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-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.
Highlights
From the tLaboratory of Intracellular Signalling, Department of Physiology, Institute of Basic Medical Sciences and the §Biotechnology Centre of Oslo, University of Oslo, N-037I Oslo, Norway
We have studied possible intracellular signaling effects of the FVIIa·Tissue factor (TF) interaction by measuring cytosolic free Ca2 + in single fura-2-10aded cells and found that 200 11M FVIIa caused Ca2 + transients in about 30% of human umbilical vein endothelial cells treated with interleukin-113 to express TF, compared to below 5% in uninduced cells
Effects ofFVIIa Binding to TF in HUVEC and J82 Cells-TF is constitutively produced in J82 cells with high surface availability
Summary
Vol 270, No., Issue of March 3, pp. 4650-4660, 1995 Printed in U.S.A. Binding of Human Factor VIla to Tissue Factor Induces Cytosolic Ca2 + Signals in J82 Cells, Transfected COS-! Cells, Madin-Darby. A gradual increase of the basal Ca2 + level was caused by binding of FVIIa. In the human bladder carcinoma cell line J82, which has a high constitutive TF activity, similar results were found. We chose to study the effect of FVIIa binding to TF on [Ca2 +]c in single cells loaded with the fluorescent Ca2+ indicator fura-2 in a digital imaging system To look for such a Ca2+ response, we took advantage of the ability of interleukin-l{3 (lL-l{:l) to induce TF in HUVEC [14]. A similar increase in Ca2 + levels was detected upon binding of FVIIa to TF in the human bladder carcinoma cell line J82 which produces TF constitutively and in monkey kidney fibroblasts (COS-I) stably transfected with a human TF construct.
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