Caspase-1–mediated pathway promotes generation of thromboinflammatory microparticles
Andrea S. Rothmeier, … , Zaverio M. Ruggeri, Wolfram Ruf
Andrea S. Rothmeier, … , Zaverio M. Ruggeri, Wolfram Ruf
Published February 23, 2015
Citation Information: J Clin Invest. 2015;125(4):1471-1484. https://doi.org/10.1172/JCI79329.
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Research Article Hematology

Caspase-1–mediated pathway promotes generation of thromboinflammatory microparticles

Abstract

Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway–dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell–induced thrombosis.

Authors

Andrea S. Rothmeier, Patrizia Marchese, Brian G. Petrich, Christian Furlan-Freguia, Mark H. Ginsberg, Zaverio M. Ruggeri, Wolfram Ruf

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Figure 2

TRXR is required for the release of highly procoagulant MPs downstream of P2RX7 activation.

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TRXR is required for the release of highly procoagulant MPs downstream o...
(A) FACS analysis of large high side scatter (SSC) and small low SSC MPs released from control and ATP-stimulated TFKI macrophages with or without DNCB treatment. MP-depleted cell supernatants of ATP-stimulated cells were analyzed as an additional control. FSC, forward scatter. (B) Detection of TF and PS on ATP-induced MP populations. (C) PS-dependent prothrombinase activity of macrophage MPs generated with or without DNCB. PS dependence was confirmed by blockade with annexin 5 (A5); mean ± SD, n = 3, ***P < 0.001, ANOVA (Bonferroni). (D) Annexin 5 staining of ATP-stimulated WT cells after 30 minutes with and without DNCB treatment showed that blocking TRXR did not prevent cell surface PS exposure; scale bar: 100 μm; mean ± SD, n = 3, **P < 0.01, t test. (E) Detection of PSGL1, TF, integrin β1, PDI, and γ-actin on ATP-induced MPs from control and DNCB-treated TFKI macrophages by Western blot. (F) Effect of DNCB on TF activity on cells and MPs from control (–) and ATP-stimulated (+) cells measured by FXa generation assay; mean ± SD, n = 13, *P < 0.05, **P < 0.01, ANOVA (Bonferroni). (G) TF activity on MPs collected 0–15 minutes and 15–30 minutes following ATP stimulation of control and DNCB-treated cells; mean ± SD, n = 3, *P < 0.05, ANOVA (Bonferroni). (H) Coomassie blue–stained gels of MP-depleted supernatants of control and ATP-stimulated TFKI macrophages with or without DNCB treatment. Six prominent protein bands were identified by mass spectrometry (see Supplemental Table 1 for results).