Cystine 186-cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption

Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, TX 75708, USA.
Blood (Impact Factor: 10.45). 03/2010; 115(21):4273-83. DOI: 10.1182/blood-2009-09-241356
Source: PubMed

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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    • "Confluent monolayers of HUVEC cultured in 100 mm dish were infected with control and TF adenovirus [34] (10 moi/cell). Two days post-infection, cells were washed with buffer A (10 mM Hepes, 0.15 M NaCl, 4 mM KCl, 11 mM glucose, pH 7.5), and then treated with calcium ionomycin (10 µM) in buffer B (buffer A containing 5 mM CaCl2 and 1 mg/ml bovine serum albumin) for 20 min at 37°C. "
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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 · 3.23 Impact Factor
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    • "Both chemical reduction of this disulfide bond [11] and tissue factor mutants lacking the Cys186-Cys209 disulfide bond results in less procoagulant activity compared to wild-type tissue factor [12] [13]. However, the involvement of the disulfide bond was questioned since mutating a cysteine residue resulted in altered cellular expression levels of tissue factor [14]. This observation was later rejected through generation of mutants with equal expression levels compared with wild-type tissue factor, confirming the importance of proper disulfide bond formation for the procoagulant activity of tissue factor [5]. "
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    ABSTRACT: It has been suggested that the main physiological trigger of coagulation, tissue factor, possesses limited procoagulant activity and occurs in an inactive or so-called encrypted state. For the conversion of encrypted into decrypted tissue factor with sufficient procoagulant activity, four distinct models have been proposed: 1; dimer formation, 2; lipid rafts, 3; disulfide bonds, and 4; phosphatidylserine exposure. Pro and cons can be given for each of these mechanisms of tissue factor encryption/decryption, however, it seems most likely that two or more mechanisms act together in activating the procoagulant activity. The exposure of phosphatidylserine in the outer layer of cell membranes supports coagulation through enhanced formation of the tenase (factors IXa, VIIIa and X) and prothrombinase (factors Xa, Va and prothrombin) complexes. The proposed role for phosphatidylserine in decryption of tissue factor could contribute to the correct orientation of the tissue factor - factor VII complex. Overall, the contribution of both tissue factor and phosphatidylserine to coagulation seems distinct with tissue factor being the physiological activator and phosphatidylserine the driving force of propagation of coagulation.
    Thrombosis Research 05/2014; 133 Suppl 1:S54-6. DOI:10.1016/j.thromres.2014.03.022 · 2.45 Impact Factor
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    • "The role of the latter (Cys186–Cys209) in the regulation of TF function and the mechanism by which it is formed on the cell surface has been the subject of debates for the last years [174–189]. The suggested range of importance for this bond is from essential [180, 189] to not having any effect on TF function [186, 188]. Bach and coworkers suggested in 1981 that preservation of disulfides is necessary for TF activity [160]. "
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    ABSTRACT: Tissue factor (TF) is an integral membrane protein that is essential to life. It is a component of the factor VIIa-TF complex enzyme and plays a primary role in both normal hemostasis and thrombosis. With a vascular injury, TF becomes exposed to blood and binds plasma factor VIIa, and the resulting complex initiates a series of enzymatic reactions leading to clot formation and vascular sealing. Many cells, both healthy, and tumor cells, produce detectable amounts of TF, especially when they are stimulated by various agents. Despite the relative simplicity and small size of TF, there are numerous contradictory reports about the synthesis and presentation of TF on blood cells and circulation in normal blood either on microparticles or as a soluble protein. Another subject of controversy is related to the structure/function of TF. It has been almost commonly accepted that cell-surface-associated TF has low (if any) activity, that is, is "encrypted" and requires specific conditions/reagents to become active, that is, "decrypted." However there is a lack of agreement related to the mechanism and processes leading to alterations in TF function. In this paper TF structure, presentation, and function, and controversies concerning these features are discussed.
    12/2012; 2012(7):964862. DOI:10.6064/2012/964862
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