Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling

Abstract

The tissue factor (TF) pathway serves both hemostasis and cell signaling, but how cells control these divergent functions of TF remains incompletely understood. TF is the receptor and scaffold of coagulation proteases cleaving protease-activated receptor 2 (PAR2) that plays pivotal roles in angiogenesis and tumor development. Here we demonstrate that coagulation factor VIIa (FVIIa) elicits TF cytoplasmic domain-dependent proangiogenic cell signaling independent of the alternative PAR2 activator matriptase. We identify a Lys-Gly-Glu (KGE) integrin-binding motif in the FVIIa protease domain that is required for association of the TF-FVIIa complex with the active conformer of integrin β1. A point mutation in this motif markedly reduces TF-FVIIa association with integrins, attenuates integrin translocation into early endosomes, and reduces delayed mitogen-activated protein kinase phosphorylation required for the induction of proangiogenic cytokines. Pharmacologic or genetic blockade of the small GTPase ADP-ribosylation factor 6 (arf6) that regulates integrin trafficking increases availability of TF-FVIIa with procoagulant activity on the cell surface, while inhibiting TF-FVIIa signaling that leads to proangiogenic cytokine expression and tumor cell migration. These experiments delineate the structural basis for the crosstalk of the TF-FVIIa complex with integrin trafficking and suggest a crucial role for endosomal PAR2 signaling in pathways of tissue repair and tumor biology.

Tissue factor at the crossroad of coagulation and cell signaling

“…Instead, the TF cytoplasmic domain recruits adaptors for signaling complexes and protein trafficking (i.e. the regulatory subunit of phosphatidylinositol 3 kinase [PI3K]) , the actin binding protein filamin and the prolyl‐isomerase Pin1 . Interaction of the TF cytoplasmic domain with Pin1 not only influences TF protein half‐life and incorporation into EV, but also Pin1‐dependent transcriptional regulation of TF expression in human smooth muscle cells .…”

“…It is currently unclear whether Pin1 regulation involves PAR2, which is known to constitutively signal independent of proteolytic cleavage . In contrast, binding of the regulatory subunit of phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) to the TF cytoplasmic domain is important for TF‐FVIIa‐PAR2 signaling that is connected to functions of cell adhesion receptors, particularly integrins of the β3 and β1 families .TF‐FVIIa complex signaling through PAR2 has been implicated in cancer cell migration, invasion, proliferation and evasion from apoptotic cell death , Blocking the TF‐integrin interaction with anti‐TF antibody 10H10, which has minimal effects on coagulation, inhibits TF‐FVIIa signaling and suppresses tumor growth . Other TF‐FVIIa inhibitors also prevent spontaneous tumor progression in syngeneic tumor models .…”

“…The molecular details of TF‐FVIIa complex formation with integrin and implications for proangiogenic and promigratory signaling have recently been further clarified. Immunoprecipitation with an antibody recognizing an active conformation of β1 integrin has shown that FVIIa induces TF complex formation with activated integrin through a specific binding site in the FVIIa protease domain remote from the catalytic cleft required for PAR2 cleavage . This site overlaps with the macromolecular substrate binding site and thereby precludes the activation of procoagulant substrates with a predicted impairment of TF‐initiated coagulation.…”

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

“…A). TF–FVIIa association with integrin β 1 is crucial for internalization and endosomal TF–FVIIa signaling . Tracking with fluorophore‐labeled anti‐FVII 12C7 and anti‐integrin β 1 TS2/16 demonstrated colocalization of FVIIa with integrin in the same endosomal compartment 30 min after FVIIa stimulation (Fig.…”

Advances in Clinical and Basic Science of Coagulation: Illustrated abstracts of the 9th Chapel Hill Symposium on Hemostasis

“…We have identified crucial residues in the FVIIa protease domain that regulate ligand binding and thereby direct the biological functions of the TF‐FVIIa complex in hemostasis and cell signaling. The integrin binding motif (KGE) residue FVII E promotes TF‐FVIIa complex formation with active integrin β1 and couples TF to integrin‐arf6 recycling required for endosomal pro‐angiogenic and pro‐migratory PAR2 signaling. In the same region of the FVIIa protease domain, the allosteric switch residue FVIIa E 154 controls the release of nascent FXa product required for TF prothrombotic activity.…”