Attila Braun

Calcium signaling in platelets

Summary.u2002 Agonist‐induced elevation in cytosolic Ca2+ concentrations is essential for platelet activation in hemostasis and thrombosis. It occurs through Ca2+ release from intracellular stores and Ca2+ entry through the plasma membrane (PM). Ca2+ store release is a well‐established process involving phospholipase (PL)C‐mediated production of inositol‐1,4,5‐trisphosphate (IP3), which in turn releases Ca2+ from the intracellular stores through IP3 receptor channels. In contrast, the mechanisms controlling Ca2+ entry and the significance of this process for platelet activation have been elucidated only very recently. In platelets, as in other non‐excitable cells, the major way of Ca2+ entry involves the agonist‐induced release of cytosolic sequestered Ca2+ followed by Ca2+ influx through the PM, a process referred to as store‐operated calcium entry (SOCE). It is now clear that stromal interaction molecule 1 (STIM1), a Ca2+ sensor molecule in intracellular stores, and the four transmembrane channel protein Orai1 are the key players in platelet SOCE. The other major Ca2+ entry mechanism is mediated by the direct receptor‐operated calcium (ROC) channel, P2X1. Besides these, canonical transient receptor potential channel (TRPC) 6 mediates Ca2+ entry through the PM. This review summarizes the current knowledge of platelet Ca2+ homeostasis with a focus on the newly identified Ca2+ entry mechanisms.

STIM2 regulates capacitive Ca2+ entry in neurons and plays a key role in hypoxic neuronal cell death.

Neurons lacking the calcium sensor STIM2 are protected from hypoxia-induced cell death. Resisting Ischemia Loss of blood flow to the brain—as can occur during a stroke—leads to the death of neurons, a process that involves a pathological increase in intracellular calcium. Berna-Erro et al. investigated the role of capacitive calcium entry (CCE), a process in which depletion of calcium from intracellular stores triggers its entry across the plasma membrane, in ischemia-induced calcium entry and neuronal death. The calcium-sensing molecule STIM1 is known to play a crucial role in mediating CCE in various cell types; in neurons, however, Berna-Erro et al. found that CCE depended instead on the closely related molecule STIM2. Neurons from mice lacking STIM2 were resistant to the effects of hypoxia in vitro; moreover, mice lacking STIM2 showed less neurological damage than did wild-type mice in a model of ischemic stroke. Thus, the authors conclude that STIM2 is critical to neuronal CCE and that CCE plays a role in neuronal death in ischemia. Excessive cytosolic calcium ion (Ca2+) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca2+ entry (CCE) is a process whereby depletion of intracellular Ca2+ stores causes the activation of plasma membrane Ca2+ channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)–resident Ca2+ sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca2+ concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca2+ accumulation in neurons. Neurons from Stim2−/− mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2−/− mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.

Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1.

In the absence of PLD1, platelets do not form stable aggregates under high shear conditions. Aggregation Regulation When damage occurs to the endothelium lining a blood vessel and exposes the underlying extracellular matrix, platelets adhere to the site of injury and aggregate to stop blood loss. However, aggregated platelets can cause ischemia if they occlude the vessel, thus creating the need for therapies that can limit platelet aggregation without increasing blood loss. Elvers et al. found that platelets from mice deficient in phospholipase D1 (PLD1) showed reduced activation of αIIbβ3 integrin, a major adhesion receptor, and did not form stable aggregates when experiencing high shear forces (such as those found in small arterioles). PLD1 deficiency conferred protection against thrombosis and cerebral ischemia in vivo, an effect that was seen with Pld1−/− mice and wild-type mice transplanted with bone marrow from Pld1−/− mice. PLD1 deficiency did not, however, increase blood loss after tail wounding. Thus, PLD1 could be a potential therapeutic target to prevent or treat stroke or other ischemic conditions. Platelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesive receptor, αIIbβ3 integrin, from a low- to high-affinity state for its ligands, a process that enables adhesion and aggregation. In response to stimulation of heterotrimeric guanosine triphosphate–binding protein or immunoreceptor tyrosine-based activation motif–coupled receptors, phospholipases cleave membrane phospholipids to generate lipid and soluble second messengers. An essential role in platelet activation has been established for phospholipase C (PLC) but not for PLD and its product phosphatidic acid. Here, we report that platelets from Pld1−/− mice displayed impaired αIIbβ3 integrin activation in response to major agonists and defective glycoprotein Ib–dependent aggregate formation under high shear conditions. These defects resulted in protection from thrombosis and ischemic brain infarction without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.

Store-operated Ca2+ entry in platelets occurs independently of transient receptor potential (TRP) C1

Changes in [Ca2+]i are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca2+ stores triggers Ca2+ entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca2+ sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1.

STIM1 is essential for Fcγ receptor activation and autoimmune inflammation

Fcgamma receptors (FcgammaRs) on mononuclear phagocytes trigger autoantibody and immune complex-induced diseases through coupling the self-reactive immunoglobulin G (IgG) response to innate effector pathways, such as phagocytosis, and the recruitment of inflammatory cells. FcRgamma-based activation is critical in the pathogenesis of these diseases, although the contribution of FcgammaR-mediated calcium signaling in autoimmune injury is unclear. Here we show that macrophages lacking the endoplasmic reticulum-resident calcium sensor, STIM1, cannot activate FcgammaR-induced Ca(2+) entry and phagocytosis. As a direct consequence, STIM1 deficiency results in resistance to experimental immune thrombocytopenia and anaphylaxis, autoimmune hemolytic anemia, and acute pneumonitis. These results establish STIM1 as a novel and essential component of FcgammaR activation and also indicate that inhibition of STIM1-dependent signaling might become a new strategy to prevent or treat IgG-dependent immunologic diseases.

Stromal interaction molecules 1 and 2 are key regulators of autoreactive T cell activation in murine autoimmune central nervous system inflammation.

Calcium (Ca2+) signaling in T lymphocytes is essential for a variety of functions, including the regulation of differentiation, gene transcription, and effector functions. A major Ca2+ entry pathway in nonexcitable cells, including T cells, is store-operated Ca2+ entry (SOCE), wherein depletion of intracellular Ca2+ stores upon receptor stimulation causes subsequent influx of extracellular Ca2+ across the plasma membrane. Stromal interaction molecule (STIM) 1 is the Ca2+ sensor in the endoplasmic reticulum, which controls this process, whereas the other STIM isoform, STIM2, coregulates SOCE. Although the contribution of STIM molecules and SOCE to T lymphocyte function is well studied in vitro, their significance for immune processes in vivo has remained largely elusive. In this study, we studied T cell function in mice lacking STIM1 or STIM2 in a model of myelin-oligodendrocyte glycoprotein (MOG35–55)-induced experimental autoimmune encephalomyelitis (EAE). We found that STIM1 deficiency significantly impaired the generation of neuroantigen-specific T cell responses in vivo with reduced Th1/Th17 responses, resulting in complete protection from EAE. Mice lacking STIM2 developed EAE, but the disease course was ameliorated. This was associated with a reduced clinical peak of disease. Deficiency of STIM2 was associated with an overall reduced proliferative capacity of lymphocytes and a reduction of IFN-γ/IL-17 production by neuroantigen-specific T cells. Neither STIM1 nor STIM2 deficiency altered the phenotype or function of APCs. These findings reveal a crucial role of STIM-dependent pathways for T cell function and activation under autoimmune inflammatory conditions, establishing them as attractive new molecular therapeutic targets for the treatment of inflammatory and autoimmune disorders.

STIM1-Independent T Cell Development and Effector Function In Vivo

Store-operated Ca2+ entry (SOCE) is believed to be of pivotal importance in T cell physiology. To test this hypothesis, we generated mice constitutively lacking the SOCE-regulating Ca2+ sensor stromal interaction molecule 1 (STIM1). In vitro analyses showed that SOCE and Ag receptor complex-triggered Ca2+ flux into STIM1-deficient T cells is virtually abolished. In vivo, STIM1-deficient mice developed a lymphoproliferative disease despite normal thymic T cell maturation and normal frequencies of CD4+Foxp3+ regulatory T cells. Unexpectedly, STIM1-deficient bone marrow chimeric mice mounted humoral immune responses after vaccination and STIM1-deficient T cells were capable of inducing acute graft-versus-host disease following adoptive transfer into allogeneic hosts. These results demonstrate that STIM1-dependent SOCE is crucial for homeostatic T cell proliferation, but of much lesser importance for thymic T cell differentiation or T cell effector functions.

STIM and Orai in platelet function

Physiological platelet activation and thrombus formation are essential to stop bleeding in case of vascular injury, whereas inadequate triggering of the same process in diseased vessels can lead to fatal thromboembolism and tissue ischemia of vital organs. A central step in platelet activation is agonist-induced elevation of the intracellular Ca(2+) concentration. This happens on the one hand through the release of Ca(2+) from intracellular stores and on the other hand through Ca(2+) influx from the extracellular space. In platelets, the major Ca(2+) influx pathway is the so-called store operated Ca(2+) entry (SOCE), induced by store depletion. Studies in the last five years discovered the molecular background of platelet SOCE. Stromal interaction molecule 1 (STIM1) and Orai1, two so far unknown molecules, got in the focus of research. STIM1 was found to be the Ca(2+) sensor in the endoplasmic reticulum (ER) membrane, whereas Orai1 was identified as the major store operated Ca(2+) (SOC) channel in the plasma membrane. These two molecules and their role in platelet function and thrombus formation are the topic of the present review with a special focus on apoptosis and apoptosis-like processes in platelet physiology.

p53 DNA Binding Cooperativity Is Essential for Apoptosis and Tumor Suppression In Vivo

Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.

Defective diacylglycerol-induced Ca2+ entry but normal agonist-induced activation responses in TRPC6-deficient mouse platelets.

Summary.u2002 Background:u2002Platelet adhesion, activation and aggregation at sites of vascular injury are essential processes for primary hemostasis. Elevation of the intracellular Ca2+ concentration is a central event in platelet activation but the underlying mechanisms are not fully understood. Store‐operated calcium entry (SOCE) through Orai1 was shown to be the main Ca2+ influx pathway in murine platelets, but there are additional non‐store‐operated Ca2+ (non‐SOC) and receptor operated Ca2+ (ROC) channels expressed in the platelet plasma membrane.Objective:u2002Canonical transient receptor potential (TRPC) channel 6 is found both in human and murine platelets and has been proposed to mediate diacylglycerol (DAG) activated ROCE but also a role in the regulation of SOCE has been suggested.Methods:u2002To investigate the function of TRPC6 in platelet Ca2+ signaling and activation, we analyzed platelets from mice deficient in TRPC6 using a wide range of in vitro and in vivo assays.Results:u2002In the mutant platelets, DAG activated Ca2+ influx was found to be abolished. However, this did not significantly affect SOCE or agonist induced Ca2+ responses. Platelet function in vitro and in vivo was also unaltered in the absence of TRPC6.Conclusion:u2002Our results indicate that DAG activated ROCE is mediated exclusively by TRPC6 in murine platelets, but this Ca2+ influx has no major functional relevance for hemostasis and thrombosis. Further, in contrast to previous suggestions, based on studies with human platelets, TRPC6 appears to play an insignificant role in the regulation of SOCE in murine platelets.