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Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1-interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow., (© 2019 by The American Society of Hematology.)

Zinc (Zn 2+ ) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn 2+ storage and release. To visualize Zn 2+ storage in human and mouse platelets, the Zn 2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d -/- mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn 2+ release upon activation. Platelets from Nbeal2 -/- mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn 2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn 2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2 -/- and Unc13d -/- mice, and the impairment could be partially restored by extracellular Zn 2+ . Altogether, we conclude that the release of ionic Zn 2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn 2+ in platelet-dependent fibrin formation.

Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development. Intravital microscopy showed that liver colonization by platelets depended primarily on Kupffer cells at early and late stages of NASH, involving hyaluronan-CD44 binding. APT reduced intrahepatic platelet accumulation and the frequency of platelet-immune cell interaction, thereby limiting hepatic immune cell trafficking. Consequently, intrahepatic cytokine and chemokine release, macrovesicular steatosis and liver damage were attenuated. Platelet cargo, platelet adhesion and platelet activation but not platelet aggregation were identified as pivotal for NASH and subsequent hepatocarcinogenesis. In particular, platelet-derived GPIbα proved critical for development of NASH and subsequent HCC, independent of its reported cognate ligands vWF, P-selectin or Mac-1, offering a potential target against NASH.

Background and Purpose- Acetylsalicylic acid and clopidogrel are the 2 main antithrombotic drugs for secondary prevention in patients with ischemic stroke (IS) without indication for anticoagulation. Because of their limited efficacy and potential side effects, novel antiplatelet agents are urgently needed. Cilostazol, a specific phosphodiesterase (PDE)-3 inhibitor, protected from IS in clinical studies comprising mainly Asian populations. Nevertheless, the detailed mechanistic role of PDE-3 inhibitors in IS pathophysiology is hardly understood. In this project, we analyzed the efficacy and pathophysiologic mechanisms of a novel and only recently described PDE-3 inhibitor (substance V) in a mouse model of focal cerebral ischemia. Methods- Focal cerebral ischemia was induced by transient middle cerebral artery occlusion in 6- to 8-week-old male C57Bl/6 wild-type mice receiving substance V or vehicle 1 hour after ischemia induction. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood-brain barrier damage, as well as the extent of local inflammatory response and cell death, was determined. Results- Inhibition of PDE-3 by pharmacological blockade with substance V significantly reduced infarct volumes and improved neurological outcome on day 1 and 7 after experimental cerebral ischemia. Reduced blood-brain barrier damage, attenuated brain tissue inflammation, and decreased local cell death could be identified as potential mechanisms. PDE-3 inhibitor treatment did neither increase the number of intracerebral hemorrhages nor affect platelet function. Conclusions- The novel PDE-3 inhibitor substance V protected mice from IS independent from platelet function. Pharmaceutical inactivation of PDE-3 might become a promising therapeutic approach to combat IS via inhibition of thromboinflammatory mechanisms and stabilization of the blood-brain barrier.

Platelets maintain hemostasis after injury, but also during inflammation. Recent studies have shown that platelets prevent inflammatory bleeding through (hem) immunoreceptor tyrosine-based activation motif-dependent mechanisms irrespective of aggregation during skin and lung inflammation. Although the exact mechanisms underlying this process remain unknown, it was speculated that mediators released from platelet granules might be involved. Maintaining cerebral hemostasis during stroke treatment is of high clinical relevance because hemorrhage may aggravate the disease state and increase mortality. Although it was shown that platelets help maintain hemostasis in the ischemic brain, their exact contribution remains ill defined. Here we show that Unc13d -/- / Nbeal2 -/- mice, which lack platelet α- and dense-granule secretion, show no signs of hemorrhage in models of skin or lung inflammation. In stark contrast, lack of platelet granule release resulted in impaired hemostasis in the ischemic brain after transient middle cerebral artery occlusion leading to increased intracranial hemorrhage and mortality. Our results reveal for the first time that platelet granule constituents are essential for maintenance of hemostasis during thrombo-inflammatory brain infarction but not experimental inflammation of the skin or lung, thereby uncovering vascular bed-specific differences in the prevention of inflammatory bleeding., (© 2017 by The American Society of Hematology.)

Platelets are essential for normal hemostasis; however, pathological conditions can also trigger unwanted platelet activation precipitating thrombosis and ischemic damage of vital organs such as the heart or brain. Glycoprotein (GP)VI- and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represents a major pathway for platelet activation. The two members of the Growth-factor receptor-bound protein 2 (Grb2) family of adapter proteins expressed in platelets – Grb2 and Grb2-related adapter protein downstream of Shc (Gads) – are part of the hem(ITAM) signaling cascade by forming an adapter protein complex with linker for activation of T cells (LAT). To date, a possible functional redundancy between these two adapters in platelet activation has not been investigated. We here generated megakaryocyte- and platelet-specific Grb2/Gads double knockout (DKO) mice and analyzed their platelet function in vitro and in vivo. The DKO platelets exhibited virtually abolished (hem)ITAM signaling whereas only partial defects were seen in Grb2 or Gads single-deficient platelets. This was based on impaired phosphorylation of key molecules in the (hem)ITAM signaling cascade and translated into impaired hemostasis and partially defective arterial thrombosis, thereby exceeding the defects in either Grb2 KO or Gads KO mice. Despite this severe (hem)ITAM signaling defect, CLEC-2 dependent regulation of blood-lymphatic vessel separation was not affected in the DKO animals. These results provide direct evidence for critically redundant roles of Grb2 and Gads for platelet function in hemostasis and thrombosis, but not development. [ABSTRACT FROM AUTHOR]

Myocardial ischemia is one of the leading health problems worldwide. Therapy consists of the restitution of coronary perfusion which is followed by myocardial inflammation. Platelet–neutrophil interaction is a crucial process during inflammation, yet its consequences are not fully understood. Here, we show that platelet–neutrophil complexes (PNCs) are increased in patients with acute myocardial infarction and that this is associated with increased levels of neuronal guidance protein semaphorin 7A (SEMA7A). To investigate this further, we injected WT animals with Sema7a and found increased infarct size with increased numbers of PNCs. Experiments in genetically modified animals identify Sema7a on red blood cells to be crucial for this condition. Further studies revealed that Sema7a interacts with the platelet receptor glycoprotein Ib (GPIb). Treatment with anti-Sema7a antibody protected from myocardial tissue injury. In summary, we show that Sema7a binds to platelet GPIb and enhances platelet thrombo-inflammatory activity, aggravating post-ischemic myocardial tissue injury. Reperfusion injury following myocardial ischemia is aggravated by inflammation and platelet–neutrophil complex formation. Here the authors show that semaphorin 7A binds to platelet GPIb, enhancing platelet–neutrophil interaction and increasing post-ischemic myocardial tissue injury, and that blockage of semaphorin 7A is protective. [ABSTRACT FROM AUTHOR]