Immunolocalization of P2Y1 and TPalpha receptors in platelets showed a major pool associated with the membranes of alpha -granules and the open canalicular system.
P2Y(1) and thromboxane-prostanoid-alpha (TPalpha) 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 P2Y(1) and a polyclonal antibody to the C-terminal domain of TPalpha revealed that while present at the platelet surface, both receptors were abundantly represented inside the platelet. Specifically, receptors were found in membranes of alpha-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 A(2) (TXA(2)) analog, I-BOP [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha)-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 P2Y(1) and TPalpha 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 P2Y(12) ADP receptor normally expressed P2Y(1) and TPalpha, 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 alpha(IIb)beta(3) at the start of secretion in a microenvironment containing agonist receptors. Pharmacologic antagonism of ADP or TXA(2) receptors in antithrombotic therapy may need to take into account blockade of internal receptor pools.
Nurden
Paquita
P
Centre National de la Recherche Scientifique (CNRS), Hôpital Cardiologique, Pessac, France. paquita.nurden@cnrshl.u-bordeaux2.fr
Poujol
Christel
C
Winckler
Joelle
J
Combrié
Robert
R
Pousseau
Nathalie
N
Conley
Pamela B
PB
Levy-Toledano
Sylviane
S
Habib
Aida
A
Nurden
Alan T
AT
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000328:Adult; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000648:ultrastructure*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000648:ultrastructure*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005340:Fibrinogen / Q000032:analysis; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000737:chemistry*; D008533:Megakaryocytes / Q000648:ultrastructure; D008854:Microscopy, Electron; D015539:Platelet Activation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D018048:Receptors, Purinergic P2 / Q000097:blood*; D058472:Receptors, Purinergic P2Y1; D017482:Receptors, Thromboxane / Q000097:blood*; D013873:Thionucleotides / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; C546382:P2RY1 protein, human; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D017482:Receptors, Thromboxane; D013873:Thionucleotides; C061750:7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid; C030812:adenosine 5'-O-(2-thiodiphosphate); D000244:Adenosine Diphosphate; D005340:Fibrinogen