Cystine 186-cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption.
ABSTRACT Tissue factor (TF) on cell surfaces resides mostly in a cryptic state. It is not entirely clear how cryptic TF differs from procoagulantly active TF and how deencryption occurs. Here, we critically evaluated the importance of cystine 186-cystine 209 (Cys186-Cys209) bond formation for TF procoagulant activity and its de-encryption. Chinese hamster ovary cells transfected with TF(C186S), TF(C209S), or TF(C186S/C209S) expressed little procoagulant activity at the cell surface. TF monoclonal antibody and activated factor VII (FVIIa) binding studies showed that little TF protein was present at the cell surface in cells expressing mutant TF. Similar data were obtained in human umbilical vein endothelial cells (HUVECs) transduced to express TF(C186S), TF(C209S), or TF(C186S/C209S). Analysis of TF activity in HUVECs expressing similar levels of wild-type TF and TF(C186S/C209S) showed that TF mutant in the presence of saturating concentrations of FVIIa exhibited similar coagulant activity as that of wild-type TF. More importantly, treatment of HUVECs expressing TF(C186S/C209S) with HgCl(2) or ionomycin increased the cell-surface TF activity to the same extent as that of the wild-type TF. Our data provide clear evidence that TF lacking the Cys186-Cys209 bond is coagulantly active once it is complexed with FVIIa, and TF de-encryption does not require Cys186-Cys209 disulfide bond formation.
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ABSTRACT: Tissue factor (TF) is a transmembrane cofactor that binds and promotes the catalytic activity of factor (F) VIIa. The TF/VIIa complex activates FX by limited proteolysis to initiate blood coagulation and helps provide the thrombin burst that is important for a stable thrombus. TF is present both in the extravascular compartment, where it functions as a hemostatic envelope, and the intravascular compartment, where it contributes to thrombus formation, particularly when endothelial disruption is minimal. The regulation of its cofactor function appears to differ in the two compartments. Intravascular TF derives predominately from leucocytes, with either monocytes or neutrophils implicated in different models of thrombosis. This TF exists mostly in a non-coagulant or cryptic form and acute events lead to local decryption of TF and FX activation. A variety of experimental observations imply that decryption of leucocyte surface TF involves both a dithiol/disulfide switch and exposure of phosphatidylserine. The dithiol/disulfide switch appears to involve the Cys186-Cys209 disulfide bond in the membrane-proximal domain of TF, although this has not been demonstrated in vivo. Activation of a purinergic receptor or complement has recently been observed to decrypt TF on myeloid cells and a dithiol/disulfide switch and the oxidoreductase, protein disulfide isomerase, have been implicated in both systems. The molecular mechanism of action of protein disulfide isomerase in TF encryption/decryption, though, remains to be determined.Journal of Thrombosis and Haemostasis 06/2013; 11(s1). DOI:10.1111/jth.12228 · 5.55 Impact Factor
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ABSTRACT: Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.PLoS ONE 12/2014; 9(12). DOI:10.1371/journal.pone.0114141 · 3.53 Impact Factor
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ABSTRACT: Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼1% expression of the mouse TF level) and high human TF mice (HTF, ∼100% of the mouse TF level) were injected with human rFVIIa (120 µg kg-1 body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40-50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF's role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process.PLoS ONE 08/2014; 9(8):e103505. DOI:10.1371/journal.pone.0103505 · 3.53 Impact Factor