Abstract
Microhemorrhages are an underestimated aspect in the pathophysiology of vulnerable plaques and aneurysms. Erythrocyte liberation within hemorrhages leads to extracellular hemoglobin accumulation and iron-containing hemin generation. Hemin induces platelet activation, thrombosis, and ferroptosis-mediated destruction of platelet membranes through GPVI/CLEC-2 signaling. Hemin-toxicity results in destruction of platelet membranes, which is caused by ferroptosis, a non-apoptotic cell death. Antiplatelet drugs have limited effect on hemin-induced activation. We evaluated the effect of hemin on platelet function using light transmission aggregometry and multipanel flow cytometry. We found that P2Y(12) and COX-1 inhibition attenuates hemin-induced aggregation only at low hemin concentrations (3.1/6.25 μM), whereas at higher concentrations (12.5/25 μM) no substantial inhibition was found. High hemin concentrations enhance phosphatidylserine exposure, procoagulant and microvesicle formation as well as ferroptosis, which was not attenuated in the presence of Src-inhibitors, indicating that membrane-disintegration is not primarily mediated via GPVI/CLEC-2 receptor-dependent ITAM-signaling. In contrast, iron chelation by deferoxamine significantly reduced microvesicle and ROS generation, loss of mitochondrial membrane potential and lipid peroxidation. Soluble recombinant Fc-GPVI scavenging of hemin protects against hemin-induced platelet activation, plasma membrane disintegration and microvesicle formation. High hemin concentrations lead to plasma membrane disintegration and ferroptosis, inhibited by iron chelation and hemin scavenging via soluble Fc-GPVI, but not by GPVI/CLEC-2 receptor-mediated ITAM signaling. We speculate that iron overload enables receptor-independent ferroptosis induction by hemin and may represent a therapeutic target to prevent platelet-driven thrombosis in microhemorrhages.