Tissue factor: mechanisms of decryption

Abstract

It is generally believed that only a small fraction of the tissue factor (TF) found on cell surfaces is active whereas the vast majority is cryptic in coagulation. It is unclear how cryptic TF differs from the coagulant active TF or potential mechanisms involved in transformation of cryptic TF to the coagulant active form. Exposure of phosphatidylserine (PS) in response to various chemical or pathophysiological stimuli has been considered as the most potent inducer of TF decryption. In addition to PS, TF self-association and association with specialized membrane domains may also play a role in TF decryption. It has been suggested recently that protein disulfide isomerase regulates TF decryption through its oxidoreductase activity by targeting Cys186-Cys209 disulfide bond in TF extracellular domain or regulating the PS equilibrium at the plasma membrane. However, this hypothesis requires further validation to become an accepted mechanism. In this article, we critically review literature on TF encryption/decryption with specific emphasis on recently published data and provide our perspective on this subject.

Figure 1. Lack of correlation between expression of TF-FVIIa procoagulant activity and FVIIa binding to TF on cell surfaces

Monolayers of OC-2008 cell line expressing TF were incubated in parallel sets with 10 nM of unlabelled or ¹²⁵I-labeled FVIIa. In one set, active site-inhibited FVIIa (FVIIai, 10 nM) were added for 1 min and the cells were washed quickly three times with ice-cold Ca²⁺-containng buffer before adding FVIIa. In all cases, at the end of specified incubation period, the cells were washed six times with ice-cold Ca²⁺-containing buffer and TF-FVIIa procoagulant activity at the cell surface was determined in factor X activation assay or FVIIa bound to TF was determined by measuring the radioactivity of ¹²⁵I-FVIIa bound to cells (adopted from Le et al.[8]).

Figure 2. A hypothetical model for tissue factor decryption

On unperturbed cells expressing TF, FVIIa binds to all available TF sites, however majority of these complexes unable to interact with substrate factor X. Upon cell perturbation that exposes to anionic phospholipids at the outer cell surface membrane, TF or TF-FVIIa complexes interact with anionic phospholipids, leading to a conformational change in TF-FVIIa that facilitate its interaction with factor X, thus transforming cryptic TF to coagulant active form.

Figure 3. PDI is localized intracellularly in endothelial cells and not secreted in response to thrombin

(A) Cellular localization of PDI by immunofluorescence confocal microscopy. HUVEC transfected with TF adenovirus were stimulated with thrombin for 10 or 30 min (0.5 U/ml) or left unperturbed. After the treatment, the cells were fixed and permeabilized with 0.25% Triton X-100. Both the intact and permeabilized cells were immunostained with rabbit anti-human TF IgG and monoclonal PDI antibody (RL90), followed by Oregon Green-labeled anti-rabbit IgG and Rhodamine Red-conjugated anti-mouse IgG as secondary reporter antibodies. The bottom panel shows staining with isotytpe controls IgG (B) HUVEC cultured in 24-well plate were treated with 500 μl of control serum-free medium or serum-free medium containing thrombin (0.5 U/ml) for varying time periods. At the end of the treatment, the whole supernatant medium was collected and concentrated by 25 times (to ~20 μl) by ultrafiltration and the cells were lysed in 100 μl of lysis buffer. Equal volumes (10 μl) of cell lysates and the concentrated supernatant media were subjected to SDS-PAGE and immunoblotted with anti-PDI antibodies.

Figure 4. The effect PDI mAb, before and after dialysis, on tissue factor procoagulant activity in endothelial cells

PDI mAb (clone #34b) was obtained from BD Biosciences, which was supplied at 250 μg/ml concentration in glycerol. A portion of the antibody was dialyzed overnight against 10 mM Hepes buffer to remove the glycerol. Confluent monolayers of HUVEC were treated with pre and post-dialyzed PDI mAb (10 μg/ml) for 30 min at room temperature. In parallel wells, HUVEC were also treated with glycerol (2 and 5%). At the end of 30 min treatment, FVII (10 nM) and FX (175 nM) was added to cells and the rate of FX activation was measured in a chromogenic assay.