Search

Tissue factor (TF) is a transmembrane protein that functions in the initiation of blood coagulation in vivo. At sites of vascular injury, TF serves as a cell-surface receptor for the serine protease factor VIIa (FVIIa), forming an enzyme--cofactor complex and enhancing the catalytic activity of FVIIa. Tissue factor, along with the receptors for alpha- and gamma-interferons, is a member of the class 2 cytokine receptor superfamily. Crystallographic analysis demonstrated that the extracellular domain of TF consists of two immunoglobulin-like domains joined by a linker region. Each domain is comprised of two antiparallel beta-sheets containing seven conserved beta-strands separated by more variable loop regions. Extensive mutagenesis has been performed in order to map the FVIIa binding site on TF. Results indicated that the discontinuous binding site for FVIIa lies at the domain--domain interface and includes residues from extended loops and beta-strands within both the N- and C-terminal domains. Our previous study provided evidence that three consecutive residues (D44, W45, K46) within the TF loop region between beta-strands C and C' of the N-terminal domain were important for interactions with FVIIa. We have presently extended our alanine-scanning mutagenesis to include the residues within the flanking beta-strands. Thirteen sTF mutants were screened for their ability to enhance FVIIa activity. Three residues within strand C (Y34, Q37, I38) and two residues within C' (K48, Y51) were shown to be important for TF cofactor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue factor (TF) is a membrane-bound glycoprotein that functions as a cofactor for coagulation factor VIIa (VIIa) and initiates blood coagulation at sites of vascular injury. On the basis of sequence alignments, TF was predicted to be a member of the cytokine receptor superfamily. Utilizing the structural information available for the cytokine receptor superfamily, we have used site-directed mutagenesis to identify the binding site on TF for VIIa. The predicted loop regions in TF were systematically replaced with the homologous loops from the gamma-interferon receptor (gamma-IFN-R), the protein most related to TF in the superfamily of cytokine receptors. Six discontinuous regions (residues 16-20, 40-46, 60-69, 101-111, 129-151, 193-207) were identified that are required for interaction with VIIa and enhancement of activity. Individual substitution of 68 residues within these loops with alanine revealed eight residues (K20, D44, W45, K46, Q110, R135, F140, V207) that are required for cofactor activity. These residues fall into two groups, those that are required only for interactions with VIIa (K46, Q110, R135, F140, V207) and those that are also required to induce the conformational change in VIIa required for enhanced activity (K20, D44, W45). The discontinuous regions of TF required for interactions with VIIa form a single binding surface for VIIa that is analogous to the interface defined by the crystal structure of the complex between growth hormone and its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Full-length tissue factor (263 rTF) and three truncated forms have been expressed in human kidney 293 cells; 1) 243 rTF, which lacks the cytoplasmic tail, is fully functional in the chromogenic assay and has a specific activity comparable with that of the full-length molecule, 263 rTF; 2) 219 rTF, which lacks both the transmembrane and cytoplasmic domains, is not functional; 3) the third variant, referred to as TF-PI, is a fusion protein containing the extracellular domain of TF (amino acids 1-219) fused to the last 37 amino acids of decay-accelerating factor which contain a signal for attachment of a phosphatidylinositol membrane anchor (PI). TF-PI is a membrane-bound protein expressed on the cell surface. The PI anchor restores TF activity lost when the transmembrane domain is deleted from the 219 rTF variant. The ability of the PI anchor to restore activity to 219 rTF clearly demonstrates that while the transmembrane domain is not required for TF activity, lipid association is required.

The possible self-association of tissue factor molecules was investigated by treating cells expressing tissue factor with bifunctional cross-linking agents. The two reagents chosen were 3,3'-dithiobis(sulfosuccinimidylpropionate) and sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate, both of which are membrane-impermeable and thiol-cleavable. A human bladder carcinoma cell line, J82, and a transfected human kidney cell line expressing high amounts of recombinant tissue factor were used in these studies. Exposure of the intact cells to the crosslinking reagents was found to result in the formation of multimeric tissue factor-containing complexes, the extent of which appeared to be dependent upon the amount of tissue factor expressed by the cell. The self-association of tissue factor was prevented in a variant tissue factor molecule harboring a non-homologous transmembrane domain.

Recombinant human tissue factor (rTF) purified from transfected mammalian cells is a glycoprotein that contains N-linked, but not O-linked oligosaccharides. Two of the three potential N-linked sites in the extracellular portion are fully glycosylated, while one site is approximately 90% utilized. These sites have complex-type oligosaccharides attached. The potential N-linked site in the cytoplasmic domain near the C-terminus is not glycosylated. Characterization of the tryptic map of rTF confirmed most of the proposed amino acid sequence. In addition, the disulfide bonds (between Cys-49 and Cys-57 and between Cys-186 and Cys-209) were demonstrated by FAB-MS analysis of cysteine-containing fragments obtained from the tryptic map.

We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.