Desensitization of the Platelet Aggregation Response to ADP: Differential Down-regulation of the P2Y1 and P2cyc Receptors
Anthony Baurand, Anita Eckly, Nadège Bari, Catherine Léon, Béatrice Hechler, Jean-Pierre Cazenave, Christian Gachet
From INSERM U.311, Etablissement Français du Sang-Alsace, Strasbourg, France
Summary Platelets activated by ADP become refractory to restimulation, butthe mechanism of this process is not well understood. A normal plateletresponse to ADP requires coactivation of the P2Y1 receptor responsiblefor shape change and the P2cyc receptor, responsible for completionand amplification of the response. The aim of the present study was tocharacterize the desensitization of platelets to ADP and to determinewhether or not these two receptors are desensitized simultaneouslythrough identical pathways when platelets become refractory to ADP. Itwas found that full inhibition of platelet aggregation in response torestimulation by ADP required the presence of ADP in the medium oruse of a high concentration (1 mM) of its non-hydrolysable analogueADPS. Platelets incubated for 1 h at 37° C with 1 mM ADPS andresuspended in Tyrode’s buffer containing apyrase displayed a stablerefractory state characterized by the inability to aggregate or changeshape in response to ADP. ADPS treated platelets loaded with fura-2/AM showed complete blockade of the calcium signal in response toADP, whereas the capacity of ADP to inhibit PGE1 stimulated cAMPaccumulation in these platelets was only diminished. Consequently,serotonin was able to promote ADP induced aggregation throughactivation of the Gq coupled 5HT2Á receptor while adrenaline had nosuch effect. These results suggested that the refractory state of ADPStreated platelets was entirely due to desensitization of the P2Y1 receptor,the P2cyc receptor remaining functional. Binding studies wereperformed to determine whether the P2Y1 and/or P2cyc binding siteswere modified in refractory platelets. Using selective P2Y1 and P2cycantagonists (A3P5P and AR-C66096 respectively), we could demonstratethat the decrease in [ 33P]2MeSADP binding sites on refractoryplatelets corresponded to disappearance of the P2Y1 sites with nochange in the number of P2cyc sites, suggesting internalization of theP2Y1 receptor. This was confirmed by flow cytometric analysis of Jurkatcells expressing an epitope-tagged P2Y1 receptor, where ADPStreatment resulted in complete loss of the receptor from the cellsurface. We conclude that the P2Y1 and P2cyc receptors are differentlyregulated during platelet activation.