Concurrent responses elicited by isolated activation of platelet Gq-coupled receptors, in vitro: a novel approach for their separation and analysis.
This study tested the hypothesis that aggregation mediated by activation of a single G(q)-coupled receptor can be studied quantitatively if four concurrent but distinct components of the observed platelet response, autocrine stimulation, shape change (SC), aggregation and deaggregation, are separately measured. Responses mediated by two G(q)-coupled receptors, the TXA(2) and the P2Y(1), were assayed by a novel, kinetics-based turbidimetric approach. Blocking the autocrine stimulation with a cocktail of receptor antagonists revealed rapid and sustained SC that largely masked the aggregation. Mathematical removal of the SC contribution from the changes in optical density indicated that selective activation of either the TXA(2) or the P2Y(1) receptor was sufficient to induce a small aggregation (DeltaOD = 0.1-0.2) that was reversed rapidly by a concurrent deaggregation. Model-derived rate constants of SC, aggregation and deaggregation described the observed complex time course of their concurrency well. Laser light scattering aggregometry confirmed the rapid deaggregation of platelet aggregates following TXA(2) or P2Y(1) receptor-mediated formation. Saturable concentration response curves of net aggregation were elicited at EC(50) values 6-15 higher than those of SC. The utility of this approach was further demonstrated by the separation of the four components of the response mediated by concurrent 5-HT(2A) and alpha(2A)-adrenoceptor activation, as well as the P2Y(1) and alpha(2A)-adrenoceptor, and the autocrine stimulation and SC mediated by the 5-HT(2A) receptor. We propose that the novel approach described here is necessary to study the receptor pharmacology and the kinetics of concurrent platelet responses in vitro as demonstrated by platelet aggregation elicited by selective activation of the TXA(2) or the P2Y(1) receptors.
The Basic Research Laboratory, Department of Anesthesiology, Box 1010, The Mount Sinai School of Medicine, New York, NY 10029-6574, USA. saul.maayani@mssm.edu
Saul
S
Maayani
Nayana D
ND
Patel
Barbara D
BD
Craddock-Royal
Todd E
TE
Schwarz
Thomas M
TM
Tagliente
D019898:Autocrine Communication; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism; D048429:Cell Size; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008962:Models, Theoretical; D015539:Platelet Activation; D010973:Platelet Adhesiveness; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism* / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology; D058472:Receptors, Purinergic P2Y1; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000378:metabolism / Q000502:physiology
C546382:P2RY1 protein, human; D043562:Receptors, G-Protein-Coupled; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11