Friedrich Reusswig

BACKGROUND: Red blood cells (RBCs) contribute to hemostasis and thrombosis by interacting with platelets via the FasL–FasR pathway to induce procoagulant activity and thrombin formation. Here, we identified a novel mechanism of platelet–RBC interaction via the CD36–TSP-1 (thrombospondin-1) signaling pathway that plays a prominent role in arterial thrombosis and abdominal aortic aneurysm (AAA) formation and progression. AAA is a life-threatening atherosclerotic-related disease, characterized by the progressive dilation of the abdominal aorta, due to chronic inflammation and extracellular matrix remodeling/degradation within the vessel wall. The objective of the present study was to elucidate a new mechanism of platelet–RBC interaction via the TSP1–CD36 axis and its significance for arterial thrombosis and the pathology of AAA. METHODS: TSP-1-deficient and CD36 cell-type-specific (RBCs and platelets) knock-out mice were analyzed in experimental mouse models of arterial thrombosis and AAA. Blood samples from patients with AAA from peripheral sites (laminar flow) and from inside the aneurysm segment (turbulent flow) were analyzed by flow cytometry and compared with age-matched controls. RESULTS: After platelet activation, platelet-released TSP-1 binds to CD36 at the RBC and platelet membrane to enhance procoagulant activity of both cells, leading to platelet aggregation and thrombosis. Patients with AAA exhibit enhanced procoagulant activity, elevated TSP-1 and CD36 plasma levels, as well as increased exposure of TSP-1 and CD36 at the RBC and platelet surface. In addition, biomechanically stress in the aneurysmal segment reinforces CD36 externalization on RBCs and platelets as well as the formation of platelet–RBC aggregates. In line, genetic deletion of either CD36 (RBC restricted) or TSP-1 protected mice against experimentally induced AAA formation. CONCLUSIONS: Our findings imply that CD36 on RBCs and platelets, as well as platelet-released TSP-1, contribute to procoagulant activity, playing a crucial role in arterial thrombosis and AAA progression.

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.

Liver failure and cirrhosis are characterized by abnormal hemostasis with aberrant platelet activation. In particular, the consequences of cholestatic liver disease and molecular mechanisms, including the role of bile acids leading to impaired platelet responses, are not well understood. Here, we demonstrate that bile acids inhibit human and murine platelet activation, adhesion and spreading, leading to reduced thrombus formation under flow conditions. We identified the G-protein coupled receptor TGR5 in platelets and provide support for its role as mediator of bile acid-induced impairment of platelet activation. In the liver, TGR5 couples to Gαs proteins, activates the adenylate cyclase to induce a transient cAMP rise and stimulates the MAPK signaling pathway to regulate cholangiocyte proliferation, hepatocyte survival and inflammation. In this report, we demonstrate that the genetic deficiency of TGR5 in mice led to enhanced platelet activation and thrombus formation, suggesting that TGR5 plays an important role in hemostasis. Mechanistically, platelet inhibition is achieved by TGR5 mediated PKA activation and modulation of AKT and ERK1/2 phosphorylation. Thus, this report provides evidence for the ability of TGR5 ligands to reduce platelet activation and identifies TGR5 agonism as a new target for the prevention of cardiovascular diseases.

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.

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.

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.

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.