Andrea S Rothmeier1, Henri H Versteeg2, Wolfram Ruf1,3. 1. Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA. 2. Einthoven Laboratory for Experimental Vascular Medicine, Department of Thrombosis and Hemostasis, Leiden, the Netherlands. 3. Center for Thrombosis and Hemostasis, Johannes Gutenberg University Medical Center, Mainz, Germany.
Abstract
Essentials Prothrombotic extracellular vesicles (EV) carry agonist pathway-specific proteomes Agonists for protease activated receptor (PAR) 2 signaling have distinct effects on EV composition PAR2 signaling rapidly generates prothrombotic EV and slowly EV with inactive tissue factor (TF) FVIIa integrin ligation restricts TF incorporation into EV from endothelial cells SUMMARY: Background Cell injury signal-induced activation and release of tissue factor (TF) on extracellular vesicles (EVs) from immune and vessel wall cells propagate local and systemic coagulation initiation. TF trafficking and release on EVs occurs in concert with the release of cell adhesion receptors, including integrin β1 heterodimers, which control trafficking of the TF-activated factor VII (FVIIa) complex. Activation of the TF signaling partner, protease-activated receptor (PAR) 2, also triggers TF release on integrin β1 + EVs from endothelial cells, but the physiological signals for PAR2-dependent EV generation at the vascular interface remain unknown. Objective To define relevant protease ligands of TF contributing to PAR2-dependent release on EVs from endothelial cells. Methods In endothelial cells with balanced expression of TF and PAR2, we evaluated TF release on EVs by using a combination of activity and antigen assays, immunocapture, and confocal imaging. Results and Conclusions PAR2 stimulation generated time-dependent release of distinct TF+ EVs with high coagulant activity (early) and high antigen levels (late). Whereas PAR2 agonist peptide and a stabilized TF-FVIIa-activated FX complex triggered TF+ EV release, stimulation with FVIIa alone promoted cellular retention of TF, despite comparable PAR2 activation. On endothelial cells, FVIIa uniquely induced formation of a complex of TF with integrin α5 β1 . Internalization of TF by FVIIa or anti-TF and activating antibodies against integrin β1 prevented PAR2 agonist-induced release of TF on EVs. These data demonstrate that intracellular trafficking controlled by FVIIa forcing interaction with integrin β1 regulates TF availability for release on procoagulant EVs.
Essentials Prothrombotic extracellular vesicles (EV) carry agonist pathway-specific proteomes Agonists for protease activated receptor (PAR) 2 signaling have distinct effects on EV composition PAR2 signaling rapidly generates prothrombotic EV and slowly EV with inactive tissue factor (TF) FVIIa integrin ligation restricts TF incorporation into EV from endothelial cells SUMMARY: Background Cell injury signal-induced activation and release of tissue factor (TF) on extracellular vesicles (EVs) from immune and vessel wall cells propagate local and systemic coagulation initiation. TF trafficking and release on EVs occurs in concert with the release of cell adhesion receptors, including integrin β1 heterodimers, which control trafficking of the TF-activated factor VII (FVIIa) complex. Activation of the TF signaling partner, protease-activated receptor (PAR) 2, also triggers TF release on integrin β1 + EVs from endothelial cells, but the physiological signals for PAR2-dependent EV generation at the vascular interface remain unknown. Objective To define relevant protease ligands of TF contributing to PAR2-dependent release on EVs from endothelial cells. Methods In endothelial cells with balanced expression of TF and PAR2, we evaluated TF release on EVs by using a combination of activity and antigen assays, immunocapture, and confocal imaging. Results and Conclusions PAR2 stimulation generated time-dependent release of distinct TF+ EVs with high coagulant activity (early) and high antigen levels (late). Whereas PAR2 agonist peptide and a stabilized TF-FVIIa-activated FX complex triggered TF+ EV release, stimulation with FVIIa alone promoted cellular retention of TF, despite comparable PAR2 activation. On endothelial cells, FVIIa uniquely induced formation of a complex of TF with integrin α5 β1 . Internalization of TF by FVIIa or anti-TF and activating antibodies against integrin β1 prevented PAR2 agonist-induced release of TF on EVs. These data demonstrate that intracellular trafficking controlled by FVIIa forcing interaction with integrin β1 regulates TF availability for release on procoagulant EVs.