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.

Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron‐containing hemin. Hemin has been shown to activate platelets and induce thrombo‐inflammation. Classical antiplatelet therapy failed to prevent hemin‐induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin‐induced platelet death (ferroptosis). We evaluated the in vitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, ex vivo thrombus formation, multiparametric flow cytometry, micro‐UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet‐derived microvesicles. Interestingly, hemin‐dependent platelet death (ferroptosis) is specifically regulated by the subtilisin‐like proprotein convertase furin. In summary, platelet undergo a non‐apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin‐induced thrombosis and thrombo‐inflammation at a site of hemolysis.

Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.