Immunolocalization of P2Y1 and TPα receptors in platelets showed a major pool associated with the membranes of α-granules and the open canalicular system

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Abstract
P2Y1 and thromboxane-prostanoid–α (TPα) receptors on platelets belong to the G-protein–coupled 7–transmembrane domain family. They transmit signals for shape change, mobilization of calcium, and platelet aggregation. Immunogold labeling with a monoclonal antibody (MoAb) to the amino-terminal domain of P2Y1 and a polyclonal antibody to the C-terminal domain of TPα revealed that while present at the platelet surface, both receptors were abundantly represented inside the platelet. Specifically, receptors were found in membranes of α-granules and elements of the open-canalicular system. A similar organization was found in mature megakaryocytes. Activation of platelets by adenosine diphosphate (ADP) and the thromboxane A2(TXA2) analog, I-BOP [1S-(1 α,2 β(5Z),3 α-(1E,3S)4 α)-7-(3-(3- hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid], increased the labeling of both P2Y1 and TPα at the surface and in intracellular pools, suggesting that activation resulted in greater antibody accessibility to the receptor. A return to a platelet discoid shape and to basal values of labeling accompanied receptor desensitization. Platelets lacking the P2Y12 ADP receptor normally expressed P2Y1 and TPα, both before and after activation. Studies with the anti–ligand-induced binding site (anti-LIBS) MoAb, AP-6, confirmed that stored fibrinogen associated with internal pools of αIIbβ3 at the start of secretion in a microenvironment containing agonist receptors. Pharmacologic antagonism of ADP or TXA2 receptors in antithrombotic therapy may need to take into account blockade of internal receptor pools.