Background: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. Methods: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. Results: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer , the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y ₁₂ (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in Dicer ^{Pf4∆/Pf4∆} mice. Conclusions: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Platelets play a significant role in atherothrombosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is critically involved in the regulation of LDL metabolism and interacts with platelet function. The effect of PCSK9 in platelet function is poorly understood. The authors of this article sought to characterize platelets as a major source of PCSK9 and PCSK9’s role in atherothrombosis. In a large cohort of patients with coronary artery disease (CAD), platelet count, platelet reactivity, and platelet-derived PCSK9 release were analyzed. The role of platelet PCSK9 on platelet and monocyte function was investigated in vitro. Platelet count and hyper-reactivity correlated with plasma LDL in CAD. The circulating platelets express on their surface and release substantial amounts of PCSK9. Release of PCSK9 augmented platelet-dependent thrombosis, monocyte migration, and differentiation into macrophages/foam cells. Platelets and PCSK9 accumulated in tissue derived from atherosclerotic carotid arteries in areas of macrophages. PCSK9 inhibition reduced platelet activation and platelet-dependent thrombo-inflammation. The authors identified platelets as a source of PCSK9 in CAD, which may have an impact on LDL metabolism. Furthermore, platelet-derived PCSK9 contributes to atherothrombosis, and inhibition of PCSK9 attenuates thrombo-inflammation, which may contribute to the reported beneficial clinical effects.

During thrombopoiesis megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca²⁺-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1^-/-) developed a significant macrothrombocytopenia as compared to wildtype mice (Pdk1^fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulted in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1^-/- BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, while the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase (LIMK) and cofilin supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1^-/-BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1^-/-BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation and thrombopoiesis. Key Points PDK1 is critically required for megakaryocyte F-actin cytoskeleton dynamics during MK maturation, podosome formation and DMS polarization. PDK1-dependent signaling in MKs during proplatelet formation involves the PAK/LIMK/cofilin phosphorylation cascade and atypical PKCζ.

During megakaryopoiesis, megakaryocytes (MKs) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here, we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and de novo lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation, resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the effect of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.