Abstract
Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell injury signals trigger the macrophages P2X7 receptor and thereby induce the release both proinflammatory IL-1β and prothrombotic MP. Prothrombotic MP carry tissue factor (TF) and high content of phosphatidylserine (PS), and can induce thrombosis causing major clinical complications in patients. We previously identified the P2X7 receptor as a crucial component of thrombosis in mice, but the mechanistic details of macrophage MP release in this thrombo-inflammatory pathway remain incompletely understood.
The the generation of these MP requires thiol-disulfide exchange-dependent activation of the inflammasome and is accompanied by the release of various soluble proteins into the extracellular space. We hypothesized that the released proteome presents regulators and structural components of the MP generation pathway and employed proteomics to unveil their identity. Amongst the most abundant proteins were γ-actin and actin cytoskeleton associated proteins, including PS-binding proteins annexin 1 and annexin 5. Cytoskeletal remodeling processes leading to formation of filopodia downstream of P2X7 receptor activation were crucial for the biogenesis of thrombo-inflammatory MP, since pharmacological inhibitors of inflammasome activation, cytoskeletal remodeling and the thioredoxin system attenuated both, filopodia formation and the release of highly procoagulant MP. Confocal microscopy demonstrated raft dependent translocation of TF onto filopodia that was prevented by the same inhibitory strategies. Surprisingly, phalloidin-staining of non-permeabilized macrophages revealed that F-actin is exposed to the cell surface decorating the base of filopodia. Positive phalloidin-staining of thrombo-inflammatory MP further demonstrated that F-actin remained associated with the MP surface. Strikingly, blocking surface actin by incubation with high concentration of phalloidin prevented the release of PS-rich MP, demonstrating that exposure of F-actin during filopodia formation is functionally linked to the biogenesis of thrombo-inflammatory MP.
As the underlying common mechanism for thiol-disulfide exchange-dependent cell surface actin exposure and MP release, we showed that blockade of the cysteine protease caspase 1, which mediates processing and release of IL-1β downstream of inflammasome activation is also required for the release of thrombo-inflammatory MP. Although caspase 1-mediated activation of caplain was required for the release of filamin A localized TF to the cell cortex, calpain was not involved in the release of thrombo-inflammatory MP release. The N-terminus of γ-actin harbors a recognition and cleavage motif for caspase 1. Residual γ-actin released from caspase 1-blocked macrophages showed decreased electrophoretic mobility, indicating prior cleavage of actin that becomes exposed on the cell surface.
We show here that the proteome released during thrombo-inflammatory activation of macrophages includes critical players in the biogenesis of MP and may provide diagnostic fingerprints in complex biological samples. Our data demonstrate an entirely unexpected role for caspase 1 and surface exposure of polymerized actin in the severing of prothrombotic MP from filopodia and thus position this protease upstream of both IL-1β processing and thrombo-inflammatory MP in cardiovascular diseases.
Disclosures
No relevant conflicts of interest to declare.
F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.
