Markus Bender

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

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.

The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction

Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction and stroke. Agonist-induced elevation of [Ca2+]i is a central step in platelet activation, but the underlying mechanisms are not fully understood. A major pathway for Ca2+ entry in nonexcitable cells involves receptor-mediated release of intracellular Ca2+ stores, followed by activation of store-operated calcium (SOC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) has been identified as the Ca2+ sensor in the endoplasmic reticulum (ER) that activates Ca2+ release–activated channels in T cells, but its role in mammalian physiology is unknown. Platelets express high levels of STIM1, but its exact function has been elusive, because these cells lack a normal ER and Ca2+ is stored in a tubular system referred to as the sarcoplasmatic reticulum. We report that mice lacking STIM1 display early postnatal lethality and growth retardation. STIM1-deficient platelets have a marked defect in agonist-induced Ca2+ responses, and impaired activation and thrombus formation under flow in vitro. Importantly, mice with STIM1-deficient platelets are significantly protected from arterial thrombosis and ischemic brain infarction but have only a mild bleeding time prolongation. These results establish STIM1 as an important mediator in the pathogenesis of ischemic cardio- and cerebrovascular events.

Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke

Summary.  Platelet adhesion, activation and aggregation on the exposed subendothelial extracellular matrix (ECM) are essential for haemostasis, but may also lead to occlusion of diseased vessels. Binding of the glycoprotein (GP)Ib‐V‐IX complex to immobilised von Willebrand factor (VWF) initiates adhesion of flowing platelets to the ECM, and thereby enables the collagen receptor GPVI to interact with its ligand and to mediate platelet activation. This process is reinforced by locally produced thrombin and platelet‐derived secondary mediators, such as adenosine diphosphate (ADP) and thromboxane A2 (TxA2). Together, these events promote a shift of β1 and β3 integrins from a low to a high affinity state for their ligands through ‘inside‐out’ signalling allowing firm platelet adhesion and aggregation. Formed platelet aggregates are stabilised by fibrin formation and signalling events between adjacent platelets involving multiple platelet receptors, such as the newly discovered C‐type lectin‐like receptor 2 (CLEC‐2). While occlusive thrombus formation is the principal pathogenic event in myocardial infarction, the situation is more complex in ischaemic stroke where infarct development often progresses despite sustained early reperfusion of previously occluded major intracranial arteries, a process referred to as ‘reperfusion injury’. Increasing experimental evidence now suggests that early platelet adhesion and activation events, orchestrate a ‘thrombo‐inflammatory’ cascade in this setting, whereas platelet aggregation and thrombus formation are not required. This review summarises recent developments in understanding the principal platelet adhesion receptor systems with a focus on their involvement in arterial thrombosis and ischaemic stroke models.

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.

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of alpha granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling.

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.

Combined In Vivo Depletion of Glycoprotein VI and C-Type Lectin-Like Receptor 2 Severely Compromises Hemostasis and Abrogates Arterial Thrombosis in Mice

Objective—Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif–bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. Approach and Results—We investigated whether both (hem)immunoreceptor tyrosine activation motif–bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2–depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. Conclusions—These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti–CLEC-2–based agents in the prevention of thrombotic diseases.

Platelet GPVI: a target for antithrombotic therapy?!

Platelet activation is a key step in the pathogenesis of ischemic cardio- and cerebrovascular diseases, which represent the leading causes of death and severe disability worldwide. Although existing antiplatelet drugs have proved beneficial in the clinic, their use is limited by their inherent effect on primary hemostasis, making the identification of novel pharmacological targets for platelet inhibition an important goal of cardiovascular research. In recent years, the central activating platelet collagen receptor, glycoprotein (GP) VI, has emerged as a promising antithrombotic target because its blockade or antibody-mediated depletion in circulating platelets was shown to effectively inhibit experimental thrombosis and thromboinflammatory disease states, such as stroke, without affecting hemostatic plug formation. In this review, we summarize the most important recent developments in understanding of GPVI function in hemostasis and thrombotic/inflammatory diseases and discuss the potential use of anti-GPVI agents to treat these pathologies in humans.

Fibrin activates GPVI in human and mouse platelets

The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability.

Genetic and antibody-induced glycoprotein VI deficiency equally protects mice from mechanically and FeCl3-induced thrombosis

At sites of vascular injury, platelet adhesion, activation and aggregation occurs on exposed subendothelial collagens and contributes to hemostasis but also to occlusion of diseased vessels leading to a myocardial infarction or ischemic stroke. The activating platelet collagen receptor, glycoprotein (GP) VI [1], is a platelet-specific transmembrane type I receptor that non-covalently associates with the Fc receptor (FcR) c-chain which contains an immunoreceptor tyrosine-based activation motif (ITAM) [2]. Upon ligand-induced GPVI clustering, the ITAM becomes tyrosine phosphorylated and initiates a series of phosphorylation events finally resulting in cellular activation [3]. GPVI has been proposed as an attractive antithrombotic target, particularly because anti-GPVI antibodies have been shown to induce irreversible downregulation of the receptor through ectodomain shedding in circulating platelets in mice, resulting in long-term antithrombotic protection but overall normal tail bleeding times [4–7]. Different mouse models of GPVI-deficiency (FcR c-chain-deficient which also lack GPVI [8], GPVI-immunodepleted or Gp6 mice) have been used to determine the functional relevance of the receptor in thrombus formation in different experimental thrombosis models (laser-, mechanically and chemically induced injuries). AlthoughGPVI is generally regarded as a critical regulator of pathological thrombus formation [9], partially contradictory results have been reported on the relative importance of GPVI in some of these models. For example, GPVI was found to play no [10,11] or only a minor [12,13] role in models of laser-induced vascular injury where thrombus formation is suggested to be largely dependent on thrombin generation, although different results were reported by others [14]. Ferric(III)chloride (FeCl3)-induced vascular injury is a widely used model in thrombosis research because it allows variable levels of injury in different vascular beds and can be monitored by microscopic visualization or blood flow measurements. Previous studies yielded evidence for a role of GPVI in FeCl3-induced thrombus formation in mice [10,15,16], but this was questioned in a very recent study assessing the mechanisms underlying thrombus formation in that model in detail [17]. The authors demonstrated that in their model, FeCl3 diffuses through all vessel wall layers and severely injures the endothelium without damaging the internal elastic lamina (IEL), and only exposes the basement membrane to the vessel lumen. They further report that in their hands GPVI-immunodepleted mice are not protected from vessel occlusion after FeCl3-induced injury of mesenteric arterioles or carotid arteries by measuring thrombus area and time to occlusion. Based on these data, the authors state that FeCl3-induced vascular injury may not be an appropriate model to study the role of subendothelial adhesive proteins and their platelet receptors in thrombosis [17]. This conclusion, if correct, may have major implications for the interpretation of a large number of previous results obtained with this model and also significantly influence its acceptance as a valid experimental system to assess the mechanisms of arterial thrombosis. Therefore, here we assessed the role of GPVI-mediated platelet activation/ adhesion in FeCl3-induced thrombus formation systematically. To do so, we subjected control, Gp6 (details of the knockout generation will be published elsewhere) and GPVIimmunodepleted (100 lg JAQ1 i.p., analysis on day 6 post injection) mice to a model of FeCl3-injury of mesenteric arterioles and carotid arteries, and monitored thrombus formation by intravital fluorescence microscopy or blood flow measurements, respectively. In addition, thrombus formation in these mice was tested in a model of mechanical injury of the abdominal aorta. A drop of 20% FeCl3 was topically applied on small mesenteric arterioles of 4to 5–week-old mice and thrombus formation of fluorescently labeled platelets was monitored for 40 min or until complete occlusion of the vessel had occurred. Kinetics of platelet adhesion (data not shown) and the beginning of first thrombus formation were similar between control, Gp6 and GPVI-immunodepleted mice (Fig. 1A, Correspondence: Bernhard Nieswandt, University Hospital Wurzburg and Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, Josef-Schneider-Str. 2, 97080 Wurzburg, Germany. Tel.: +49 931 31 80405; fax: +49 931 201 61652. E-mail: bernhard.nieswandt@virchow.uni-wuerzburg.de