Identification Of The Coagulation Factor VIIa Integrin Binding Site Essential For Pro-Angiogenic Tissue Factor Signaling
Authors: Rothmeier Andrea S , Mueller Barbara M , Ostergaard Henrik , Ruf Wolfram 
Published: October 2019

The cellular initiator of the coagulation cascade, tissue factor (TF), plays pivotal roles in primary hemostasis and wound repair. In vitro evidence point to specific functions of the TF-FVIIa complex in inducing a pro-angiogenic and migratory program in keratinocytes dependent on activation of G protein-coupled signaling of protease activated receptor 2 (PAR2). Accordingly, mice with low levels of FVIIa have delayed cutaneous wound healing and pathological angiogenesis in genetic mouse models of cancer progression is impaired in mice that either lack PAR2 or the TF cytoplasmic domain.

Our previous data demonstrated that TF cytoplasmic domain signaling regulates α3β1-dependent migration and that FVIIa promotes the association of TF with β1 integrin heterodimers, but the functional contributions of this interaction to TF-dependent signaling remained unclear. Here, we identified a KGE motif in the FVIIa protease domain that is crucial for FVIIa-dependent complex formation of TF with integrins. Mutation of the negatively charged E26 to A affected neither the catalytic activity of FVIIa, nor significantly FXa generation in a purified system and on cell surfaces. Wild-type FVIIa induced co-immunoprecipitation of TF with keratinocyte-expressed α3β1 integrin. In contrast, FVIIa E26A was severely impaired in promoting association of TF with integrins in a pull-down assay with an antibody that recognized the active conformational state of the integrin β1. Previous transcriptome analysis has indicated that TF-FVIIa produces signaling distinct from activation of PAR2 with agonist peptide. In keratinocytes, wild-type FVIIa induced PI-3 kinase-dependent AKT and ERK phosphorylation, whereas ERK was phosphorylated independent of PI-3 kinase in cells stimulated with the PAR2 agonist SLGIRL. Mutation of FVIIa E26 required for integrin ligation abolished AKT phosphorylation and attenuated ERK phosphorylation, indicating that activation of the PI-3 kinase pathway specifically depends on complex formation of TF with integrins in the context of PAR2 cleavage. Consistently, FVIIa E26A inefficiently supported the PI-3 kinase-, ERK- and PAR2-dependent induction of the pro-angiogenic cytokine IL-8.

Mutation of FVIIa E26 similarly reduced complex formation with integrin β1 and attenuated PI-3 kinase- and ERK-dependent IL-8 induction in melanoma cells. Furthermore, the mutation impaired stimulation of migration in melanoma and breast cancer cells. In addition, deletion of the cytoplasmic domain-deleted TF transfected into TF-negative melanoma cells resulted in significantly reduced IL-8 induction by FVIIa in comparison to controls. However, the TF cytoplasmic domain was not required for FVIIa-induced complex formation with integrins, demonstrating direct contributions of the TF cytoplasmic domain to pro-angiogenic signaling. Taken together, these data elucidate the molecular details by which ligand binding promotes complex formation between TF and integrins and provide a novel mechanism of protease selective signaling of a promiscuous PAR in angiogenesis and wound repair.

Disclosures:

Ostergaard: Novo Nordisk A/S: Employment.