TXA2 and purinergic receptor cross-talk in human platelets: desensitisation at the level of calcium signalling

Barton, JF, Poole, AW, and Mundell, SJ. Department of Pharmacology, University of Bristol, Bristol, UK.

Thromboxane A2 (TXA2) and ADP are both major positive feedback mediators of platelet activation, produced and then released from platelets in response to activating agonists. Both are involved in the further activation and recruitment of platelets to the primary haemostatic plug. These two agonists operate through G-protein-coupled receptors (GPCR) on the platelet surface: TPα and TPβ for TXA2 and P2Y1 and P2Y12 for ADP. There is increasing evidence that GPCRs do not operate in isolation but that activation of one class of receptor may modulate the activity of another class. Since TP and P2Y receptors are both targeted by drugs used in the prevention of myocardial infarction we have studied the potential cross-talk between these receptors.

Whole blood was drawn from healthy, drug-free volunteers under informed consent and ethical approval from the Local Research Ethics Committee, United Bristol Healthcare Trust. Washed platelets were prepared and changes in Gq-coupled receptor activity assessed by measurement of agonist-induced increases in cytosolic free calcium using the fluorescent Ca2+ indicator Fura-2 as previously described (Hardy et al., 2005).

At the level of the calcium response, U46619 (1μM; 30 sec) pretreatment reduces the ADP response by ~50% (P<0.001), and increases the EC50 from 441±154nM to 1.93±1.54μM. Similarly, ADP (10μM; 30 sec) pretreatment reduces the maximal U46619 response to ~80% of its control (P<0.01), and increases the EC50 from 71.4±10.2nM to 106±13.9nM. ADP mediates its effects through P2Y1, as pretreatment with the P2Y1 antagonist A3P5P (1mM; 5 min) and ADP results in no significant change (P>0.05) in the U46619-mediated calcium response, whereas pretreatment with the P2Y 12 antagonist AR-C69931MX (1μM; 5 min) with ADP still altered the U46619 calcium response (P<0.0001). Pretreatment with either the non-specific protein kinase inhibitor staurosporine (1μM), or the non-specific protein kinase C inhibitor GF109203X (1μM), for 5 minutes, significantly attenuates the TXA2 receptor-mediated decrease in the purinergic receptor-induced [Ca2+] i rise (P>0.05, compared with the non-pretreated control), implicating a role for PKC in heterologous desensitisation. However, P2Y-mediated desensitisation of the TXA2 receptor-mediated calcium response is not affected by GF109203X or staurosporine (P<0.05), suggesting a PKC- and possibly a protein kinase-independent mechanism. All data are mean ± SEM (n=3), and are analysed using PRISM 3.0 by 2-way ANOVA (dose-response curves) or 1-way ANOVA with Bonferroni’s multiple comparison test.

This is the first evidence of purinergic- TXA2 receptor cross-talk in human platelets and may have major implications for the therapy of thrombosis using drugs that modulate these two sets of receptors.

Hardy et al. (2005). Blood 105, 3552-3560