thromboplastin and Chromosome-Deletion

The activation of human blood coagulation factor VII can occur by the feedback activity of either factor VIIa (autoactivation) or factor Xa. Both of these reactions are known to be enhanced by the presence of tissue factor, an integral membrane protein and the cofactor for factor VIIa. We examine here the activation of 125I-factor VII by both factor VIIa and factor Xa employing a mutant soluble form of tissue factor which has had its transmembrane and cytoplasmic domains deleted (sTF1-219). This mutant soluble tissue factor retains cofactor activity toward factor VIIa in a single-stage clotting assay but shows a strong dependence on initial plasma levels of factor VIIa (from 1 to 10,000 ng/ml) when compared to wild-type tissue factor. We show that this dependence is due to a deficiency of sTF1-219 in ability to both promote autoactivation and enhance the factor Xa-catalyzed activation of 125I-factor VII. sTF1-219 does not, however, inhibit the tissue factor-independent activation of 125I-factor VII by factor Xa. The results strongly suggest that the phospholipid anchoring region of tissue factor is essential for autoactivation and beneficial for factor Xa-catalyzed activation of 125I-factor VII. In addition, when taken together with the dependence of clotting times on initial factor VIIa levels observed with sTF1-219, these results indicate that factor VII autoactivation may be of greater importance in the initiation of blood coagulation via tissue factor than has been previously realized.

Topics: Cell Line; Chromatography, Affinity; Chromatography, Ion Exchange; Chromosome Deletion; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Factor VII; Factor VIIa; Factor Xa; Feedback; Humans; Kinetics; Molecular Weight; Mutation; Recombinant Proteins; Thromboplastin

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.

Topics: Amino Acid Sequence; Base Sequence; Cell Line; Cell Membrane; Chromosome Deletion; Factor VII; Fluorescent Antibody Technique; Genetic Vectors; Glycolipids; Glycosylphosphatidylinositols; Humans; Molecular Sequence Data; Phosphatidylinositols; Plasmids; Restriction Mapping; Thromboplastin; Transfection

We have cloned a serum- and cycloheximide-inducible mRNA from AKR-2B murine fibroblasts which encodes a protein with significant sequence similarity to human tissue factor, a cellular initiator of the blood coagulation cascade. Information derived from this clone was used to establish the presence of a virtually identical sequence in mouse brain. Most importantly, cDNA-directed expression in a quail fibroblast cell line produced high levels of tissue factor procoagulant activity, confirming the identity of this protein as murine tissue factor. Additional studies demonstrate that transforming growth factor type beta 1 stimulates tissue factor gene transcription and is a potent inducer of tissue factor procoagulant activity in fibroblasts. Other tested mitogens such as platelet-derived growth factor, epidermal growth factor, and insulin were weak inducers. These results may reflect a role for transforming growth factor beta 1 in the maintenance of hemostasis or, alternatively, a role for tissue factor in cellular functions unrelated to blood coagulation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Brain; Cell Line; Chromosome Deletion; Cloning, Molecular; Cycloheximide; Gene Expression Regulation; Gene Library; Humans; Kinetics; Mice; Mice, Inbred AKR; Molecular Sequence Data; Oligonucleotide Probes; RNA, Messenger; Sequence Homology, Nucleic Acid; Thromboplastin; Transfection; Transforming Growth Factor beta

Tissue factor (TF) is the primary initiator of the coagulation protease cascades. This cell surface glycoprotein is the receptor and essential cofactor for the serine protease factor VIIa. TF is constitutively expressed in some extravascular cell types and is transiently induced in monocytes, endothelial cells, and fibroblasts. Inducible expression is implicated in cellular immune responses, inflammation, and intravascular coagulation. Transcriptional regulation of the TF promoter was analyzed in COS-7 cells under conditions of (i) high-level expression and (ii) serum induction. The region comprising nucleotides -209 to +121 (relative to the transcription start site) supports high-level transcriptional activity and can be divided into two distinct regions: a region (-111 to +121) that exhibited low promoter activity and a region (-209 to -112) that enhanced transcriptional activity to a high level. The role of further upstream sequences is still to be established, although two consensus binding sites for the transcriptional activator protein AP-1 did enhance low-level promoter activity. In serum-starved COS-7 cells TF expression was transiently increased 20-fold by serum. All transcriptionally active constructs were responsive to serum, indicating the presence of at least one serum response element, whose function was retained in the immediate 5' aspect of the gene, at -111 to +14. Based on this functional map, we propose that the elaborate pattern of TF expression by cells results from a relatively complex promoter.

Topics: Animals; Base Sequence; Cell Line; Chromosome Deletion; Enhancer Elements, Genetic; Exons; Gene Expression Regulation; Humans; Kinetics; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Promoter Regions, Genetic; Thromboplastin; Transfection