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.
View: Text | PDF
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

×

Figure 9

F-actin is exposed on the cell surface during MP generation.

Options: View larger image (or click on image) Download as PowerPoint
F-actin is exposed on the cell surface during MP generation. (A) Surface...
(A) Surface F-actin was detected by phalloidin staining of nonpermeabilized ATP-stimulated cells (middle panel) using phalloidin–Alexa 633 conjugates, and counterstained for total F-actin (right panel) with phalloidin–Alexa 488 conjugates and nuclei with Hoechst after fixation. Cells were pretreated with DNCB or YVAD, as indicated; scale bar: 20 μm. (B) Detection of F-actin on ATP-induced MP populations by FACS using phalloidin–Alexa 633 conjugates. (C) Confocal images of ATP-stimulated cells in the presence of phalloidin–Alexa 546 concentrations used for cell staining (66 nM) or MP release blocking experiments (500 nM) demonstrate that higher concentration of phalloidin did not inhibit filopodia formation. (D) Abundance of ATP-induced MPs detected by FACS from control cells and from cells stimulated in the presence of cell-impermeable phalloidin to block extracellular actin. TFKI macrophages were used for FACS analysis and confocal imaging.