Protease-activated Receptor 2-dependent Phosphorylation of the Tissue Factor Cytoplasmic Domain

Abstract

Tissue factor (TF) is the physiological activator of the coagulation cascade that plays pathophysiological roles in metastasis, angiogenesis, and inflammation. Downstream in coagulation, thrombin is the central protease that signals through G protein-coupled, protease-activated receptors (PARs). However, the TF-VIIa-Xa complex upstream in coagulation also activates PAR1 and 2. Here, we address the question of whether signaling of the TF initiation complex is a relevant pathway that leads to TF cytoplasmic domain phosphorylation. In heterologous expression systems and primary endothelial cells, we demonstrate that the ternary TF-VIIa-Xa complex induces TF phosphorylation specifically by activating PAR2 but not through PAR1 signaling. In addition, TF cytoplasmic domain phosphorylation is induced only by TF-dependent signaling but not by other coagulation factors in endothelial cells. Phosphorylation of the Prodirected kinase target site Ser 258 is dependent on prior phosphorylation of Ser 253 by protein kinase C (PKC) ␣. TF phosphorylation is somewhat delayed and coincides with sustained PKC␣ activation downstream of PAR2 but not PAR1 signaling. Phosphatidylcholine-dependent phospholipase C is the major pathway that leads to prolonged PKC␣ recruitment downstream of PAR2. Thus, PAR2 signaling specifically phosphorylates TF in a receptor cross-talk that distinguishes upstream from downstream coagulation protease signaling.

The Endothelial Protein C Receptor Supports Tissue Factor Ternary Coagulation Initiation Complex Signaling through Protease-activated Receptors

“…HUVECs were maintained and transduced with TF and PAR2 adenoviral constructs as described previously (36). Human HaCaT immortalized keratinocytes were maintained in DMEM, 10% FBS.…”

Regulation of Tissue Factor–Mediated Initiation of the Coagulation Cascade by Cell Surface Grp78

“…20,21 bEND.3 and RAW cells grown to 50% to 60% confluence were transduced with TF (50 virus particles per cell) for 3 hours, and grown for 48 hours. Cells were solubilized with 50 mmol/L n-Octyl-␤-D-glucopyranoside and incubated with antiGrp78 M , Hsc70, (Santa Cruz) or 10H10 anti-TF antibody 22 for 12 to 16 hours at 4°C followed by immunoprecipitation using protein A/G beads (Amersham Biosciences, Uppsala, Sweden).…”

Cryptic messages: Is noncoagulant tissue factor reserved for cell signaling?

“…Is PDI able to bridge the interaction between TF and PAR2, or is there a direct TF:PAR2 interaction? Ruf and colleagues (14,15) have suggested another regulatory switch whereby TF cytoplasmic tail phosphorylation promotes PAR2 signaling and simultaneously releases TF from a complex with integrins. How does PDI and TF disulfide switching fit with this model?…”