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

Abstract

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 BackgroundCell 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. ObjectiveTo define relevant protease ligands of TF contributing to PAR2‐dependent release on EVs from endothelial cells. MethodsIn 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 ConclusionsPAR2 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 α5β1. 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.

A proposal for managing bleeding in patients on therapeutic factor XI(a) inhibitors

“…Furthermore, rFVIIa may cleave protease-activated receptor 1 to induce extracellular vesicle release that further potentiates thrombin generation. 45 However, we suspect that low-dose rFVIIa may appear to be so effective partly because antifibrinolytic therapy alone is adequate for achieving hemostasis in many cases. This is consistent with the impression that FXI primarily counters fibrinolysis, and that its importance is diminished when fibrinolysis is inhibited.…”

“…In the absence of FXI, rFVIIa may promote hemostasis by enhancing fibrin resistance to fibrinolysis through formation of more tightly packed fibrin fibers, 44 as well as through thrombin‐activatable fibrinolysis inhibitor activation. Furthermore, rFVIIa may cleave protease‐activated receptor 1 to induce extracellular vesicle release that further potentiates thrombin generation 45 . However, we suspect that low‐dose rFVIIa may appear to be so effective partly because antifibrinolytic therapy alone is adequate for achieving hemostasis in many cases.…”

Extracellular Vesicles and Thrombogenicity in Atrial Fibrillation

“…Second, the presence of polyphosphate (polyP) in cell-derived EVs may promote thrombosis through a tissue factor-independent route, whereas these effects were initially described in cancer-associated thrombosis [37] and then extrapolated to others. Yet, the pro-coagulant activity to EVs that originated from mature endothelial cells was found to be up-regulated by the induction of the expression of adhesion proteins and several encapsulated pro-inflammatory cytokines, mainly IL-8 and tumor necrosis factor-alpha (TNF-alpha), on mother cells [38]. These mechanisms are rigorously regulated by the protease-activated receptor (PAR) 2 signaling pathway, which, in turn, mediates the rapid generation of pro-thrombotic components, such as inactive tissue factor/factor VII and integrin α5 β1 into the EVs secreted by endothelial cells [39].…”

The Effect of Extracellular Vesicles on Thrombosis

“…The transport and release of TFs on EVs coincide with the release of cell adhesion receptors, including integrin beta1 heterodimers, which control the transport of the TF-activating factor VIIa (FVIIa) complex. Activation of the TF signaling chaperone PAR2 also induces the release of integrin beta1 and TF-enriched EVs from ECs [ 61 ]. On ECs, FVIIa specifically induces the formation of TF complexes with integrin α5β1 [ 61 ].…”