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 5

P2rx7–/– mice are protected from thrombosis in the FeCl3-induced carotid artery model.

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P2rx7–/– mice are protected from thrombosis in the FeCl3-induced caroti...
(A) Carotid artery injury was induced with 8% or 10% FeCl3•6H20 in C57BL/6 WT and P2rx7–/– mice; where indicated, anti-FXI 14E11 (65 ng/g) or anti-TF 21E10 (25 μg/g body weight) were also given prior to injury. (B) At 5 minutes before injury with 8% FeCl3•6H20, mice were injected into the contralateral jugular vein with anti-FXI 14E11 (65 ng/g, calculated to inhibit approximately 30% of plasma FXI) with addition of anti-TF 21E10 (25 μg/g body weight), anti-PDI RL90 (2 or 4 μg/g), or corresponding amounts of irrelevant mouse monoclonal IgG. Results for individual mice are shown; horizontal lines and error bars denote median and interquartile range of the time to first occlusion (defined as flow <10% of that measured in the artery before injury) and mean ± SEM of the flow index (ratio between total volume of arterial blood flow after injury and expected volume based on preinjury flow). Flow index of 1 indicates completely unimpeded flow throughout the observation period. Differences in occlusion time were evaluated with the nonparametric Kruskal-Wallis test followed by paired comparisons with the Dunn test; differences in flow index were assessed with 1-way ANOVA followed by Bonferroni test. *P < 0.05; **P < 0.01.