David Stegner

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.

Platelet receptor signaling in thrombus formation

Platelet activation and subsequent thrombus formation at sites of vascular injury is crucial for normal hemostasis, but it can also cause myocardial infarction and stroke. The initial capture of flowing platelets to the injured vessel wall is mediated by the interaction of the glycoprotein (GP) Ib-V-IX complex with von Willebrand factor immobilized on the exposed subendothelial extracellular matrix. Tethered platelets are then able to bind to collagens through the immunoglobulin-like receptor GPVI and to initiate cellular activation, a process that is reinforced by G protein-coupled receptors stimulated by locally produced thrombin and soluble mediators released from activated platelets. These signaling events lead to a rise in the cytosolic Ca2+ concentration, rearrangement of the cytoskeleton, release of granule content, and functional upregulation of integrin adhesion receptors allowing firm adhesion and thrombus growth. Fully activated platelets also undergo a procoagulant conversion thereby facilitating coagulation and thrombus stabilization. This review summarizes the most important receptor systems and signaling mechanisms involved in platelet activation and thrombus formation with special focus on recent discoveries.

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.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.

Differentially regulated GPVI ectodomain shedding by multiple platelet–expressed proteinases

Glycoprotein VI (GPVI) mediates platelet activation on exposed subendothelial collagens at sites of vascular injury and thereby contributes to normal hemostasis, but also to the occlusion of diseased vessels in the setting of myocardial infarction or stroke. GPVI is an attractive target for antithrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and/or ectodomain shedding. Metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family have been proposed to mediate this ectodomain shedding, but direct evidence for this is lacking. Here, we studied GPVI shedding in vitro and in vivo in newly generated mice with a megakaryocyte-specific ADAM10 deficiency and in Adam17(ex/ex) mice, which lack functional ADAM17. We demonstrate that GPVI cleavage in vitro can occur independently through either ADAM10 or ADAM17 in response to distinct stimuli. In contrast, antibody (JAQ1)-induced GPVI shedding in vivo occurred in mice lacking both ADAM10/ADAM17 in their platelets, suggesting the existence of a third GPVI cleaving platelet enzyme. This was supported by in vitro studies on ADAM10/ADAM17 double-deficient platelets. These results reveal that ectodomain shedding of GPVI can be mediated through multiple differentially regulated platelet-expressed proteinases with obvious therapeutic implications.

Endothelial TWIK-related potassium channel-1 (TREK1) regulates immune-cell trafficking into the CNS

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal basement membrane ensheathed by pericytes, smooth muscle cells and a layer of astrocyte end feet. Circulating blood cells, such as leukocytes, complete the NVU. BBB disruption is common in several neurological diseases, but the molecular mechanisms involved remain largely unknown. We analyzed the role of TWIK-related potassium channel-1 (TREK1, encoded by KCNK2) in human and mouse endothelial cells and the BBB. TREK1 was downregulated in endothelial cells by treatment with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Blocking TREK1 increased leukocyte transmigration, whereas TREK1 activation had the opposite effect. We identified altered mitogen-activated protein (MAP) kinase signaling, actin remodeling and upregulation of cellular adhesion molecules as potential mechanisms of increased migration in TREK1-deficient (Kcnk2−/−) cells. In Kcnk2−/− mice, brain endothelial cells showed an upregulation of the cellular adhesion molecules ICAM1, VCAM1 and PECAM1 and facilitated leukocyte trafficking into the CNS. Following the induction of experimental autoimmune encephalomyelitis (EAE) by immunization with a myelin oligodendrocyte protein (MOG)35–55 peptide, Kcnk2−/− mice showed higher EAE severity scores that were accompanied by increased cellular infiltrates in the central nervous system (CNS). The severity of EAE was attenuated in mice given the amyotrophic lateral sclerosis drug riluzole or fed a diet enriched with linseed oil (which contains the TREK-1 activating omega-3 fatty acid α-linolenic acid). These beneficial effects were reduced in Kcnk2−/− mice, suggesting TREK-1 activating compounds may be used therapeutically to treat diseases related to BBB dysfunction.

Mechanistic explanation for platelet contribution to cancer metastasis

Cancer-associated mortality is frequently caused by metastasis, however, our understanding of this process remains incomplete and therapeutic options are limited. Metastasis is a dynamic multi-step process involving intravasation of tumor cells into the hosts blood and lymphatic vessels, their dissemination within the circulation, and finally arrest and extravasation in a distant organ where they establish secondary tumors. It is generally conceived that platelets contribute to all steps of hematogenous tumor dissemination. In this review, we provide an overview of the current knowledge of the platelet receptors involved in tumor cell-induced platelet aggregation, an essential immune surveillance escape mechanism of circulating tumor cells. We discuss how platelets prevent immunological attack, contribute to tumor cell extravasation and thereby facilitate colonization of distant organs.