Agonist-independent constitutive trafficking of the P2Y12 purinoceptor - relevance in platelets?

Ian Stanton, Alastair Poole, Stuart Mundell. School of Physiology and Pharmacology, University of Bristol, Bristol, BS8 1TD, United Kingdom.

The platelet P2Y12 purinoceptor plays a critical role in haemostasis with the level of surface expression of receptor having an important effect on platelet responsiveness. Work from our laboratory has shown that P2Y12 purinoceptor responsiveness is rapidly and reversibly modulated in human platelets (Mundell et al., 2008) with ADP stimulation promoting receptor internalization and subsequent recycling required to maintain receptor responsiveness. Although agonist-induced internalisation of P2Y12 is a relatively well characterised process there has been little investigation into constitutive, agonist-independent internalisation.

In these studies we used both Chinese Hamster Ovary (CHO) cells stably transfected with N-terminal haemaglutanin (HA)-tagged P2Y12 receptor as previously described (Mundell et al., 2008) or human platelets. In CHO cells changes in surface receptor expression were assessed by an ELISA assay or immunofluorescent microscopy as is standard in our laboratory (Mundell et al., 2008). In human platelets endogenous P2Y12 surface receptor expression was assessed by immunofluorescent microscopy in fixed platelets making use of a P2Y12 specific N-terminal antibody.

In our initial studies we found that the P2Y12 purinoceptor internalises in the absence of agonist in human platelets. Further studies in CHO cells revealed significant agonist-independent internalization both by ELISA (17.2% ±3.4 of total surface receptor (n = 5)) and immunofluorescent microscopy. In cell lines blockade of arrestin (by dominant negative mutant) and dynamin (by Dynasore - 80µM) resulted in a reduction in internalisation of up to 30% of total surface receptor number (Arrestin: control 31.9% ±9.2, DNM 8.7% ±6.2; Dynamin: control 16.8%, Dynasore -14.6%). Interestingly, unlike agonist-induced internalisation, the constitutive process is not dependent upon the presence of an intact PSD-95/drosophila discs large/ZO-1 (PDZ) binding motif found at the extreme C-terminus of the receptor. This motif is the site of mutations which result in abnormal bleeding phenotypes in patients. Further study is ongoing to dissect the particular region of the C-terminus which regulates the process of constitutive internalisation as well as further characterization of the interacting partners involved.

In conclusion we have discovered that the P2Y12 purinoceptor receptor internalises in human platelets and CHO cells in an agonist-independent manner. The functional significance of this process in relation to both P2Y12 purinoceptor signalling and platelet function is now under investigation.

Mundell, S.J., J.F. Barton, M.B. Mayo-Martin, A.R. Hardy and A.W. Poole (2008) Rapid resensitization of purinergic receptor function in human platelets. J Thromb Haemost. 6:1393-404.

Work supported by BHF Studentship to IGS