Abstract 70: P2X7-Induced Thiol-Disulfide Exchange is Critical for the Coupling of Coagulation and Inflammation.

Abstract

Macrophages are important players in the maintenance of tissue homeostasis, but promote inflammation through the release of IL1β triggered by danger signals in form of extracellular ATP that activates the purinergic P2X7 receptor. We found that P2X7 signaling also contributes to thrombosis by inducing thiol-dependent tissue factor (TF) activation coupled to procoagulant microparticles (MP) release. In the present study, we identified thiol-regulated proteins released on MP and based on this information delineated key steps in the P2X7-induced generation of prothrombotic TF + MP. We find that TF procoagulant activity of LPS/IFNγ primed macrophages is controlled by internalization through the arf6/integrin-recycling pathway. Activation of P2X7 inactivates arf6 and prevents TF internalization, but additional steps are required to generate highly procoagulant MP carrying TF and integrin β1. Imaging of cell surface TF by confocal microscopy shows translocation of TF onto filopodia that form in response to P2X7 activation. Blocking raft mobility does not inhibit filopodia formation, but rather specifically prevents TF and integrin β1 trafficking and release on MP. We show that filopodia formation is dependent on thioredoxin reductase (TRXR). Remarkably, thioredoxin (TRX), the direct substrate of TRXR, is entirely released from the cytosol. Pharmacological inhibition of TRXR blocks both TRX release and reductive changes on the cell surface and MP, identifying the molecular events that change the extracellular redox environment. TRXR-mediated externalization of TRX was also required for activation of the inflammasome and caspase1 leading to IL1β processing and release. These data elucidate the molecular events required for the generation of highly procoagulant TF + MP and identifies TRXR-TRX dependent thiol-disulfide exchange as common upstream regulator responsible for the induction of inflammation and coagulation in innate immune cells.