Thrombin receptors on human platelets - Initial localization and subsequent redistribution during platelet activation

Platelet responses to thrombin are at least partly mediated by a G-protein-coupled receptor whose NH2 terminus is a substrate for thrombin, In the present studies we have examined the location of thrombin receptors in resting platelets and followed their redistribution during platelet activation, The results reveal several new aspects of thrombin receptor biology, 1) On resting platelets, approximately two-thirds of the receptors were located in the plasma membrane, The remainder were present in the membranes of the surface connecting system, 2) When platelets were activated by ADP or a thromboxane analog, thrombin receptors that were initially in the surface connecting system were exposed on the platelet surface, increasing the number of detectable receptors by 40% and presumably making them available for subsequent activation by thrombin, 3) Platelet activation by thrombin rapidly abolished the binding of the antibodies whose epitopes are sensitive to receptor cleavage and left the platelets in a state refractory to both thrombin and the agonist peptide, SFLLRN, This was accompanied by a 60% decrease in the binding of receptor antibodies directed COOH-terminal to the cleavage site irrespective of whether the receptors were activated proteolytically by thrombin or nonproteolytically by SFLLRN, 4) The loss of antibody binding sites caused by thrombin was due in part to receptor internalization and in part to the shedding of thrombin receptors into membrane microparticles, especially under conditions in which aggregation was allowed to occur, However, at least 40% of the cleaved receptors remained on the platelet surface, 5) Lacking the ability to synthesize new receptors and lacking an intracellular reserve of preformed receptors comparable to that found in endothelial cells, platelets were unable to repopulate their surface with intact receptors following exposure to thrombin, This difference underlies the ability of endothelial cells to recover responsiveness to thrombin rapidly while platelets do not, despite the presence on both of the same receptor for thrombin.