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 5

Macrophage-derived MPs are prothrombotic in flowing blood.

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Macrophage-derived MPs are prothrombotic in flowing blood. Three-dimensi...
Three-dimensional reconstruction of platelet aggregates and fibrin on surface-adherent macrophages was generated from confocal z-sections serially collected before and after blood perfusion. Images of green and red autofluorescent cells before flow were combined and converted to blue for superposition to corresponding images after flow. (A) Top left: LPS-primed macrophages exposed to recalcified flowing blood containing mepacrine (green) to visualize platelets and Alexa 546–labeled anti-fibrin antibody (red); merging of green, red, and blue yields the colors shown schematically at the center of the figure. Note that cells vary from blue to aqua owing to variable mepacrine incorporation from blood. Top right: Only the red component (fibrin) of the image on the left is shown. Bottom panels: Same as top, except that macrophages were stimulated with ATP (5 mM) before blood perfusion. (B) Mouse blood, treated as in A, was perfused over ATP-stimulated TF-deficient macrophages from Tffl/fl Lysm-Cre mice. (C) Top panels: MPs from ATP-stimulated TFKI macrophages (expressing human TF) were added to blood before perfusion over TF-deficient, unstimulated macrophages. Note the appearance of fibrin strands. Middle panels: Species-specific anti–human TF antibody 5G9 was added with MPs to the blood before perfusion. No fibrin strands formed. Bottom panels: MPs from filipin-treated TFKI macrophages were perfused in blood over TF-deficient, unstimulated cells. In B and C, images on the right show a magnification of the area delimited by the white rectangle in the images on the left.