An ex vivo standardized assay to measure human platelet cGMP

INTRODUCTION: Nitric oxide (NO) acts a pleiotropic biomodulator in several systems, including the cardiovascular, nervous and immune systems. The intracellular levels of cyclic guanylate monophosphate (cGMP) can be increased by NO or by inhibiting the breakdown to 5'cGMP operated by the cyclic nucleotide phosphodiesterases (PDEs). Platelets are anuclear circulating cells that are rich in both soluble guanylyl cyclase and PDEs. The purpose of this study was to standardize cGMP determination in human platelets. METHODS: Fresh blood samples were obtained from a group of healthy volunteers in order to obtain washed platelets. Platelet (3×10(4)/μl or 5×10(5)/μl) cGMP levels were measured in basal and stimulated conditions. To stimulate platelets two different NO-donors were used: sodium nitroprusside (SNP; 10, 100, 1000 μM) or diethylamine NONOate (DEA-NONOate; 1, 10, 100 μM). Different times of incubation were also studied (5, 15 and 30 min). As positive control has been used ODQ a well known inhibitor of guanylyl cyclase. Platelet cGMP accumulation was measured by using a standard ELISA kit using different sample dilutions. RESULTS: The optimal stimulus was DEA-NONOate, the optimal washed platelet concentration was 5×10(5)/μl, incubation time was 30 min and dilution to be used was 1:2. DISCUSSION: Platelets represent a valuable marker to investigate the effect of drugs interfering with the cGMP cascade. This standardized assay allows to measure ex vivo the inhibitory (PDE inhibitors) or stimulatory effect (NO donors) of drugs given in vivo to humans.