration [18] and cardiomyocyte hypertrophy [19], suggesting the signaling of STIM1 associated store-operated Ca 2+ channel activation could act as new therapeutic targets. "
    [Show abstract] [Hide abstract]
    ABSTRACT: STIM1 is a Ca(2+) sensing molecule. Once the Ca(2+) stores are depleted, STIM1 moves towards the plasma membrane (PM) (translocation), forms puncta (clustering), and triggers store-operated Ca(2+) entry (SOCE). Although this process has been regarded as a main mechanism for store-operated Ca(2+) channel activation, the STIM1 clustering is still unclear. Here we discovered a new phenomenon of STIM1 clustering, which is not triggered by endoplasmic reticulum (ER) Ca(2+) depletion. STIM1 subplasmalemmal translocation and clustering can be induced by ER Ca(2+) store depletion with thapsigargin (TG), G-protein-coupled receptor activator trypsin and ryanodine receptor (RyR) agonists caffeine and 4-chloro-3-ethylphenol (4-CEP) in the HEK293 cells stably transfected with STIM1-EYFP. The STIM1 clustering induced by TG was more sustained than that induced by trypsin and RyR agonists. Interestingly, 4-CEP-induced STIM1 clustering also happened in the cytosol without ER Ca(2+) store depletion. Application of some pharmacological regulators including flufenamic acid, 2-APB, and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) at concentrations without affecting ER Ca(2+) store also evoked cytosolic STIM1 clustering. However, the direct store-operated ORAI channel blockers (SKF-96365, Gd(3+) and diethylstilbestrol) or the signaling pathway inhibitors (genistein, wortmannin, Y-27632, forskolin and GF109203X) did not change the STIM1 movement. Disruption of cytoskeleton by colchicine and cytochalasin D also showed no effect on STIM1 movement. We concluded that STIM1 clustering and translocation are two dynamic processes that can be pharmacologically dissociated. The ER Ca(2+) store-independent mechanism for STIM1 clustering is a new alternative mechanism for regulating store-operated channel activity, which could act as a new pharmacological target.
    Biochemical pharmacology 07/2012; 84(8):1024-35. DOI:10.1016/j.bcp.2012.07.013 · 4.65 Impact Factor
Show more