Cryptic Vs. Active Tissue Factor: Cystine186-Cystine 209 Disulfide Bond Switch Is Not the Answer.

Abstract 333

Tissue factor (TF) plays the key role in initiation of blood coagulation by allosterically activating coagulation factor VIIa (FVIIa). TF is essential for hemostasis but aberrant expression would lead to thrombotic disorders. TF on cell surfaces resides mostly in a cryptic state. At present it is not entirely clear how cryptic TF differs from procoagulant active TF and how de-encryption occurs. Recent studies have suggested that cryptic and active TF may have different conformations, i.e., cryptic TF contains unpaired cysteine thiols at Cys 186 and Cys 209 in the membrane proximal domain whereas procoagulant active TF contains the oxidized Cys186-Cys209 disulfide bond. It has been suggested that the catalytic switching of this disulfide bond between reduced and oxidized states by protein disulfide isomerase (PDI) might modulate the activity status of TF. Although the validity of this hypothesis has been questioned, more recent reports have supported the contention that PDI-catalyzed disulfide bond formation of Cys186-Cys209 in TF plays a role in regulation of TF activity both in vitro and in vivo. To reconcile the conflicting data in the recent literature, we re-examined in the present study the central dogma on which the hypothesis was originated, i.e., a TF mutant lacking Cys186-Cys 209 disulfide bond is functionally inactive (cryptic) and that TF activation requires formation of the Cys186-Cys209 disulfide bond. CHO cells were transfected with wild type TF or TF mutants that selectively preclude Cys186-Cys209 disulfide bond formation, i.e., TF_C186S, TF_C209S, or TF_C186S/C209S, and TF procoagulant and antigen expression levels at the cell surface were quantified accurately by measuring TF procoagulant activity in a factor × activation assay and TF antigen levels in radioligand binding studies utilizing two different TF mAb (10H10 and 5G9) and FVIIa. CHO cells transfected with TF_C186S, TF_C209S or TF_C186S/C209S expressed very little procoagulant activity (less than 1% of CHO cells transfected with wild-type TF under identical experimental conditions). However, TF mAb and FVIIa binding studies revealed that CHO cells transfected with TF_C186S, TF_C209S or TF_C186S/C209S expressed very little TF protein on the cell surface (∼ 1 to 2% of CHO cells transfected with wild-type TF). Similar results were obtained whether we mutated Cys 186 or Cys 209 to Ser or Ala or whether we used transient or stable transfections. To confirm that the lower expression of TF_C186S, TF_C209S or TF_C186S/C209S is neither an artifact nor limited to CHO cell model system, we extended these studies to endothelial cells. Human umbilical vein endothelial cells (HUVECs) were transduced with adenovirus particles (25 moi/cell) encoding wild-type TF, TF_C186S, TF_C209S or TF_C186S/C209S and TF antigen levels at the cell surface and in cell lysates were measured using multiple methods - confocal microscopy, FACS, TF antigen assay, immuno blot analysis, TF mAb binding and FVIIa binding. HUVEC transduced with adenoviral particles encoding TF_C186S, TF_C209S or TF_C186S/C209S expressed very low levels of TF antigen both at the cell surface ( 1 to 5%) as well as in total (10 to 15%) (TF_C186S > TF_C209S >TF_C186S/C209S) compared to HUVEC transduced with the same number of adenovirus particles expressing wild-type TF. Cell surface TF activity measured in the presence of saturating concentrations of FVIIa revealed that the TF procoagulant activity of the mutants correlate well with the amount of TF antigen present on the cell surface, i.e., there were no significant differences in the specific activity of TF mutants and wild-type TF (TF procoagulant activity/amount of TF antigen, pM FXa formed/femto mole TF: wild-type TF, 68 ± 6; TF_C186S, 55 ± 10; TF_C209S, 57 ± 17; and TF_C186S/C209S, 64 ± 0.2). More importantly, treatment of HUVEC with the thiol-oxidizing agent HgCl₂ or with ionomycin increased the cell surface TF activity to the same extent (200 to 400% increase over HUVEC treated with control vehicle) in HUVEC expressing TF_C186S/C209S or wild-type TF. In summary the present data provide clear evidence for that TF lacking Cys186-Cys209 bond is coagulantly active and a lower TF procoagulant activity in cells expressing the mutant TF that precludes Cys186-Cys209 disulfide bond formation is due to severe impairment in TF protein synthesis/processing and not because the mutant TF assumes the cryptic conformation. Our data also show that TF de-encryption does not require the formation of Cys186-Cys209 disulfide bond.