Measuring P2X1 receptor activity in washed platelets in the absence of exogenous apyrase

摘要

Purinergicreceptorsignalingeventsinplateletsareamajordeterminantinplateletfunction.However,investigatingtheATP-sensitiveP2X1plateletreceptorisdifficultduetoitsrapiddesensitizationinthewashedplateletsamplematrix.Tominimizedesensitization,moststudiesinvolvingP2X1activityinwashedplateletsrequireapyraseinthesampletoreducematrixATPlevels.Unfortunately,theapyrasewillalsorapidlydegradeanyATPaddedexogenouslyduringthestudies.Here,wedescribeamethodthatemploysthereportedP2X1inhibitorNF449tosensitizewashedplateletsintheabsenceofanyaddedapyrase.SensitizationisverifiedbyspectrofluorometricdeterminationofCa2+entryintotheplateletsafterstimulationwithconcentrationsofATPrangingfrom0.625μMto5μM.ResultssuggestthatsensitizationoftheP2X1receptorbyNF449isnotnecessarilydependentupontheinhibitorconcentration,butrathertheratiooftheinhibitortoexogenously-addedATPconcentrations.WitharatioofATPagonisttoNF440concentrationof～5?:?1,theresultingpercentchangeinfluorescenceduetoCa2+entryintotheplateletis39.3±0.8%;however,ataratioof1?:?8ATPtoNF449thepercentchangeisreducedto13.1±2.2%.Thesensitizingeffectisalsoinvestigatedasafunctionoftime.TheresultsobtainedverifythatNF449canbehaveasaconcentration-dependentinhibitorandsensitizeroftheplateletP2X1receptorinwashedplateletsamples,dependingontheATPconcentrationinthematrix...