Red blood cells mediate the onset of thrombosis in the ferric chloride murine model

Application of ferric chloride (FeCl3) to exposed blood vessels is widely used to initiate thrombosis in laboratory mice. Because the mechanisms by which FeCl3 induces endothelial injury and subsequent thrombus formation are little understood, we used scanning electron and brightfield intravital microscopy to visualize endothelial damage and thrombus formation occurring in situ. Contrary to generally accepted belief, FeCl3 does not result in appreciable subendothelial exposure within the time frame of thrombosis. Furthermore, the first cells to adhere to FeCl3-treated endothelial surfaces are red blood cells (RBCs) rather than platelets. Energy dispersive x-ray spectroscopy demonstrated that ferric ions predominantly localize to endothelial-associated RBCs and RBC-derived structures rather than to the endothelium. With continuing time points, RBC-derived structures rapidly recruit platelets, resulting in large complexes that subsequently enlarge and coalesce, quickly covering the endothelial surface. Further studies demonstrated that neither von Willebrand factor nor platelet glycoprotein Ib-alpha receptor (GPIb-alpha) is required for RBCs to adhere to the endothelium, and that deficiency of GPIb-alpha greatly abrogated the recruitment of platelets to the endothelial-associated RBC material. These findings illuminate the mechanisms of FeCl3-mediated thrombosis and reveal a previously unrecognized ability of RBCs to participate in thrombosis by mediating platelet adhesion to the intact endothelial surface.