As noted in earlier studies,23,30 cells transfected with TFC186S, TFC209S, or TFC186S/C209S exhibited low TF procoagulant activity, approximately 1% or less compared with cells expressing wild-type TF (Physique 2A). FVIIa, and TF de-encryption does not require Cys186-Cys209 disulfide bond formation. Introduction Tissue factor (TF), a plasma membrane glycoprotein, plays a key role in the initiation of blood coagulation by allosterically activating coagulation factor VIIa (FVIIa). TF is essential for hemostasis, but the aberrant expression of it prospects to thrombosis and contributes to inflammation and malignancy.1C6 Thus, the Rabbit polyclonal to LDLRAD3 proper regulation of TF expression is critical not only for maintenance of the hemostatic sense of balance but also for health in general. It Ziprasidone hydrochloride monohydrate is well known that TF on cell surfaces exists in 2 different populations: a minor populace of coagulant-active TF, which binds FVIIa and the resultant TF-FVIIa complexes cleave macromolecular substrates, factor X (FX) and factor IX, and a major populace of cryptic TF, which also binds FVIIa but the resultant TF-FVIIa complexes are incapable of activating macromolecular substrates.7C10 Although FVIIa appears to bind preferentially to active TF, the differences between FVIIa binding to active and cryptic TF are not readily distinguishable, and the binding studies often showed a single class of high-affinity binding sites for FVII or FVIIa, suggesting that FVII and FVIIa form stable high-affinity associations with both decrypted and encrypted TF.7,11C13 It is unclear at present how the coagulantly active TF differs from your cryptic form and how the cryptic TF is converted to the active form. Studies from our laboratory14C16 and others8,9 exhibited that exposure of cells to calcium ionophore or other stimuli, which increase the negatively charged phospholipids at the outer leaflet of cell-surface membrane, enhanced the TF coagulant activity at the cell surface, suggesting that availability of negatively charged phospholipids within the vicinity of TF converts the cryptic TF to active TF. In addition to negatively charged phospholipids, dimerization of TF17 and association of TF with cholesterol and lipid rafts18C20 were also shown to modulate TF procoagulant activity (observe review in Egorina et al21). Recent studies suggest that cryptic and active TF exist in different conformations because the cryptic form of TF contains unpaired cysteine thiols at cystine 186 and cystine 209 in the membrane-proximal domain name, whereas the active form of TF is usually thought to have an oxidized cystine 186Ccystine 209 (Cys186-Cys209) disulfide bond.22,23 It was further suggested that protein disulfide isomerase (PDI) regulates TF activity by targeting this disulfide Ziprasidone hydrochloride monohydrate bond.23 The validity of the proposal that TF encryption/de-encryption involves PDI-mediated disulfide isomerization has been questioned.15 It has recently been suggested that differences in cell-model systems might have contributed to opposing conclusions around the importance of disulfide isomerization in TF encryption/de-encryption.24 However, this suggestion has been repudiated.25 Despite this unresolved controversy, it has recently been reported that PDI plays a critical role in thrombus formation in vivo.26,27 Although these studies provide no direct evidence that TF actually exists in the reduced form in vivo and PDI de-encrypts TF by forming the Cys186-Cys209 disulfide bond or that this de-encrypted TF is responsible for thrombus formation, it was strongly implied that this appears to be the mechanism responsible for thrombus formation. Despite enthusiasm for this new model, other investigators Ziprasidone hydrochloride monohydrate in the field, in addition to us, have raised questions about the validity of this model.28,29 The very idea that the Cys186-Cys209 disulfide bond is critical for TF coagulant activity and the cryptic TF contains unpaired cysteine thiols at this position comes from the earlier observation that ablation of the Cys186-Cys209 disulfide bond by mutating both cysteine residues severely impaired the procoagulant activity of TF.30 However, there was no direct evidence in this or other reports22,23 that TF lacking the Cys186-Cys209 disulfide bond actually behaves like cryptic TF, that is, binds to FVIIa, but the resultant TF-FVIIa complexes fail to activate FX. Furthermore, you will find no published studies that examined whether a TF mutant in which Cys186-Cys209 disulfide bond formation is usually precluded is usually resistant to de-encryption. In the present study, we provide clear evidence that diminished procoagulant activity of TF in cells expressing TF mutants lacking the ability to form a Cys186-Cys209 disulfide bond Ziprasidone hydrochloride monohydrate results from diminished antigen expression at the cell surface and the mutant’s lower affinity for FVIIa. Our data show that TF mutants lacking the Cys186-Cys209 disulfide bond exhibit the same specific coagulant activity as the wild-type TF in the presence of saturating concentrations of FVIIa. More importantly, the TF mutant.