Kinetics of thrombin receptor cleavage on intact cells. Relation to signaling

摘要: Thrombin, a protease generated at sites of vascular injury, signals cellular responses vital for hemostasis and thrombosis. How thrombin, an enzyme rather than classical ligand, effects graded concentration-dependent in its target cells has been long-standing question. Thrombin activates receptor by cleaving off activation peptide to unmask tethered ligand. We utilized thrombin with epitope-tagged directly demonstrate cleavage examine the kinetics on intact cells. The rate was proportional concentration over physiologic range, but low concentrations ultimately cleaved activated all receptors. Cumulative phosphoinositide hydrolysis response correlated precisely cumulative cleavage. These data strongly suggest that each produces "quantum" phosphatidylinositol hydrolysis, then shuts off. Surprisingly, this shut occurred despite continued presence "activated" receptors cell surface time when were refractory sensitive agonist peptide, suggesting novel mechanism may have evolved deal Unlike case ligands, thus cannot detect differences as fractional occupancy must sense different rates activation. Because generates quantum second messenger, magnitude cell's be determined balance between messenger clearance.