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

Myocardial injury is associated with release of heme proteins such as myoglobin and liberation of free heme/hemin into the microvasculature. Enhanced levels of hemin are connected with intravascular thrombus formation and microcirculatory impairment. Conventional antiplatelet drugs (acetylsalicylic acid or P₂Y₁₂ inhibitors) are poor inhibitors of hemin-induced thrombus formation. Hemin and free iron react with hydrogen peroxide to generate highly oxidizing species capable of initiating lipid peroxidation and cell membrane destruction (ferroptosis). Lipid peroxidation is a hallmark of ferroptosis and directly destroys cellular membranes. The purpose of our study was to disclose mechanisms and functional consequences of platelet ferroptosis in response to free hemin.

We found that hemin reduces BODIPY 581/591 C11 fluorescence in a dose dependent manner (3.1 to 25 µM) indicating enhanced lipid peroxidation. Lipid peroxidation was paralleled by an increase in formation of reactive oxygen species (ROS), loss of mitochondrial membrane potential and increased phosphatidylserine presentation (Figure A). Multipanel flow cytometry (CD42b, PAC-1, AnnexinV) revealed enhanced formation of procoagulant and ferroptotic platelet subtypes (AnnexinV⁺) indicating substantial changes of the plasma membrane (Figure B). Especially in comparison to platelet activation through dual stimulation by CRP-XL (collagen-related peptide, 5 µg/ml) and Thrombin (0.1 U/ml), whereby the aggregatory subpopulation were predominant. Further, the formation of platelet microvesicles/ferroptotic bodies was significantly enhanced in response to hemin (Figure C). In addition, adhesion and spreading on fibrinogen-coated coverslips of hemin-treated platelets was significantly reduced compared to untreated platelets (Figure D). To disclose changes of platelet lipids in response to hemin, a mass spectrometry approach (UHPLC–ESI-QTOF-MS/MS) was applied.  After pre-processing and structural annotation of the lipids detected by MS, we could detect 13 oxylipins from isolated platelets. Compared to untreated controls, hemin induced primarily lipids associated with lipid peroxidation and ferroptosis (12-HHT, TXB2, 8-HETE, 11-HETE, 5-HETE, FA20:4, 15-HETE)(Figure E)     
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 (BODIPY), loss of mitochondrial membrane potential (TMRE), ROS production (H₂DCFDA) and phosphatidylserine presentation was substantially attenuated. Further, pretreatment of platelet with the furin inhibitor SSM3 prevents formation of aggregatory/procoagulant platelets and microvesicles/ferroptotic bodies as well as reduced platelet adhesion and spreading. The rescue of hemin-induced platelet destruction was associated with reduction of specific ferroptotic lipids (12-HHT, TXB2, 8-HETE, 11-HETE, 5-HETE, FA20:4, 15-HETE) in the presence of the furin inhibitor.

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 thrombus formation in areas of tissue destruction with enhanced release of heme-carrying proteins.