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 P2Y1 receptor in vitro

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 vitro 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. G_i-coupled receptor (α_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 selective P2Y₁ receptor antagonist, A3P5P, was independent of the response measured and is consistent with 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.