Activation of G i -coupled receptors releases a tonic state of inhibited platelet aggregation

Abstract

Single-receptor pharmacology does not satisfactorily explain the physiology of the ADP-induced platelet aggregation response. It has been shown that, in addition to Gq-coupled receptor activation, one Gi-coupled receptor, either the ADP P2T or the alpha2-adrenoceptor, is required for elicitation of aggregation. The underlying mechanism of this action, however, has not been elucidated. By systematically assaying the entire time course of the aggregation and its fade using two methods of aggregometry, we have investigated the role of graded activation of these two Gi-coupled receptors. We demonstrate that constant activation of either of two Gq-coupled receptors, the ADP P2Y1 or the 5-HT2A, and incremental activation of either of the two Gi-coupled receptors, tightly regulates the aggregation response in vitro, through the apparent release of a tonic inhibition of platelet aggregation. This tightly regulated release of inhibition, which appears analogous to the phenomena of disinhibition observed in the central nervous system, may be instrumental for the continuous adaptation of the aggregation response to variable physiological conditions.

96-well plate-based aggregometry

“…Most groups have studied aggregation in response to ADP, collagen and thrombin receptor-activating peptide (TRAP)-6 (or thrombin in washed platelets), while others have also investigated the effects of epinephrine, serotonin, the thromboxane (Tx) A 2 mimetic U46619, and ristocetin (10–13). There is little consensus from these various groups with regard to the specific concentrations of agonists to be used.…”

“…Krause et al ., studying the effects of glycoprotein IIb/IIIa antagonists, and Maayani et al . also used an assay volume of 150 μl, albeit with different ratios of PRP to agonist (10,16). A few groups have used an assay volume of more than 150 μl (15,17,19), but most have reduced volumes of PRP or washed platelets to around 100 μl (9,11,20–22).…”

“…These shakers come in different varieties, e.g. linear (8,16,23), orbital (17,21) and kinetic (10,11,15). In LTA, a stir bar is present in each sample which creates a simple reproducible vortex in the cuvette whereas the internal shakers in plate readers are hugely variable.…”

“…The majority of this work concerns the characterization of platelet reactivity in the presence of antiplatelet therapies such as GPIIb/IIIa inhibitors (16,23) or dual anti-platelet therapy with ADP P2Y 12 antagonists plus aspirin (10–13,17,21,22,25,26). Using these techniques, it is possible to produce concentration-response curves which can be used to evaluate the pharmacological action of these therapies.…”

Activation of the 2A –adrenoceptor mediates deceleration of the deaggregation component of the response to ADP or 5-HT in human platelets in vitro

“…6 4 We also suggested that release of the inhibition of aggregation, or disinhibition, is necessary to produce an aggregation response. 64 Disinhibition may result from a deceleration of deaggregation elicited by activation of the G i -coupled ADP P2Y 12 or a 2A -adrenoceptor subtypes. In this study, we confirmed that in the presence of P2Y 12 receptor blockade, activation of the P2Y 1 or the 5-HT 2A receptor subtypes requires a 2 A -adrenoceptor activation to produce an aggregation response.…”