Abstract 43: Recycling Controls Procoagulant and Cell Signaling Functions of Tissue Factor
Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell danger signals in form of high level of extracellular ATP trigger the macrophage P2X7 receptor leading to secretion of pro-inflammatory IL-1β and the release of pro-thrombotic tissue factor (TF) positive microparticles (MP). P2X7 receptor signaling uncouples the thioredoxin-thioredoxin reductase system and induces inflammasome activation leading to decryption of cell-surface TF and its trafficking onto filopodia for incorporation into MP. These MP have a distinct composition of thiol-regulated proteins and a high surface density of procoagulant PS. Besides its well-established function in IL-1β processing and secretion, we found an entirely unexpected role for caspase 1 in the release of TF from the final severing of highly thrombotic TF-positive MP. Thus, caspase 1 directly couples the release of pro-inflammatory and pro-thrombotic mediators.
P2X7 activation not only causes MP release, but also activates cell surface TF that remains non-coagulant without secondary signals on primed macrophages. We observed that cell detachment rapidly induced full TF procoagulant activity, pointing to an important role of cell adhesion in the maintenance of a non-thrombotic state. Stable cell adhesion is controlled by integrin recycling pathways and the small GTPase arf6 is one of the key regulators of these processes. We found that blocking receptor internalization retains TF on the cell surface and markedly enhanced TF procoagulant activity. Under these conditions, TF was associated with integrin β1 and arf6, and importantly, P2X7 receptor activation promoted arf6-GTP hydrolysis to arf6-GDP, indicating interruption of this recycling pathway following stimulation. In addition, increasing arf6-GDP levels, either by pharmacological inhibitor or overexpression of an arf6-mutant, prevented TF-dependent signaling though protease activated receptor 2 (PAR2) and simultaneously increased cellular procoagulant TF activity in epithelial and in melanoma cells. These findings demonstrate an unexpected role for arf6-controlled recycling in the regulation of TF procoagulant and signaling functions.
Author Disclosures: A.S. Rothmeier: None. W. Ruf: None.
- © 2015 by American Heart Association, Inc.