BINDING OF HUMAN FACTOR VIIA TO TISSUE FACTOR INDUCES CYTOSOLIC CA2-1CELLS, MADIN-DARBY CANINE KIDNEY-CELLS AND IN HUMAN ENDOTHELIAL-CELLSINDUCED TO SYNTHESIZE TISSUE FACTOR( SIGNALS IN J82 CELLS, TRANSFECTED COS)

Tissue factor (TF) is the most potent trigger of blood clotting known, It activates factor VII (FVII) thereby initiating a cascade of proteo lytic reactions resulting in thrombin production, The cloning of TF re vealed its structural characteristics to be those of a receptor relate d to the class 2 cytokine receptor superfamily, but until now no intra cellular signal has been discovered related to binding of the ligand ( FVIIa) to the putative receptor, We have studied possible intracellula r signaling effects of the FVIIa-TF interaction by measuring cytosolic free Ca2+ in single fura-2-loaded cells and found that 200 nM FVIIa c aused Ca2+ transients in about 30% of human umbilical vein endothelial cells treated with interleukin-1 beta to express TF, compared to belo w 5% in uninduced cells, A gradual increase of the basal Ca2+ level wa s 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 respons e rate to control levels, COS-1 cells which do not make TF did not res pond to FVIIa as opposed to COS-1 cells expressing TF after transfecti on with a human TF cDNA construct, The canine kidney cell line MDCK, a constitutive TF producer, responded especially well; up to 100% of th e cells examined showed Ca2+ oscillations which were dose dependent wi th regard to frequency, latency, maximal amplitude, and recruitment of responding cells, The frequency was reduced by inhibition of Ca2+ inf lux with 100 mu M LaCl3, In confluent MDCK cells the Ca2+ oscillations were synchronous, constituting the first evidence of a synchronous cy tosolic Ca2+ oscillator generated by global application of agonist, Th us, TF mediates a cytosolic Ca2+ signal upon interaction with its liga nd FVIIa, thereby suggesting a more complex biological role for TF.