RESUMO
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).
RESUMO
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.