Agonist concentration-dependent differential responsivity of a human platelet purinergic receptor: pharmacological and kinetic studies of aggregation, deaggregation and shape change responses mediated by the purinergic P2Y₁receptorin vitro

Abstract

Platelet shape change (SC), aggregation and deaggregation responses are integral components of hemostasis that are elicited and modulated in vivo by the simultaneous activation of several membrane receptors. Selective activation of the purinergic P2Y1 receptor in vivo elicits a sustained SC and a small, transient aggregation response that is reversed rapidly by a robust deaggregation response (Platelets 2003; 14: 89). Using a kinetics-based turbidimetric approach to study the modulation of these concurrent components of human platelet responses, we demonstrate that these P2Y1 receptor-related responses and a number of their kinetic and steady-state characteristics are differentially elicited and modulated. P2Y1 receptor agonist concentrations that elicited aggregation (pEC50 for ADP, 2-MeSADP; 5.88, 6.69) were 10-fold greater than those that elicited SC (7.33, 7.67). The magnitude of the aggregation response was agonist concentration-dependent, saturable and was associated with an agonist concentration-dependent deceleration of the deaggregation response. Gi-coupled receptor (alpha 2A-adrenoceptor, EP3 and P2Y12 receptors) agonists also enhanced aggregation through deceleration of the deaggregation response, and an inhibitor of PI3K activity (wortmannin) inhibited aggregation through acceleration of the deaggregation response. Neither treatment affected the extent or the kinetics of the SC response. The aggregation but not the SC response was rapidly desensitized by P2Y1 receptor activation by ADP. The affinity of the presence of a single P2Y1 receptor subtype. The differential characteristics and modulation of the SC and aggregation responses by a single receptor support the idea that different signaling pathways activated at different occupancy states of the same receptor underlie the two responses. P2Y1 receptor-mediated platelet aggregation and SC responses provide a convenient model for studying the phenomenon of agonist-directed signaling by differential occupancy of the same membrane receptor.

The role of prostanoid receptors in mediating the effects of PGE2on human platelet function

“…Promotion of platelet aggregation by sulprostone via an effect at EP3 receptors is already well established [7,10,11,[13][14][15][16][17], as is the ability of EP3 to link with adenylyl cyclase via G i and potentiate platelet aggregation via a decrease in cAMP [10,11,13,17]. We have demonstrated that sulprostone is able to decrease basal VASP phosphorylation and the concentrations needed to bring this about compare very well with the concentrations required to potentiate platelet aggregation.…”

“…There is good evidence that potentiation of platelet function is via effects at EP3 receptors linked to G i and inhibition of adenylyl cyclase [7,10,11,[13][14][15][16][17]. It is currently believed that inhibition of platelet aggregation may be via interaction with IP receptors linked to G s and stimulation of adenylyl cyclase [10,11], although this is mainly based on experiments conducted using mouse platelets [10] and the involvement of other receptors has not been fully explored.…”

Detection of P2Y14 protein in platelets and investigation of the role of P2Y14 in platelet function in comparison with the EP3 receptor

“…We compared the effectsofUDP-glucosewith thoseofthe EP 3 receptor agonist sulprostone, aprostaglandin E 2 (PGE 2 )analogue (18).The stimulated EP 3 receptor potentiates plateletfunction by coupling to inhibitoryGp roteins, whichr esults in inhibitiono fa denylate cyclase (19)(20)(21). Sulprostone has been shown to potentiate primarya ggregation responses caused by several primary plateletagonistsincluding ADP,plateletactivating factor (PAF), 11,9-epoxymethano PGH 2 (U-46619),c ollagen, thrombin receptor activating peptide (TRAP)a nd 5-hydroxytryptamine (18,(22)(23)(24). It wasusedhere as apositivecontrol for the studies on the functionalityofthe P2Y 14 receptor.…”