Release of Tissue Factor-bearing microparticles by human dendritic cells induced by stimulation of membrane P2X7 receptors

Introduction: Dendritic cells (DCs), critical for antigen capture, are present in the atherosclerotic plaques and express P2 receptors (P2R). The P2X7R subtypes, stimulated with extracellular nucleotides, cause release of microparticles (MPs). In vivo observations provide evidence for ATP release at foci of tissue injury and inflammation. Elevated levels of tissue factor (TF)-bearing MPs were found to be associated with cardiovascular disease, with a potential role in thrombus formation after plaque rupture. Methods: Monocyte-derived human DCs were stimulated with ATP or benzoyl ATP (BzATP). MPs were obtained by ultracentrifugation of cell supernatants. MPs-TF was evaluated by Western blot and for cofactor activity toward rFVIIa in a FXa generation assay. TF release after stimulation was also analyzed by confocal microscope analysis. TF mRNA was investigated by RT PCR. Results: Quiescent DCs express the mRNA for the full-length and the alternatively spliced (as) TF. Membrane-bound and soluble TF forms were detected by Western blot analyses. The vast majority of TF disappeared upon stimulation of DC, as shown by immunoblot and confocal microscopy analysis. TF antigen and rFVIIa cofactor activity appeared in the vesicular fraction. Only the full length TF was found in MPs, suggesting that the asTF was directly released into extracellular space upon DC stimulation. rFVIIa cofactor activity of MPs-TF was compared to that of recombinant TF and inhibited in a dose-dependent manner by a specific anti-human TF antibody. Conclusions: Stimulated DCs release MPs endowed with full length TF, characterized by FVIIa cofactor activity. MPs are quantitatively important carriers for the dissemination of TF from DCs. A large fraction of TF, and of its procoagulant/signalling potential, may be deliverable after physiological or pathological stimuli. These findings might have implications for triggering and propagating coagulation in healthy and atherosclerotic vessels.