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 8

Caspase-1 activity is required for thromboinflammatory MP generation.

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Caspase-1 activity is required for thromboinflammatory MP generation. (A...
(A) Effects of inhibitors of TRXR (DNCB), caspase-1 (YVAD), and calpain (ALLN) on ATP-induced activation of caspase-1 and IL-1β, and secretion of TRX detected by Western blotting. (B) Streptavidin blots of MPB-labeled free thiol groups on cells and MPs generated in the presence or absence of YVAD or DNCB. (C) Effects of YVAD on ATP-induced filopodia formation and TF translocation. TF was stained with anti–TF–Alexa 647 conjugate (red) before stimulation. Fixed and permeabilized cells were counterstained for F-actin (green) and nuclei (blue); scale bar: 10 μm. Length of filopodia and appearance of TF on filopodia were assessed; mean ± SD, n = 4, ***P < 0.001, t test. (D) ATP-induced MPs from control and YVAD-pretreated cells detected by FACS. (E) Confocal imaging of continuously MPB-labeled cells stimulated with ATP with or without YVAD. MPB was detected with Texas Red–labeled streptavidin (red) and nuclei with DAPI (blue) after fixation; scale bar: 10 μm. (F) Coomassie blue staining of proteins in the MP-depleted supernatants of ATP-stimulated cells with or without YVAD or ALLN treatment. TFKI macrophages were used for FACS analysis and confocal imaging.