COAGULATION-FACTOR-VII AND COAGULATION-FACTOR-X INDUCE CA2-DARBY CANINE KIDNEY-CELLS ONLY WHEN PROTEOLYTICALLY ACTIVE( OSCILLATIONS IN MADIN)

We have recently reported that the activated serine protease and blood coagulation Factor VII (FVIIa) can induce Ca2+ oscillations in Madin- Darby canine kidney cells. We now demonstrate a similar response by Ma din-Darby canine kidney cells to the active coagulation Factor X (FXa) , which is also a serine protease and a substrate of the tissue factor (TF)-FVIIa complex in the initiation of the coagulation cascade. The phosphatidyl inositol-specific phospholipase C inhibitor U73122 inhibi ted the signals elicited by both FVIIa and FXa. Lack of sensibility to the tyrosine kinase inhibitors herbimycin A, genistein, and the tyrph ostin AG18 and discordance between TF expression and FVIIa responsiven ess argued against TF acting as a cytokine-like receptor, with tyrosin e kinase-mediated activation by FVIIa. As demonstrated using the prote ase inhibitor benzamidine and by specific active site inhibition with 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone, both FVIIa and FXa lost th eir ability to elicit a calcium response when devoid of their proteoly tic activity. Consistent with this, the native (zymogen) form of Facto r X did not induce Ca2+ transients. Homologous but not heterologous in hibition of FVIIa- and FXa-evoked Ca2+ signals by 1,5-dansyl-Glu-Gly-A rg chloromethyl ketone-inactivated FVIIa and FXa suggested that each f actor had its own specific cell surface anchoring receptor. The two co agulation factors did not show homologous desensitization as seen for thrombin stimulation. Studies with hirudin excluded involvement of the established activation pathway through thrombin itself. Lack of desen sitization of the response to FVIIa or FXa by thrombin ruled out any i nvolvement of proteinase activated receptor-1 (PAR-1), the thrombin re ceptor. We speculate that FXa and FVIIa may work via a receptor (possi bly common) analogous to PAR-I or its functional homologue PAR-2. Alth ough TF is essential for the FVIIa-induced signaling event; its role i n the phosphatidyl inositol-specific phospholipase C-mediated Ca2+ sig nal may be in anchoring FVIIa to the cell surface rather than in trans membrane signal mediation.