Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA.

Abstract

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. X Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin Kdependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).Blood coagulation can be initiated by a complex of tissue factor (TF), a membrane-bound glycoprotein, and factor VII, a plasma coagulation factor (for reviews, see refs. 1 and 2). The physiological significance of this extrinsic pathway can be judged by the severe bleeding frequently observed in individuals who are markedly deficient in factor VII (3, 4). In contrast, individuals who have deficiencies or abnormalities in proteins that are involved in the early steps of the intrinsic pathway of coagulation-i.e., high molecular weight kininogen, prekallikrein, and factor XII-are asymptomatic (5). The TF-factor VII complex activates factor IX, a component of the intrinsic pathway, as well as factor X (6). Thus, it is reasonable that the association of TF and factor VII may be the crucial event triggering the initiation of clotting in vivo.The cDNAs for all of the proteins involved in TF-initiated coagulation, with the exception of TF itself, have already been cloned and sequenced (7-13). The TF apoprotein has been purified from both bovine and human sources (14-16). Approximately 50-70% of the amino acid sequence of both species has now been determined, and this has permitted us to select suitable amino acid sequences to serve as a basis for constructing oligonucleotide probes. This in turn has enabled the isolation and characterization of two human placental TF cDNA clones that contain the entire coding region of the mature protein. The nucleotide sequence of these clones, together with amino acid sequence data, has allowed us to formulate a primary structure for the human TF apoprotein §. MATERIALS AND METHODSTF Purification and Sequencing. A monoclonal antibody (17) prepared against human TF that had been purified by using factor VII affinity columns (15) was used for immunoaffinity isolation of TF. Briefly, TF was extracted from human brain or placental tissue acet...

Comparison of natural and recombinant tissue factor proteins: new insights

“…The N-terminal analysis showed that both recombinant and placental proteins exist in two forms, a full-length form composed of 263 amino acid residues and a truncated form composed of 261 residues, where the first two amino acids on the N-terminus of the protein are missing. This observation was consistent with previously published data (Spicer et al , 1987 ;Paborsky et al , 1989 ). The fractional abundance of each form in the two proteins is different.…”

“…TF is a 263-amino-acid lipoprotein containing three domains ( Figure 1 ): (1) an extracellular domain representing the amino-terminal part of the molecule (residues 1 -219); (2) a trans-membrane domain, which anchors TF to the membrane (residues 220 -242); and (3) a cytoplasmic carboxy-terminal domain (residues 243 -263) (Spicer et al , 1987 ). The extracellular domain of TF participates in interactions with factor VIIa at multiple sites forming a complex enzyme.…”

Increased expression of T‐plastin gene in cisplatin‐resistant human cancer cells: identification by mRNA differential display

“…Nucleotide sequences of clones dd72 (A) and dd78 (B) aligned with the corresponding regions of human T-plastin and human tissue factor (TF) genes, respectively. Dashes represent identical residues, flanking primer sequences are underlined, and numbering of T-plastin [37] and tissue factor [38] sequences is as previously described.…”

Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation.

“…The retroviral vector LXSN(12) was used to construct all of the vectors for transfecting normal human TF cDNA (13) and two mutant TF cDNAs into melanoma cells (Fig. 1).…”