Cross regulation of the P2Y12 and TXA2 receptors in human platelets

Mark I. Butler1, Andrew D. Mumford2, Johanna F. Barton1, Alastair W. Poole1, Thomas W. Johnson2, Stuart J. Mundell1. 1School of Physiology and Pharmacology University of Bristol, BS8 1TD Bristol, United Kingdom, 2Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, BS1 3NU Bristol, United Kingdom.

Platelets play a central role in the development of the arterial thrombosis in heart disease. They respond to a variety of extracm b bellular stimuli to undergo a rapid aggregation response, releasing active granule contents and leading to a rapidly growing thrombus. Central amongst these agonists are thromboxane A2 and ADP which operate through G protein-coupled receptors (GPCRs) on the platelet surface: TPα and TPβ for thromboxane A2 and P2Y1 and P2Y12 for ADP. Two of the major therapeutic approaches in the treatment of arterial thrombosis, aspirin and clopidogrel, target platelet receptors for these two major activatory agonists, with aspirin treatment reducing TP receptor signalling and clopidogrel blocking P2Y12 receptor activation. Unfortunately a significant number of patients (∼30%) display a degree of resistance (i.e. they suffer a thrombotic event) to either therapy. A recent study from our laboratory revealed cross-desensitization between ADP and thromboxane receptor signaling in human platelets (Barton et al., 2008). In this study we sort to investigate if pretreatment of human platelets with either aspirin or clopidogrel, which will reduce TP and P2Y12 responsiveness respectively could also alter the sensitivity of other receptor systems. (Representative volunteer: 14% increase in maximum aggregation to 10µM U44619 following aspirin treatment. mnmMean increase in treated subject’s calcium response to ADP compared to control subjects 57% ±40.93, n = 3.)

All experiments were undertaken in platelets taken from volunteers as previously described (Barton et al., 2008) or human astrocytoma 1321N1 cells stably transfected with N-terminal haemaglutanin (HA)-tagged P2Y12 or P2Y1 receptors as previously described (Mundell et al, 2008) Receptor and platelet function was assessed in volunteers by light transmission aggregometry and intracellular calcium measurements as previously described.

In an initial patient study we discovered that there was increased platelet aggregation in response to a maximal concentration of the TP receptor agonist U46619 (10 µM) following patient treatment with clopidogrel. Further studies revealed that in patient’s resistant to clopidogrel treatment there was no significant change in TP receptor function. Importantly in patients with effective P2Y12 blockade a number (2 out of 5) did display a significant increase in TP receptor responsiveness. In a parallel study we investigated if P2Y receptor responsiveness was altered in patients administered aspirin. Importantly we discovered that ADP-stimulated P2Y platelet receptor function was increased (10-20%) in patients following aspirin treatment. In both of these studies patient numbers were too low to place any statistical significance to our findings. (Representative non-responder: 0.5% decrease in maximum ADP aggregation and 2.4% decrease in maximum U46619 aggregation; representative responder: 31.7% decrease in maximum ADP aggregation and 10.8% increase in maximum U46619 aggregation following clopidogrel treatment.)

We showed that U46619 mediates a greater level of P2Y12 receptor internalisation in cell lines expressing the tagged receptor then ADP. (Following 60 minutes incubation with 10µM ADP there was 18.95% ±7.31 receptor loss compared with 51.41% ±10.84 for 10µM U46619, n = 4.)

In conclusion, our preliminary results suggest that blockade of TP receptor responsiveness in human platelets can increase P2Y signalling whilst P2Y12 purinoceptor blockade increases TP receptor responsiveness. Such changes in receptor activity have potentially significant implications for the therapeutic use of aspirin and clopidogrel. For example is aspirin resistance due in part to increased P2Y purinoceptor signalling producing platelets hyper-responsive to ADP? Further work is ongoing to demonstrate the significance of our observations and to define the molecular mechanisms underlying these phenomena.

Barton JF, Hardy AR, Poole AW, Mundell SJ (2008). Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation. J Thromb Haemost 6(3): 534-543.

Mundell, S. J., J. F. Barton, et al. (2008). “Rapid resensitization of purinergic receptor function in human platelets.” J Thromb Haemost 6(8): 1393-1404.