Tissue Factor Activation: Is Disulfide Switching a General Regulatory Mechanism?.
Authors: Pendurthi Usha , Ghosh Samit , Mandal Samir , Rao L. Vijaya Mohan 
Published: October 2019

Abstract
Tissue factor (TF) is the cellular receptor for plasma clotting factor VIIa, and the formation of TF-VIIa complexes on cell surfaces trigger the coagulation cascade and cell signaling. It is a well-known fact that only a small fraction of TF at the cell surface is coagulantly active whereas a majority of TF on the cell surface is non-functional (cryptic). However, it is unclear, at present, how the coagulant active TF differs from the cryptic form, and mechanisms involved in TF activation. Recent studies show that a thiol oxidizing agent, HgCl2, increases TF coagulant activity on the surface of HL-60 cells by several fold (Chen et al., Blood vol 106, abstract #684, 2005). Further, TF is shown to associate with protein disulfide isomerase (PDI) in HaCaT cells (Ahamed et al., Blood vol 106, abstract #685, 2005). Based on these and other observations, it has been proposed that switching between cryptic and coagulant TF involves cleavage and formation of allosteric disulfide bond (Cys186-Cys209) and PDI has been implicated in controlling the conversion of cryptic TF to the coagulant form and to act as a switch between TF-mediated signaling and coagulation. Although these data are interesting and novel, there is no fail-proof evidence that disulfide switching alone and not other potential changes, such as exposure of anionic phospholipids, at the cell surface is responsible for the TF activation associated with various treatments. Therefore we have examined the effect of HgCl2 and other treatments on TF activation in MDA 231 cells in relation to anionic phospholipids and also characterized the cellular expression of PDI in this and other cell types. As reported earlier, the HgCl2 treatment increased the cell surface TF coagulant activity (5-fold or higher). However, the HgCl2 treatment also increased the prothombinase activity by 3-fold. More importantly, annexin V, which binds to anionic phospholipids, markedly reduced the increased TF coagulant activity associated with the HgCl2 treatment whereas it had only minimal and insignificant effect on TF activity of the control cells. Further, pretreatment of cells with 5,5′-dithio-bis(2-nitronezoic acid) (DTNB), a sulfhydryl reagent that reacts with thiol groups and thus can block disulfide switching, failed to prevent the increase in TF activity associated with the HgCl2 treatment. Interestingly, we found that treatment of MDA 231 cells with glutathione (5 to 100 mM), a cell impermeable reducing agent, also increased the surface TF activity by about 2- to 3-fold. In additional studies, we found that PDI antibodies had no effect on either the TF coagulant activity or TF-mediated cell signaling. Further, we found no evidence for the expression of PDI at the cell surface in immunofluorescence confocal microscopy as both monoclonal and polyclonal PDI antibodies failed to stain nonpermeabilized cells whereas they brightly stained intracellular PDI in permeabilized cells. In contrast, TF antibodies stained intensely the surface of both nonpermeabilized and permeabilized cells. Exposure of tumor cells to various proteases failed to transport the intracellular PDI to the cell surface. The present data raise a valid question whether disulfide switching by PDI plays the predominant and general regulatory role in controlling the TF coagulant activity and signaling functions. Our data also suggest that other cellular changes, including increase in anionic phospholipids, may be responsible for increased TF coagulant activity associated with the thiol oxidizers and other treatments.