Red blood cells (RBCs) influence rheology, and release ADP, ATP, and nitric oxide, suggesting a role for RBCs in hemostasis and thrombosis. Here, we provide evidence for a significant contribution of RBCs to thrombus formation. Anemic mice showed enhanced occlusion times upon injury of the carotid artery. A small population of RBCs was located to platelet thrombi and enhanced platelet activation by a direct cell contact via the FasL/FasR (CD95) pathway known to induce apoptosis. Activation of platelets in the presence of RBCs led to platelet FasL exposure that activated FasR on RBCs responsible for externalization of phosphatidylserine (PS) on the RBC membrane. Inhibition or genetic deletion of either FasL or FasR resulted in reduced PS exposure of RBCs and platelets, decreased thrombin generation, and reduced thrombus formation in vitro and protection against arterial thrombosis in vivo. Direct cell contacts between platelets and RBCs via FasL/FasR were shown after ligation of the inferior vena cava (IVC) and in surgical specimens of patients after thrombectomy. In a flow restriction model of the IVC, reduced thrombus formation was observed in FasL-/- mice. Taken together, our data reveal a significant contribution of RBCs to thrombosis by the FasL/FasR pathway.

Objective: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GPIb. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GPVI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. Conclusions: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.