Influence of hemin on biophysical properties of membranes

Hemin released from erythrocytes activates platelets through both receptor-dependent and receptor-independent mechanisms. While lower hemin concentrations predominantly activate the immune receptor tyrosine-based activation motif (ITAM) signaling via glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), higher concentrations induce pronounced receptor-independent cellular alterations, including loss of mitochondrial membrane potential, generation of reactive oxygen species (ROS), lipid peroxidation, and plasma membrane fragmentation. However, the mechanism of receptor-independent effect of hemin is still unclear. To test whether hemin can directly interact with and remodel membranes, we employed giant unilamellar vesicles composed of different lipid compositions as model membrane systems and treated them with unlabeled and fluorescently-labeled hemin. We analyzed how hemin treatment alters membrane biophysical properties using fluorescence microscopy, spectral imaging, fluorescence correlation spectroscopy (FCS), and flow cytometry. We found that fluorophore-labeled hemin can be directly visualized in lipid bilayers. Furthermore, high concentrations of hemin increased membrane order in all lipid compositions tested. The strongest effect was observed in more fluid membranes, whereas highly ordered membranes were less affected. Interestingly, in cholesterol-containing membranes, even the lowest hemin concentration produced changes comparable to those induced by the highest concentration, suggesting that cholesterol enhances the influence of hemin on lipid bilayers. Finally, we observed strong increase in permeability of membranes upon hemin treatment. Overall, our findings demonstrate that hemin can directly accumulate in lipid membranes, increase membrane order, reduce membrane fluidity and alter membrane integrity, resulting in enhanced membrane permeability.