Platelets Induce Cell Apoptosis of Cardiac Cells via FasL after Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Cell apoptosis in the myocardium plays an important role in ischemia and reperfusion (I/R) injury, leading to cardiac damage and dysfunction. Platelets are major players in hemostasis and play a crucial role in vessel occlusion, inflammation, and cardiac remodeling after I/R. Here, we studied the impact of platelets on cell apoptosis in the myocardium using a close-chest mouse model of AMI. We found caspase-3-positive resident cardiac cells, while leukocytes were negative for caspase-3. Using two different mouse models of thrombocytopenia, we detected a significant reduction in caspase-3 positive cells in the infarct border zone after I/R injury. Further, we identified platelet FasL to induce cell apoptosis via the extrinsic pathway of Fas receptor activation of target cells. Mechanistically, hypoxia triggers platelet adhesion to FasR, suggesting that platelet-induced apoptosis is elevated after I/R. Platelet-specific FasL knock-out mice showed reduced Bax and Bcl2 expression, suggesting that platelets modulate the intrinsic and extrinsic pathways of apoptosis, leading to reduced infarct size after myocardial I/R injury. Thus, a new mechanism for how platelets contribute to tissue homeostasis after AMI was identified that should be validated in patients soon.

PACSIN2 regulates platelet integrin ß1 hemostatic function.

BACKGROUND: Upon vessel injury, platelets adhere to exposed matrix constituents via specific membrane receptors, including the von Willebrand factor receptor glycoprotein (GP)Ib-IX-V complex and integrins ß1 and ß3. In platelets, the Fes/CIP4-homology Bin-Amphiphysin-Rvs protein PACSIN2 associates with the cytoskeletal and scaffolding protein filamin A (FlnA), linking GPIbα and integrins to the cytoskeleton. OBJECTIVES: Here we investigated the role of PACSIN2 in platelet function. METHODS: Platelet parameters were evaluated in mice lacking PACSIN2 and platelet integrin ß1. RESULTS: Pacsin2-/- mice displayed mild thrombocytopenia, prolonged bleeding time, and delayed thrombus formation in a ferric chloride-mediated carotid artery injury model, which was normalized by injection of control platelets. Pacsin2-/- platelets formed unstable thrombi that embolized abruptly in a laser-induced cremaster muscle injury model. Pacsin2-/- platelets had hyperactive integrin ß1, as evidenced by increased spreading onto surfaces coated with the collagen receptor α2ß1-specific peptide GFOGER and increased binding of the antibody 9EG7 directed against active integrin ß1. By contrast, Pacsin2-/- platelets had normal integrin αIIbß3 function and expressed P-selectin normally following stimulation through the collagen receptor GPVI or with thrombin. Deletion of platelet integrin ß1 in Pacsin2-/- mice normalized platelet count, hemostasis, and thrombus formation. A PACSIN2 peptide mimicking the FlnA-binding site mediated the pull-down of a FlnA rod 2 construct by integrin ß7, a model for integrin ß-subunits. CONCLUSIONS: Pacsin2-/- mice displayed severe thrombus formation defects due to hyperactive platelet integrin ß1. The data suggest that PACSIN2 binding to FlnA negatively regulates platelet integrin ß1 hemostatic function.

The NMDA receptor regulates integrin activation, ATP release and arterial thrombosis through store-operated Ca2+ entry in platelets.

Introduction: Platelet activation and thrombus formation is crucial for hemostasis, but also trigger arterial thrombosis. Calcium mobilization plays an important role in platelet activation, because many cellular processes depend on the level of intracellular Ca2+ ([Ca2+](i)), such as integrin activation, degranulation, cytoskeletal reorganization. Different modulators of Ca2+ signaling have been implied, such as STIM1, Orai1, CyPA, SGK1, etc. Also, the N-methyl-D-aspartate receptor (NMDAR) was identified to contribute to Ca2+ signaling in platelets. However, the role of the NMDAR in thrombus formation is not well defined. Methods: In vitro and in vivo analysis of platelet-specific NMDAR knock-out mice. Results: In this study, we analyzed Grin1fl/fl-Pf4-Cre+ mice with a platelet-specific knock-out of the essential GluN1 subunit of the NMDAR. We found reduced store-operated Ca2+ entry (SOCE), but unaltered store release in GluN1-deficient platelets. Defective SOCE resulted in reduced Src and PKC substrate phosphorylation following stimulation of glycoprotein (GP)VI or the thrombin receptor PAR4 followed by decreased integrin activation but unaltered degranulation. Consequently, thrombus formation on collagen under flow conditions was reduced ex vivo, and Grin1fl/fl-Pf4-Cre+ mice were protected against arterial thrombosis. Results from human platelets treated with the NMDAR antagonist MK-801 revealed a crucial role of the NMDAR in integrin activation and Ca2+ homeostasis in human platelets as well. Conclusion: NMDAR signaling is important for SOCE in platelets and contributes to platelet activation and arterial thrombosis. Thus, the NMDAR represents a novel target for anti-platelet therapy in cardiovascular disease (CVD).

Constitutive Activation of gp130 in T Cells Results in Senescence and Premature Aging.

IL-6 family members contribute to host defense through the stimulation of acute-phase signaling, hematopoiesis, immune reactions, and regenerative processes. To investigate essential mechanisms that are linked toward a constitutively activated gp130 signaling, we generated and characterized a mouse model that reflects a constitutive and cytokine-independent activation of JAK/STAT3 signaling by Lgp130 in CD4- and CD8-positive T cells. Lgp130 is an engineered form of gp130 in which dimerization and activation are forced by a leucine zipper. T cell-specific Lgp130 activation resulted in massive phenotypical abnormalities, including splenomegaly, lymphadenopathy, and an upregulation of innate immune system components shown by hyperinflammatory signatures in several organs. Moreover, T cell-restricted expression of Lgp130 resulted in increased numbers of cytotoxic and regulatory T cells, especially in lymph nodes. Consistent with this, we found an elevated platelet production and increase in megakaryocytes in the spleen and bone marrow that are causative for an acute thrombocytosis accompanied by anemia. Due to a shortened life span of T cell-specific Lgp130 mice, we could also show that next to an overall increase in regulatory cell-cycle genes, an activation of p53 and increased expression of p21 provide evidence for a senescence-like phenotype. Together, these data suggest that T cell-restricted gp130 activation is not only involved in autoimmune processes but also in senescence-associated aging. Therefore, Lgp130 expression in T cells might be a suitable model to study inflammation and disease.

Platelet pannexin-1 channels modulate neutrophil activation and migration but not the progression of abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a common disease and highly lethal if untreated. The progressive dilatation of the abdominal aorta is accompanied by degradation and remodeling of the vessel wall due to chronic inflammation. Pannexins represent anion-selective channels and play a crucial role in non-vesicular ATP release to amplify paracrine signaling in cells. Thus, pannexins are involved in many (patho-) physiological processes. Recently, Panx1 channels were identified to be significantly involved in abdominal aortic aneurysm formation through endothelial derived Panx1 regulated inflammation and aortic remodeling. In platelets, Panx1 becomes activated following activation of glycoprotein (GP) VI. Since platelets play a role in cardiovascular diseases including abdominal aortic aneurysm, we analyzed the contribution of platelet Panx1 in the progression of abdominal aortic aneurysm. We detected enhanced Panx1 plasma levels in abdominal aortic aneurysm patients. In experimental abdominal aortic aneurysm using the pancreatic porcine elastase (PPE) mouse model, a major contribution of platelet Panx1 channels in platelet activation, pro-coagulant activity of platelets and platelet-mediated inflammation has been detected. In detail, platelets are important for the migration of neutrophils into the aortic wall induced by direct cell interaction and by activation of endothelial cells. Decreased platelet activation and inflammation did not affect ECM remodeling or wall thickness in platelet-specific Panx1 knock-out mice following PPE surgery. Thus, aortic diameter expansion at different time points after elastase infusion of the aortic wall was unaltered in platelet-specific Panx1 deficient mice suggesting that the modulation of inflammation alone does not affect abdominal aortic aneurysm formation and progression. In conclusion, our data strongly supports the role of platelets in inflammatory responses in abdominal aortic aneurysm via Panx1 channels and adds important knowledge about the significance of platelets in abdominal aortic aneurysm pathology important for the establishment of an anti-platelet therapy for abdominal aortic aneurysm patients.

Platelets modulate cardiac remodeling via the collagen receptor GPVI after acute myocardial infarction.

Introduction: Platelets play an important role in cardiovascular diseases. After acute myocardial infarction, platelets display enhanced activation and migrate into the infarct zone. Furthermore, platelets trigger acute inflammation and cardiac remodeling leading to alterations in scar formation and cardiac function as observed in thrombocytopenic mice. GPVI is the major collagen receptor in platelets and important for platelet activation and thrombus formation and stability. Antibody induced deletion of GPVI at the platelet surface or treatment of mice with recombinant GPVI-Fc results in reduced inflammation and decreased infarct size in a mouse model of AMI. However, the role of GPVI has not been fully clarified to date. Methods/Results: In this study, we found that GPVI is not involved in the inflammatory response in experimental AMI using GPVI deficient mice that were analyzed in a closed-chest model. However, reduced platelet activation in response to GPVI and PAR4 receptor stimulation resulted in reduced pro-coagulant activity leading to improved cardiac remodeling. In detail, GPVI deficiency in mice led to reduced TGF-ß plasma levels and decreased expression of genes involved in cardiac remodeling such as Col1a1, Col3a1, periostin and Cthrc1 7 days post AMI. Consequently, collagen quality of the scar shifted to more tight and less fine collagen leading to improved scar formation and cardiac function in GPVI deficient mice at 21d post AMI. Conclusion: Taken together, this study identifies GPVI as a major regulator of platelet-induced cardiac remodeling and supports the potential relevance of GPVI as therapeutic target to reduce ischemia reperfusion injury and to improve cardiac healing.

Only Acute but Not Chronic Thrombocytopenia Protects Mice against Left Ventricular Dysfunction after Acute Myocardial Infarction.

BACKGROUND: Platelets are major players of thrombosis and inflammation after acute myocardial infarction (AMI). The impact of thrombocytopenia on platelet-induced cellular processes post AMI is not well defined. METHODS: The left anterior descending artery was ligated in C57/Bl6 mice and in two thrombocytopenic mouse models to induce AMI. RESULTS: Platelets from STEMI patients and from C57/Bl6 mice displayed enhanced platelet activation after AMI. This allows platelets to migrate into the infarct but not into the remote zone of the left ventricle. Acute thrombocytopenia by antibody-induced platelet depletion resulted in reduced infarct size and improved cardiac function 24 h and 21 days post AMI. This was due to reduced platelet-mediated inflammation after 24 h and reduced scar formation after 21 days post AMI. The collagen composition and interstitial collagen content in the left ventricle were altered due to platelet interaction with cardiac fibroblasts. Acute inflammation was also significantly reduced in Mpl-/- mice with chronic thrombocytopenia, but cardiac remodeling was unaltered. Consequently, left ventricular function, infarct size and scar formation in Mpl-/- mice were comparable to controls. CONCLUSION: This study discovers a novel role for platelets in cardiac remodeling and reveals that acute but not chronic thrombocytopenia protects left ventricular function post AMI.

The Proteoglycan Biglycan Modulates Platelet Adhesion and Thrombus Formation in a GPVI-Dependent Manner.

BACKGROUND: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. METHODS: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. RESULTS: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. CONCLUSIONS: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

Genetic Labeling of Cells Allows Identification and Tracking of Transgenic Platelets in Mice.

BACKGROUND: The use of knock-out mouse models is crucial to understand platelet activation and aggregation. METHODS: Analysis of the global double fluorescent Cre reporter mouse mT/mG that has been crossbred with the megakaryocyte/platelet specific PF4-Cre mouse. RESULTS: Platelets show bright mT (PF4-Cre negative) and mG (PF4-Cre positive) fluorescence. However, a small proportion of leukocytes was positive for mG fluorescence in PF4-Cre positive mice. In mT/mG;PF4-Cre mice, platelets, and megakaryocytes can be tracked by their specific fluorescence in blood smear, hematopoietic organs and upon thrombus formation. No differences in platelet activation and thrombus formation was observed between mT/mG;PF4-Cre positive and negative mice. Furthermore, hemostasis and in vivo thrombus formation was comparable between genotypes as analyzed by intravital microscopy. Transplantation studies revealed that bone marrow of mT/mG;PF4-Cre mice can be transferred to C57BL/6 mice. CONCLUSIONS: The mT/mG Cre reporter mouse is an appropriate model for real-time visualization of platelets, the analysis of cell morphology and the identification of non-recombined platelets. Thus, mT/mG;PF4-Cre mice are important for the analysis of platelet-specific knockout mice. However, a small proportion of leukocytes exhibit mG fluorescence. Therefore, the analysis of platelets beyond hemostasis and thrombosis should be critically evaluated when recombination of immune cells is increased.

Efficiently Restored Thrombopoietin Production by Ashwell-Morell Receptor and IL-6R Induced Janus Kinase 2/Signal Transducer and Activator of Transcription Signaling Early After Partial Hepatectomy.

BACKGROUND AND AIMS: Thrombocytopenia has been described in most patients with acute and chronic liver failure. Decreased platelet production and decreased half-life of platelets might be a consequence of low levels of thrombopoietin (TPO) in these patients. Platelet production is tightly regulated to avoid bleeding complications after vessel injury and can be enhanced under elevated platelet destruction as observed in liver disease. Thrombopoietin (TPO) is the primary regulator of platelet biogenesis and supports proliferation and differentiation of megakaryocytes. APPROACH AND RESULTS: Recent work provided evidence for the control of TPO mRNA expression in liver and bone marrow (BM) by scanning circulating platelets. The Ashwell-Morell receptor (AMR) was identified to bind desialylated platelets to regulate hepatic thrombopoietin (TPO) production by Janus kinase (JAK2)/signal transducer and activator of transcription (STAT3) activation. Two-thirds partial hepatectomy (PHx) was performed in mice. Platelet activation and clearance by AMR/JAK2/STAT3 signaling and TPO production were analyzed at different time points after PHx. Here, we demonstrate that PHx in mice led to thrombocytopenia and platelet activation defects leading to bleeding complications, but unaltered arterial thrombosis, in these mice. Platelet counts were rapidly restored by up-regulation and crosstalk of the AMR and the IL-6 receptor (IL-6R) to induce JAK2-STAT3-TPO activation in the liver, accompanied by an increased number of megakaryocytes in spleen and BM before liver was completely regenerated. CONCLUSIONS: The AMR/IL-6R-STAT3-TPO signaling pathway is an acute-phase response to liver injury to reconstitute hemostasis. Bleeding complications were attributable to thrombocytopenia and platelet defects induced by elevated PGI2 , NO, and bile acid plasma levels early after PHx that might also be causative for the high mortality in patients with liver disease.

Molecular Drivers of Platelet Activation: Unraveling Novel Targets for Anti-Thrombotic and Anti-Thrombo-Inflammatory Therapy.

Cardiovascular diseases (CVDs) are the leading cause of death globally-partly a consequence of increased population size and ageing-and are major contributors to reduced quality of life. Platelets play a major role in hemostasis and thrombosis. While platelet activation and aggregation are essential for hemostasis at sites of vascular injury, uncontrolled platelet activation leads to pathological thrombus formation and provokes thrombosis leading to myocardial infarction or stroke. Platelet activation and thrombus formation is a multistage process with different signaling pathways involved to trigger platelet shape change, integrin activation, stable platelet adhesion, aggregation, and degranulation. Apart from thrombotic events, thrombo-inflammation contributes to organ damage and dysfunction in CVDs and is mediated by platelets and inflammatory cells. Therefore, in the past, many efforts have been made to investigate specific signaling pathways in platelets to identify innovative and promising approaches for novel antithrombotic and anti-thrombo-inflammatory strategies that do not interfere with hemostasis. In this review, we focus on some of the most recent data reported on different platelet receptors, including GPIb-vWF interactions, GPVI activation, platelet chemokine receptors, regulation of integrin signaling, and channel homeostasis of NMDAR and PANX1.

Phospholipase D1 regulation of TNF-alpha protects against responses to LPS.

Sepsis is a systemic inflammatory disorder with organ dysfunction and represents the leading cause of mortality in non-coronary intensive care units. A key player in septic shock is Tumor Necrosis Factor-alpha (TNF-α). Phospholipase (PL)D1 is involved in the regulation of TNF-α upon ischemia/reperfusion injury in mice. In this study we analyzed the impact of PLD1 in the regulation of TNF-α, inflammation and organ damage in experimental sepsis. PLD1 deficiency increased survival of mice and decreased vital organ damage after LPS injections. Decreased TNF-α plasma levels and reduced migration of leukocytes and platelets into lungs was associated with reduced apoptosis in lung and liver tissue of PLD1 deficient mice. PLD1 deficient platelets contribute to preserved outcome after LPS-induced sepsis because platelets exhibit an integrin activation defect suggesting reduced platelet activation in PLD1 deficient mice. Furthermore, reduced thrombin generation of PLD1 deficient platelets might be responsible for reduced fibrin formation in lungs suggesting reduced disseminated intravascular coagulation (DIC). The analysis of Pld1fl/fl-PF4-Cre mice revealed that migration of neutrophils and cell apoptosis in septic animals is not due to platelet-mediated processes. The present study has identified PLD1 as a regulator of innate immunity that may be a new target to modulate sepsis.

Defective Platelet Activation and Bleeding Complications upon Cholestasis in Mice.

BACKGROUND/AIMS: Platelets are essential mediators of hemostasis to avoid excessive blood loss. Cirrhosis and chronic liver diseases are characterized by alterations in hemostasis. Alterations in the secondary hemostasis have been well studied, while defects in primary hemostasis, especially the consequences of cholestatic liver disease on platelet function are not well defined. METHODS: After bile duct ligation (BDL) platelet activation and thrombus formation were analyzed in mice. RESULTS: BDL in mice had a moderate effect on platelet counts; however, intrinsic platelet activation was strongly reduced upon activation of the collagen receptor GPVI at early time points. 7 days after bile duct ligation, platelets displayed an almost complete loss of activation with reduced agonist-triggered release of alpha and dense granules and expression of integrin αIIbß3 on the platelet surface. This activation defects resulted in strongly reduced thrombus formation under flow, reduced platelet adhesion to fibrinogen and bleeding complications in BDL mice as measured by tail bleeding experiments. Mechanistically, elevated nitric oxide and prostacyclin levels induced phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), an established inhibitor of platelet activation. Furthermore increased tissue plasminogen activator in plasma of BDL mice led to enhanced plasmin levels that might be responsible for reduced glycoprotein expression of BDL platelets. Besides, high amounts of bile acids contribute to defective signal transduction as shown in platelets from mice fed with a cholic acid diet. CONCLUSIONS: Cholestatic liver disease induces multiple platelet activation defects and impairs thrombus formation responsible for bleeding complications at least in mice.