KINETICS OF THROMBIN RECEPTOR CLEAVAGE ON INTACT-CELLS - RELATION TO SIGNALING

Thrombin, a protease generated at sites of vascular injury, signals ce llular responses vital for hemostasis and thrombosis. How thrombin, an enzyme rather than a classical ligand, effects graded and concentrati on-dependent responses in its target cells has been a long-standing qu estion. Thrombin activates its receptor by cleaving off an activation peptide to unmask a tethered peptide ligand. We utilized a thrombin re ceptor with an epitope-tagged activation peptide to directly demonstra te thrombin receptor cleavage and to examine the kinetics of receptor activation on intact cells. The rate of thrombin receptor cleavage was proportional to thrombin concentration over the physiologic range, bu t low thrombin concentrations ultimately cleaved and activated all rec eptors. Cumulative phosphoinositide hydrolysis in response to thrombin correlated precisely with cumulative receptor cleavage. These data st rongly suggest that each cleaved and activated thrombin receptor produ ces a ''quantum'' of phosphatidylinositol hydrolysis, then shuts off. Surprisingly, this shut off occurred despite the continued presence of cleaved and ''activated'' receptors on the cell surface and at a time when the cells were refractory to thrombin but sensitive to agonist p eptide, suggesting that a novel shut off mechanism may have evolved to deal with the tethered ligand. Unlike the case with classical ligands , cells thus cannot detect differences in thrombin concentrations as d ifferences in fractional occupancy but rather must sense different rat es of receptor activation. Because each cleaved thrombin receptor gene rates a quantum of second messenger, the magnitude of the cell's respo nse to thrombin must be determined by the balance between rates of rec eptor activation and second messenger clearance.