Binding of Human Factor VIIa to Tissue Factor Induces Cytosolic Ca2+ Signals in J82 Cells, Transfected COS-1 Cells, Madin-Darby Canine Kidney Cells and in Human Endothelial Cells Induced to Synthesize Tissue Factor (∗)
Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions resulting in thrombin production. The cloning of TF revealed its structural characteristics to be those of a receptor related to the class 2 cyt...
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Published in | The Journal of biological chemistry Vol. 270; no. 9; pp. 4650 - 4660 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
03.03.1995
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Subjects | |
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Abstract | Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions resulting in thrombin production. The cloning of TF revealed its structural characteristics to be those of a receptor related to the class 2 cytokine receptor superfamily, but until now no intracellular signal has been discovered related to binding of the ligand (FVIIa) to the putative receptor. We have studied possible intracellular signaling effects of the FVIIa-TF interaction by measuring cytosolic free Ca2+ in single fura-2-loaded cells and found that 200 nM FVIIa caused Ca2+ transients in about 30% of human umbilical vein endothelial cells treated with interleukin-1β to express TF, compared to below 5% in uninduced cells. A gradual increase of the basal Ca2+ level was also caused by binding of FVIIa. In the human bladder carcinoma cell line J82, which has a high constitutive TF activity, similar results were found. An antibody neutralizing TF activity decreased the response rate to control levels. COS-1 cells which do not make TF did not respond to FVIIa as opposed to COS-1 cells expressing TF after transfection with a human TF cDNA construct. The canine kidney cell line MDCK, a constitutive TF producer, responded especially well; up to 100% of the cells examined showed Ca2+ oscillations which were dose dependent with regard to frequency, latency, maximal amplitude, and recruitment of responding cells. The frequency was reduced by inhibition of Ca2+ influx with 100 μM LaCl3. In confluent MDCK cells the Ca2+ oscillations were synchronous, constituting the first evidence of a synchronous cytosolic Ca2+ oscillator generated by global application of agonist. Thus, TF mediates a cytosolic Ca2+ signal upon interaction with its ligand FVIIa, thereby suggesting a more complex biological role for TF. |
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AbstractList | Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions resulting in thrombin production. The cloning of TF revealed its structural characteristics to be those of a receptor related to the class 2 cytokine receptor superfamily, but until now no intracellular signal has been discovered related to binding of the ligand (FVIIa) to the putative receptor. We have studied possible intracellular signaling effects of the FVIIa-TF interaction by measuring cytosolic free Ca2+ in single fura-2-loaded cells and found that 200 nM FVIIa caused Ca2+ transients in about 30% of human umbilical vein endothelial cells treated with interleukin-1β to express TF, compared to below 5% in uninduced cells. A gradual increase of the basal Ca2+ level was also caused by binding of FVIIa. In the human bladder carcinoma cell line J82, which has a high constitutive TF activity, similar results were found. An antibody neutralizing TF activity decreased the response rate to control levels. COS-1 cells which do not make TF did not respond to FVIIa as opposed to COS-1 cells expressing TF after transfection with a human TF cDNA construct. The canine kidney cell line MDCK, a constitutive TF producer, responded especially well; up to 100% of the cells examined showed Ca2+ oscillations which were dose dependent with regard to frequency, latency, maximal amplitude, and recruitment of responding cells. The frequency was reduced by inhibition of Ca2+ influx with 100 μM LaCl3. In confluent MDCK cells the Ca2+ oscillations were synchronous, constituting the first evidence of a synchronous cytosolic Ca2+ oscillator generated by global application of agonist. Thus, TF mediates a cytosolic Ca2+ signal upon interaction with its ligand FVIIa, thereby suggesting a more complex biological role for TF. Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions resulting in thrombin production. The cloning of TF revealed its structural characteristics to be those of a receptor related to the class 2 cytokine receptor superfamily, but until now no intracellular signal has been discovered related to binding of the ligand (FVIIa) to the putative receptor. We have studied possible intracellular signaling effects of the FVIIa-TF interaction by measuring cytosolic free Ca2+ in single fura-2-loaded cells and found that 200 nM FVIIa caused Ca2+ transients in about 30% of human umbilical vein endothelial cells treated with interleukin-1 beta to express TF, compared to below 5% in uninduced cells. A gradual increase of the basal Ca2+ level was also caused by binding of FVIIa. In the human bladder carcinoma cell line J82, which has a high constitutive TF activity, similar results were found. An antibody neutralizing TF activity decreased the response rate to control levels. COS-1 cells which do not make TF did not respond to FVIIa as opposed to COS-1 cells expressing TF after transfection with a human TF cDNA construct. The canine kidney cell line MDCK, a constitutive TF producer, responded especially well; up to 100% of the cells examined showed Ca2+ oscillations which were dose dependent with regard to frequency, latency, maximal amplitude, and recruitment of responding cells. The frequency was reduced by inhibition of Ca2+ influx with 100 microM LaCl3. In confluent MDCK cells the Ca2+ oscillations were synchronous, constituting the first evidence of a synchronous cytosolic Ca2+ oscillator generated by global application of agonist. Thus, TF mediates a cytosolic Ca2+ signal upon interaction with its ligand FVIIa, thereby suggesting a more complex biological role for TF. |
Author | Iversen, Jens-Gustav Enden, Tone R⊘ttingen, John-Arne Prydz, Hans Camerer, Eric |
Author_xml | – sequence: 1 givenname: John-Arne surname: R⊘ttingen fullname: R⊘ttingen, John-Arne organization: Laboratory of Intracellular Signalling, Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, N-0371 Oslo, Norway – sequence: 2 givenname: Tone surname: Enden fullname: Enden, Tone organization: Biotechnology Centre of Oslo, University of Oslo, N-0371 Oslo, Norway – sequence: 3 givenname: Eric surname: Camerer fullname: Camerer, Eric organization: Biotechnology Centre of Oslo, University of Oslo, N-0371 Oslo, Norway – sequence: 4 givenname: Jens-Gustav surname: Iversen fullname: Iversen, Jens-Gustav organization: Laboratory of Intracellular Signalling, Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, N-0371 Oslo, Norway – sequence: 5 givenname: Hans surname: Prydz fullname: Prydz, Hans organization: Biotechnology Centre of Oslo, University of Oslo, N-0371 Oslo, Norway |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/7876236$$D View this record in MEDLINE/PubMed |
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Snippet | Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions... |
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SubjectTerms | Animals Antibodies Binding Sites Calcium - metabolism Cell Line Cells, Cultured Cytosol - metabolism Dogs Endothelium, Vascular - cytology Endothelium, Vascular - metabolism Factor VIIa - metabolism Humans Kidney - cytology Kidney - metabolism Protein Binding Signal Transduction Thromboplastin - biosynthesis Thromboplastin - metabolism Transfection |
Title | Binding of Human Factor VIIa to Tissue Factor Induces Cytosolic Ca2+ Signals in J82 Cells, Transfected COS-1 Cells, Madin-Darby Canine Kidney Cells and in Human Endothelial Cells Induced to Synthesize Tissue Factor (∗) |
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