P2×7 Signaling Triggers Tissue Factor Activation and Protein Disulfide Isomerase-Dependent Release of TF+ Microparticles In Thrombosis
Authors: Freguia Christian Furlan , Marchese Patrizia , Gruber Andras , Ruggeri Zaverio M. , Ruf Wolfram 
Published: October 2019

Abstract
Abstract 344
Thrombosis induced by the tissue factor (TF) pathway remains a major cause for morbidity and mortality in cardiovascular diseases. The cellular mechanisms of TF activation in primary cells implicated in pathological thrombosis are poorly understood and a controversy exists about the importance of thiol exchange involving protein disulfide isomerase (PDI) versus cell surface exposure of procoagulant anionic phosphatidylserine (PS). Myeloid cells are a source for TF positive (+) procoagulant microparticles (MP). We found that primed bone marrow-derived macrophages had undetectable TF activity. Upon stimulation of the purinergic P2X7 receptor with ATP, TF was readily decrypted. P2X7 signaling induced both the activation of TF cell surface procoagulant activity and thiol- and PDI-dependent generation of procoagulant MP in a pathway that was independent of cell surface PS exposure. Specifically, thiol blockade with dithio-bis-2-nitrobenzoic-acid (DTNB) prevented ATP-triggered release of procoagulant MP without effects on baseline or agonist-induced PS exposure. Similarly, anti-PDI antibody RL90, previously shown to have antithrombotic activities in vivo, attenuated the ATP-induced release of redox active TF+ MP without appreciable effects on cell surface TF activity or PS exposure. Importantly, P2X7 signaling also regulated TF activation and MP release from smooth muscle cells, implicated in TF thrombogenic pathways. Accordingly, P2X7 signaling-deficient mice displayed an attenuated TF-dependent thrombogenic response to an 8% FeCl3·6H2O carotid artery injury. In a more severe 10% injury, P2X7 deficient (-/-) mice eventually formed occluding thrombi that were inhibited by anti-FXI antibody given at a dose without effect in wild-type controls. In contrast, anti-TF did not prevent thrombosis, implying that P2X7-/- mice under these conditions rely on the intrinsic pathway for vascular occlusion and are generally impaired in TF-dependent thrombosis. Bone marrow transplantation experiments showed that P2X7 inactivation on both hematopoietic and vessel wall cells was required for protection from thrombosis. In further studies on this pathway, we identified another anti-PDI monoclonal antibody with unique activating effects that bypassed defective signaling of P2X7-/- cells, directly induced cell surface TF activity, and restored the thiol pathway leading to release of procoagulant MP from P2X7-/- smooth muscle cells and macrophages. The MP released from P2X7-/- macrophages by this antibody were remarkably similar to ATP-induced MP from wild-type mice and carried the thrombus targeting receptor P-selectin glycoprotein ligand-1 (PSGL-1), integrin b1, and PDI. In vivo, the activating anti-PDI antibody induced stable arterial occlusion in P2X7-/- mice challenged with 8% FeCl3·6H2O, and confirmed to be TF dependent since occlusion under these conditions was prevented by a function blocking anti-TF antibody. In conclusion, this study provides novel evidence that cell signaling is required for the activation of prothrombotic TF in vivo, thus challenging the current thinking that loss of vascular integrity or cell injury and PS exposure are the primary triggers for TF-dependent thrombosis. Our findings position cell surface PDI at a central node that controls P2X7-dependent TF cellular decryption and MP release. This novel activation pathway is critical for the generation of prothrombotic TF by vessel wall and blood cells and may be targeted as an alternative strategy to conventional anticoagulant therapy directed towards coagulation proteases.

Disclosures:
No relevant conflicts of interest to declare.