Tissue Factor Internalization and Trafficking in Fibroblasts: Involvement of Protease Activated Receptors-Mediated Cell Signaling.
Authors: Mandal Samir K. , Pendurthi Usha R. , Rao L. Vijaya Mohan 
Published: October 2019

Abstract
Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa) and the formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. TF is constitutively expressed in many extravascular cells, including fibroblasts and pericytes in and surrounding blood vessel walls, and lung epithelial cells. Our recent studies (Blood2006; 107:4746–4753) show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. FVIIa binding to cell surface TF induces the internalization of TF, and interestingly, mobilizes the Golgi TF pool and transports it to the outer cell surface. This process is dependent on FVIIa protease activity. This present study is aimed to elucidate potential mechanisms involved in TF internalization and the mobilization from the Golgi. Since studies from our laboratory and others showed that TF-FVIIa could activate protease-activated receptor (PAR)-mediated cell signaling and FVIIa protease activity is required for FVIIa-dependent internalization and trafficking of TF, we hypothesize that TF-VIIa activation of PAR1 or PAR2 plays a role in TF internalization and trafficking. To test this hypothesis, we first examined the role of PAR activation in TF-internalization and trafficking. Lung fibroblasts (WI-38 cells) were exposed to a variety of PAR activators, PAR activating peptide agonists (AP) and various proteases, and TF internalization and trafficking was evaluated by measuring the cell surface TF antigen and activity levels, internalization of cell surface TF (by using biotinylation of cell surface receptors and immunoprecipitation techniques) and mobilization of TF from the Golgi (by immunofluorescence confocal microscopy). PAR1 AP and PAR2 AP treatments increased the TF activity and antigen levels at the cell surface by 20 to 50% whereas PAR3 AP and PAR4 AP had no effect on cell surface TF activity and antigen levels. Cell surface TF activity and antigen levels were also increased slightly in fibroblasts exposed to thrombin and trypsin. Confocal microscopic image analysis of distribution of TF and the Golgi protein (golgin-97) revealed that about 85% of the untreated cells possess intact Golgi TF pool with high degree of colocalization with golgin-97 whereas as only 20–30% of FVIIa, thrombin, trypsin, PAR1 AP or PAR2 AP-treated cells had TF pool in the Golgi. Plasmin and FXa had moderate effect on TF mobilization from the Golgi. No detectable differences were found between control (untreated) cells and cells treated with either FFR-FVIIa, APC, PAR3 AP or PAR4 AP. Next, we investigated the role of PAR1 and PAR2 activation in FVIIa-mediated TF internalization and trafficking. Pretreatment of fibroblasts with PAR2 but not PAR1 activation blocking antibodies attenuated FVIIa-mediated Golgi TF mobilization. Consistent with these data, silencing PAR2 receptor by siRNA technique completely blocked FVIIa-mediated Golgi TF mobilization whereas PAR1 siRNA transfection had no effect (in control studies, we showed PAR1 antibodies or PAR1si RNA transfection blocked thrombin-mediated TF mobilization). Additional studies showed a significant internalization of TF in cells exposed to FVIIa which was completely blocked by silencing PAR2 but not PAR1. Overall the data provided herein suggest a novel mechanism by which tissue factor expression is regulated at the cell surface.