RESUMO
The mechanism of ADP-mediated platelet activation has been difficult to unravel due to the large number of receptors for extracellular nucleotides (P2 receptors). mRNA levels in circulating platelets are very low, but have been shown to be translationally active. By optimizing mRNA extraction and using real time (RT)-PCR we were able to establish a protocol for highly sensitive platelet mRNA quantification in human regular blood samples. In platelets from healthy volunteers, only P2X1, P2Y1 and P2Y12 were found in significant levels, with the following order of expression: P2Y12 >> P2X1 > P2Y1. Other P2 receptors (P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, P2X4, P2X7) had very low expression. As a control measurement to exclude contamination, P2 receptors in buffy coat were quantified but had a completely different profile. Incubation in vitro revealed a more rapid degradation rate for P2X1 receptor mRNA than for P2Y1 and P2Y12, indicating that the level of P2X1 may be relatively higher in newly released platelets and in megacaryocytes. In conclusion, we have developed the first protocol for quantifying mRNA expression in human platelets limiting the P2 receptor drug development targets to P2Y12, P2Y1 and P2X1. Furthermore, the method could be used to study platelet expression for any gene in human materials.
