Molecular Switch between Tissue Factor-Mediated Signaling and Coagulation.

Tissue factor (TF) forms a catalytic complex with coagulation factor VIIa and thus triggers coagulation as well as cell signaling through protease activated receptor (PAR) 2. The molecular switch between these two functions remains elusive. We demonstrate in growth arrested keratinocytes that TF’s coagulant function remains unchanged when TF expression levels and TF-VIIa signaling are concordantly downregulated. Thus, coagulation and signaling are mediated by two distinct and differentially regulated pools of TF. TF-VIIa signaling, but not coagulation, is blocked by bacitracin, an inhibitor of protein disulfide isomerase, and TF co-immunoprecipitates protein disulfide isomerases. To test the role of isomerization, we expressed single thiol containing mutants of the cross-stranded, surface exposed TF Cys¹⁸⁶-Cys²⁰⁹ disulfide bond. Retaining Cys¹⁸⁶, but not Cys²⁰⁹, preserves signaling properties with high affinity for VIIa, but eliminates the TF-dependent coagulation. Cys¹⁸⁶-containing TF is surface expressed predominantly in a dimeric form, indicating a trapped isomerization intermediate. Signaling by wild-type, but not the Cys¹⁸⁶ dimer, is inhibited by blocking free thiols indicating that thiol-isomerization plays an essential role in regulating TF signaling. Furthermore, we identified a monoclonal antibody that blocks TF-VIIa signaling of wild-type, but not Cys¹⁸⁶ dimer TF, suggesting interference with the dynamics of TF isomerization. This human TF specific antibody does not interfere with coagulation, but inhibits the growth of human xenograft tumors in mice. Thus, TF-VIIa signaling, not coagulation, is the primary pathway for TF-driven tumorigenesis. These experiments resolve the molecular mechanism that switches TF-VIIa from coagulation to signaling and show feasibility to block signaling TF for the therapeutic intervention in cancer.