Vijay Kondreddy1, Usha R Pendurthi1, Xiao Xu2, John H Griffin2, L Vijaya Mohan Rao1. 1. From the Department of Cellular and Molecular Biology, The University of Texas Health Science Center, Tyler (V.K., U.R.P., L.V.M.R.). 2. Department of Molecular Medicine, Scripps Research Institute, La Jolla, CA (X.X., J.H.G.).
Abstract
OBJECTIVE: Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1-mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on β-arrestin1. CONCLUSIONS: Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.
OBJECTIVE: Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1-mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results:Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on β-arrestin1. CONCLUSIONS: Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.
Authors: Kaushik Das; Shiva Keshava; Shabbir A Ansari; Vijay Kondreddy; Charles T Esmon; John H Griffin; Usha R Pendurthi; L Vijaya Mohan Rao Journal: Blood Date: 2021-06-17 Impact factor: 25.476
Authors: Silvio Antoniak; Kohei Tatsumi; Clare M Schmedes; Grant J Egnatz; Alyson C Auriemma; Vanthana Bharathi; Tracy Stokol; Melinda A Beck; John H Griffin; Joseph S Palumbo; Nigel Mackman Journal: J Thromb Haemost Date: 2021-02-19 Impact factor: 16.036
Authors: Elena Pompili; Valerio De Franchis; Claudia Giampietri; Stefano Leone; Elena De Santis; Francesco Fornai; Lorenzo Fumagalli; Cinzia Fabrizi Journal: Biomolecules Date: 2021-11-10
Authors: Magdalena L Bochenek; Rajinikanth Gogiraju; Stefanie Großmann; Janina Krug; Jennifer Orth; Sabine Reyda; George S Georgiadis; Henri M Spronk; Stavros Konstantinides; Thomas Münzel; John H Griffin; Philipp Wild; Christine Espinola-Klein; Wolfram Ruf; Katrin Schäfer Journal: JCI Insight Date: 2022-07-22