The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Platelet Ferroptosis

The porphyrin-derivative hemin is liberated in various pathophysiological situations such as intramyocardial haemorrhages occurring after reperfusion of ischemic areas following acute myocardial ischemia, intraplaque haemorrhages or intracerebral haemorrhages. In these situations, extravascular red blood cells undergo haemolysis leading to a disintegration of haemoglobin and a liberation of extracellular heme, which is further oxidized to free iron-containing hemin. Hemin is described to induce platelet activation and to trigger an iron-mediated cell death known as ferroptosis in platelets. Conventional antiplatelet therapy has failed to prevent hemin-induced platelet activation, highlighting the need for alternative pharmacological targets. The purpose of our study was to disclose mechanisms and functional consequences of platelet ferroptosis in response to free hemin.

We found that hemin induces lipid peroxidation, formation of reactive oxygen species (ROS), increased phosphatidylserine presentation and loss of mitochondrial membrane potential (TMRE). Multi-colour flow cytometry (CD42b, PAC-1, AnnexinV) revealed enhanced formation of aggregatory, procoagulant and ferroptotic platelet subtypes after hemin stimulation. To disclose changes of platelet lipids in response to free hemin, a mass spectrometry approach (Micro-UHPLC-MS/MS) was applied. Compared to untreated controls, hemin induced primarily lipids associated with ferroptosis and inflammation (TXB2, 5-HETE, 8-HETE, 11-HETE, 15-HETE). Lipid peroxidation and generation of oxylipins is connected with a destabilization of the plasma membrane. Therefore, we used scanning ion conductance microscopy (SICM) to evaluated platelet metamorphosis in response to hemin. We found that hemin stimulation results in a rupture of the plasma membrane leading to a vast generation of platelet-derived microvesicles. Membrane rupture is accompanied by degradation of cytoskeleton components indicated by a loss of F-actin. These hemin-induced microvesicles are aggregatory and inducing thrombus formation. We further validated and illustrated hemin-induced platelet ferroptosis by transmission electron microscopy highlighting platelet membrane destruction and microvesicle generation.

Recently, we found that the subtilisin-like protease furin hemin-specifically regulates platelet surface receptor shedding. Thus, we asked whether furin also modulates platelet ferroptosis and platelet destruction. In the presence of the specific furin inhibitor SSM3 (25 µM) hemin-dependent lipid peroxidation, loss of mitochondrial membrane potential, ROS production and phosphatidylserine presentation was substantially attenuated. Further, furin inhibition prevents formation of aggregatory, procoagulant and ferroptotic platelets. The rescue of hemin-induced platelet destruction by furin inhibition was associated with a reduction in the generation of the specific ferroptotic lipids as well as a decrease in the cytoskeleton degradation. Further generation of microvesicles was significantly reduced.

We conclude that the subtilisin-like protease furin is a critical regulator of platelet ferroptosis and might offer a therapeutic strategy to control hemin-dependent platelet activation and ferroptosis.