Upon activation, platelets undergo a change from a discoid to a spherical shape and the production of pseudopodia (Paul et al., 1999). ADP, an important mediator of platelet activation, activates two platelet GPCRs, P2Y₁ and P2Y₁₂(Gachet, 2001). It has become accepted that P2Y₁, which couples to Gq, is solely responsible for the rise in intracellular calcium and platelet shape change elicited by ADP (Jin et al., 1998). P2Y₁ is known to cause platelet shape change through the activation of two distinct pathways – a rise in cytosolic calcium, and activation of Rho-kinase (Wilde et al., 2000). However, recently, P2Y₁₂ has been shown to couple to the activation of Rho-kinase when heterologously expressed in CHO cells (Soulet et al., 2004) as well as the potentiation of the calcium response (Hardy et al., 2004). Due to the importance of ADP in platelet activation, we sought to characterise a role for P2Y₁₂ in ADP-induced shape change.

Human platelets were isolated from drug-free volunteers on the day of experiment . For measurements of cytosolic calcium, platelets were loaded with Fura 2-AM. For measurements of myosin light chain phosphorylation, platelets were loaded with 1 mCi/ml H₃[³²P]O₄. For measurements of actin polymerisation, platelets were labelled with TRITC-phalloidin and fluorescence measured by flow cytometry. For scanning electron microscopy (SEM), fixed, dehydrated, gold-coated platelets were viewed on a Philips 501b SEM. For all experiments stimulations were made at 30 °C in the presence of 1 mM EGTA to block platelet aggregation, other than measurements of cytosolic calcium where stimulations were made at room temperature in the absence of EGTA. Statistical comparison of data was made by Student’s t-test.

As measured by the qualitative techniques optical aggregometry and SEM, although shape change can be completely ablated with the P2Y₁-specific antagonist, A3P5P (1 mM) in agreement that it is a P2Y₁-dependent phenomenon, the P2Y₁₂-specific antagonists AR-C69931MX (ARC; 1 µM) and 2MeSAMP (10 µM), inhibit the shape change response in a similar fashion to the Rho kinase inhibitor Y27632, by inhibiting maintenance of the response, and there was no additive effect when Y27632 and ARC were combined (n=3).

Y27632 reduced myosin phosphorylation from 3.5 ± 0.4 fold-over-basal (fob) to 1.62 ± 0.07 fob (n=3), which was insignificantly different from a reduction caused by ARC (p>0.5) and from the reduction caused by the combination of Y27632 with ARC (p>0.5). Measurements of actin polymerisation showed that Y27632 reduced actin polymerisation from 25.4 ± 5 % to 9.8 ± 2.8 % (n=3; p<0.05), whereas ARC reduced actin polymerisation to 11.9 ± 2.6 % (n=3; p<0.05), and the combination of Y27632 with ARC resulted in no additional effect compared with Y27632 alone (n=3; p>0.5). Thus P2Y₁₂ antagonism inhibits myosin phosphorylation and actin polymerisation similar to Rho-kinase inhibition with no summation between the two inhibitors. Taken together, these data indicate that P2Y₁₂ couples to the Rho-kinase pathway resulting in myosin phosphorylation and actin polymerisation, and a potentiation of the shape change response. Thus Rho-kinase may represent a newly-identified point of GPCR crosstalk in the control of platelet shape change, which is likely to be of considerable importance for our understanding of platelet activation.

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