P2X7 receptor signaling contributes to tissue factor–dependent thrombosis in mice
Christian Furlan-Freguia, … , Zaverio M. Ruggeri, Wolfram Ruf
Christian Furlan-Freguia, … , Zaverio M. Ruggeri, Wolfram Ruf
Published June 13, 2011
Citation Information: J Clin Invest. 2011;121(7):2932-2944. https://doi.org/10.1172/JCI46129.
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Research Article Hematology

P2X7 receptor signaling contributes to tissue factor–dependent thrombosis in mice

Abstract

Thrombosis is initiated by tissue factor (TF), a coagulation cofactor/receptor expressed in the vessel wall, on myeloid cells, and on microparticles (MPs) with variable procoagulant activity. However, the molecular pathways that generate prothrombotic TF in vivo are poorly defined. The oxidoreductase protein disulfide isomerase (PDI) is thought to be involved in the activation of TF. Here, we found that in mouse myeloid cells, ATP-triggered signaling through purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7 receptor; encoded by P2rx7) induced activation (decryption) of TF procoagulant activity and promoted release of TF+ MPs from macrophages and SMCs. The generation of prothrombotic MPs required P2X7 receptor–dependent production of ROS leading to increased availability of solvent-accessible extracellular thiols. An antibody to PDI with antithrombotic activity in vivo attenuated the release of procoagulant MPs. In addition, P2rx7–/– mice were protected from TF-dependent FeCl3-induced carotid artery thrombosis. BM chimeras revealed that P2X7 receptor prothrombotic function was present in both hematopoietic and vessel wall compartments. In contrast, an alternative anti-PDI antibody showed activities consistent with cellular activation typically induced by P2X7 receptor signaling. This anti-PDI antibody restored TF-dependent thrombosis in P2rx7–/– mice. These data suggest that PDI regulates a critical P2X7 receptor–dependent signaling pathway that generates prothrombotic TF, defining a link between inflammation and thrombosis with potential implications for antithrombotic therapy.

Authors

Christian Furlan-Freguia, Patrizia Marchese, András Gruber, Zaverio M. Ruggeri, Wolfram Ruf

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Figure 7

P2X7 receptor signaling regulates TF activation and TF+ MP release from SMCs.

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P2X7 receptor signaling regulates TF activation and TF+ MP release from ...
(A) TF and SMA expression by isolated lung-derived SMCs. Scale bar: 20 μm. (B) Western blot of integrin β1 and TF in MPs from ATP-stimulated SMCs relative to the corresponding cell lysates. Lanes for cell lysates were run on the same gel but were noncontiguous. (C) P2X7 receptor expression by WT and P2rx7–/– lung SMCs, determined by Western blotting. A slower migrating, possibly glycosylation or splice isoform of the P2X7 receptor was absent from cells of P2rx7–/– mice. No difference in expression of integrin β1, TF, and actin was observed between WT and P2rx7–/– cells. (D and E) Protein composition and TF activity of MPs released from ATP-stimulated WT and P2rx7–/– lung-derived SMCs. P2X7 receptor in WT MPs had a mobility corresponding to the slower migrating isoform in C. (F) P2X7 receptor stimulation induced aortic SMC uptake of YO-PRO (10 μM), added during ATP stimulation. Scale bar: 250 μm. (G) TF protein content and TF-dependent FXa generation in MPs from ATP-stimulated aortic WT SMCs (n ≥ 3). SMCs were isolated from transgenic mice expressing GFP ubiquitously in all cells in order to monitor MP release based on packaging of cytosolic content. *P < 0.05 vs. control.