Abstract
Tissue factor (TF) is the cellular cofactor for the serine protease coagulation factor VIIa (FVIIa). The TF-FVIIa complex formed on the cell surface initiates the coagulation cascade. It is believed that most of the TF molecules on the cell surface of a resting cell exist in an encrypted state with very little procoagulant activity. Encrypted TF must undergo decryption to become fully active. The exact mechanisms by which TF activity on the cell surface is regulated are unknown. Exposure of phosphatidylserine (PS) to the outer leaflet of the cell membrane is thought to play a critical role in TF decryption. Recent studies of molecular dynamics simulation of TF ectodomain in solution and on the surface of anionic phospholipids suggested a direct interaction of PS headgroups with specific residues in TF. At present, the role of the putative lipid interactive residues of TF in TF decryption is unknown. In the present study, we investigated the potential role of TF direct interaction with the cell surface lipids on basal TF activity as well as enhanced TF activity following the decryption using different TF mutants. Plasmids or adenoviral constructs encoding wild-type or mutant TF (mutations in the putative lipid binding region) were used to transduce TF expression in CHO-K1 or monocytic THP-1 cells, respectively. TF protein expression level at the cell surface and FVIIa binding to the cell surface TF were evaluated by radioligand binding studies using 125I-labeled TF mAb or FVIIa, respectively. TF-FVIIa coagulant activity on the cell surface was determined in FX activation assay. Data of these studies showed that all TF mutants were capable of interacting with FVIIa with no apparent defect. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A-exhibited a similar or slightly higher TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A and TFK166A, was reduced substantially to a range of 40% - 70% of that of wild-type TF. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% loss of TF specific activity. Mutation of all nine lipid binding residues together (DLBR) did not further decrease the specific activity of TF anymore than that of mutation of G164 alone. Comparison of the present data with the published data on these mutants revealed that some of the TF residues that are critical for regulating TF activity on liposomes are not crucial for TF activity on the cell surface. To address whether the decreased FXa generation seen with the select TF variants is caused by changes in TF-membrane interaction or by the substrate interaction with TF/FVIIa complex, we performed Michaelis-Menten kinetics of FX activation for two of TF mutants (TFS163A and TFG164A). Results of this study suggested that there were no significant differences in Km values between wild-type TF and TF mutants (wild-type TF, 51 ± 14.6 nM; TFS163A, 68 ± 19.5 nM; TFG164A, 39 ± 18.4 nM, n=4). Interestingly, mutation of the selective residues in the lipid binding region failed to abrogate the PS-dependent TF decryption. The fold-increase in TF activity in cells expressing wild-type TF or TF variants was similar following cell activation with either HgCl2 or calcium ionomycin treatment. Annexin V markedly diminished the increased TF-FVIIa activation of FX in cells expressing wild-type TF as well as cells expressing the TF mutant (DLBR mutant). Overall, our data suggest that the regulation of TF activity at the cell surface milieu may be different from that of PC/PS vesicles and TF region other than earlier identified LBR may be responsible for enhancing TF activity following the PS exposure.
Disclosures
No relevant conflicts of interest to declare.
Characterization of the Coagulant Activity of Cultured Human Fibroblasts