Neither DTNB nor RL90 experienced an effect on baseline PS exposure or the known exteriorization of PS by ATP (40), even though PS staining appeared to be more apical in DNTB-treated versus ATP-stimulated cells (Physique ?(Physique2F),2F), presumably reflecting differences in cell morphology. activities consistent with cellular activation typically induced by P2X7 receptor signaling. This anti-PDI antibody restored TF-dependent thrombosis in mice. These data suggest that PDI regulates a critical P2X7 receptorCdependent signaling pathway that generates prothrombotic TF, defining a link between inflammation and thrombosis with potential implications for antithrombotic therapy. Introduction In the current view of thrombus formation, exposure of tissue factor (TF) in the hurt vessel wall is a crucial event that initiates thrombosis in high-flow vessels, such as the carotid artery (1, 2). In vitro studies demonstrate that TF procoagulant activity is usually tightly regulated, and TF is usually switched from a cryptic, nonactive state to a decrypted, active state by a number of cellular agonists or manipulations (3), but the mechanisms of TF decryption and the relevance of these in vitro findings for thrombosis remain a matter of ongoing argument (4C10). The cell surface TF procoagulant activity is usually regulated by chaperones, subcellular localization, and possibly dimerization and glycosylation (11C16) and is enhanced by the exposure of procoagulant phosphatidylserine (PS) (3). However, PS exposure alone cannot explain the decryption of TF in all cellular models (8, 9, 17). Protein disulfide isomeraseCdependent (PDI-dependent) redox regulation of TFs extracellular allosteric Cys186-Cys209 disulfide bond was proposed as an alternative mechanism that directly alters macromolecular substrate acknowledgement required for coagulation, while preserving the cell signaling function of TF (7, 8). PDI is NADP an oxidoreductase localized mainly in the endoplasmic reticulum (ER), but around the cell surface, PDI is crucial NADP for protein S-nitrosylation and nitric oxide uptake (18, 19). PDI may influence TF procoagulant function through thiol- and nitric oxideCdependent mechanisms, since TF is usually susceptible to S-nitrosylation and glutathionation (7, 20, 21). Antibody inhibition NADP of PDI has antithrombotic effects in mice (21, 22), and it was proposed that PDI released from hurt cells activates cryptic TF on microparticles (MPs) (21). Myeloid cells represent a source for circulating MPs, and the generation and targeting of these MPs to thrombi is dependent on the conversation between P-selectin and P-selectin glycoprotein ligandC1 (PSGL-1) (23C27). Although activation of purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7 receptor; encoded by in mice), by ATP has been shown to release TF+ MPs from dendritic cells of potential relevance for immune responses or sepsis (28), the mechanisms that generate prothrombotic TF+ MPs are poorly comprehended. The P2X7 receptor is usually expressed on myeloid and vascular cells (29). P2X7 receptor is essential for maturation and release of IL-1 (30) and is unique among the ATP-gated channels for its ability to open transiently a membrane pore permeable to highCmolecular excess weight dyes including pannexin-1 hemichannel-dependent and -impartial mechanisms (31C34). The P2X7 receptor is usually further connected to integrins and stimulates cytoskeletal rearrangements (35). In the present study, we showed that this P2X7 receptor is crucial for TF activation and release on MPs transporting integrin 1, PSGL-1, and PDI from myeloid cells as well as for the release of procoagulant MPs from SMCs. TF-dependent thrombosis was reduced in mice, and P2X7 receptor signaling in hematopoietic and vessel wall cells supported thrombosis. Our findings delineate what we believe to be a novel role for thiol pathways as crucial regulators of procoagulant TF+ MP generation downstream of P2X7 receptor signaling. Results ATP stimulation of the P2X7 receptor decrypts TF activity on macrophages. TF procoagulant activity was investigated in BM-derived macrophages primed with interferon- and Rabbit Polyclonal to OGFR LPS to induce TF expression (36). Cell surface TF activity on intact cells was found to be very low and required activation, e.g., with ionomycin (3), that was thiol-dependent and poorly correlated with the surface exposure of PS (Supplemental Physique 1; supplemental material available online with this short article; doi: 10.1172/JCI46129DS1). Although ionomycin is not a physiological agonist,.