Tissue Factor Prothrombotic Activity Is Regulated by Integrin-arf6 Trafficking

Abstract

Objective Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine (PS) and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a non-coagulant state and to generating prothrombotic TF remains incompletely understood. Approach and Results Here we study activation of TF in primary macrophages by a combination of pharmacological, genetic and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. Following cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles (MV). TF cell surface availability for release onto MV is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, MV proteome analysis identifies activation of Gαi2 as a participating factor in the release of MV with prothrombotic activity in flowing blood. ATP not only prevents TF and PS internalization, but furthermore induces TF conversion to a conformation with high affinity for its ligand, FVIIa. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant PS, the resulting TF+ MV distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for MV generated by thrombo-inflammatory P2rx7 activation. Conclusions These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic MV from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional MV-based diagnostic approaches.

Tissue factor at the crossroad of coagulation and cell signaling

“…Whereas epithelial cell‐expressed TF has clearly been linked to the prevention of hemorrhage in a murine acute lung injury model and arterial thrombosis following ferric chloride (FeCl 3 ) injury induced in mice involves vessel wall TF expressed by smooth muscle cells , the cellular sources of TF within the vascular compartment are diverse and TF prothrombotic activity is typically controlled by cellular activation, including for TF expressed by smooth muscle cells . Although TF expression by platelets has been discussed controversially, generation of platelet‐like particles from in vitro differentiated human megakaryocytes demonstrates that platelets can carry both TF protein and mRNA in distinct subpopulations and platelet activation can induce splicing of intron‐retained TF mRNA for TF protein translation .…”

Extracellular vesicles in cardiovascular homeostasis and disease

“…Immediately following atherosclerotic plaque rupture leads to thrombotic vessel occlusion associated with platelet activation and activation of the coagulation cascade. Progression of the coagulation cascade requires the presence and activation of several enzymes that aid in the generation of thrombin, and many of the rate limiting enzymatic reactions begin at the surface of EVs [24]. Tissue factor, a key enzyme that initiates the coagulation cascade, is found on subpopulations of EVs derived from vascular SMCs [25*], endothelial cells [26], monocytes/macrophages [27], and platelets [28].…”

Factor VIIa‐induced interaction with integrin controls the release of tissue factor on extracellular vesicles from endothelial cells

“…D). We have previously shown that integrin α 4 β 1 , but not integrin α 5 β 1 , is associated with thromboinflammatory EVs generated by ATP stimulation of macrophages , indicating that different agonist pathways can produce EVs with different integrin repertoires.…”

“…Monocyte and vessel wall smooth muscle cell tissue factor (TF) activated by cell injury signals contributes to venous and arterial thrombosis. TF activated by thiol–disulfide exchange‐dependent complement activation or cell injury‐derived ATP is frequently released on highly procoagulant extracellular vesicles (EVs) that carry specific integrin β 1 heterodimers . Although the pathological roles of endothelial cell‐expressed TF were thought to be limited to neovascularization , endothelial TF, through protease‐activated receptor (PAR) 2 signaling, contributes to vascular inflammation associated with sickle cell disease .…”

“…Monocyte [1,2] and vessel wall smooth muscle cell [3,4] tissue factor (TF) activated by cell injury signals [5] contributes to venous and arterial thrombosis. TF activated by thiol-disulfide exchange-dependent complement activation or cell injury-derived ATP [6,7] is frequently released on highly procoagulant extracellular vesicles (EVs) that carry specific integrin b 1 heterodimers [8].…”