Guanine nucleotides and Ca 2+ ‐dependent lysosomal secretion in electropermeabilised human platelets

1 Metabolically stable analogues of GTP, e.g. guanosine 5′‐[γ‐thio]trio]triphosphate (GTP[S]) and guanosine 5′‐[β‐γ‐imido]triphosphate (pp[NH]pG), enhance the extent of Ca 2+ ‐dependent secretion of β‐ N ‐acetylgluco‐saminidase and β‐galactosidase from electropermeabilised human platelets in the presence of less than 5 μM Ca 2+ .A similar effect is observed on addition either of 1,2‐dioctanoin or of GTP in the presence or absence of thrombin. 2 In the presence of higher Ca 2+ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca 2+ ] of 30–40 μM. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca 2+ ‐dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides, thrombin or 1,2‐dioctanoin. 3 The concentration of GTP[S] or pp[NH]pG required to obtain half‐maximal enhancement of lysosomal secretion is dependent on [Ca 2+ ] for secretion of 5‐hydroxytryptamine, β‐ N ‐acetylglucosaminidase and β‐galactosidase. At two fixed [Ca 2+ ] the median effective concentration (EC 50 ) values for GTP[S] and pp[NH]pG which characterise enhancement of 5‐hydroxytryptamine secretion are significantly different from those characterising enhancement of the secretion of β‐ N ‐acetylglucosaminidase and β‐galactosidase. 4 In the presence of a saturating concentration of GTP[S] marked 5‐hydroxytryptamine and β‐ N ‐acetylglucosaminidase secretion is observed at nanomolar [Ca 2+ ] and these responses show little dependence on [Ca 2+ ] over the attainable range. Secretion of β‐ N ‐acetylglucosaminidase is also induced at nanomolar Ca 2+ concentrations by addition of activators of protein kinase C. 5 Guanosine 5′‐[β‐thio]diphosphate inhibits enhancement of β‐ N ‐acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca 2+ when added alone. 6 Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca 2+ ‐dependent platelet lysosomal secretion by activating a guanine‐nucleotide‐binding protein (G E ) located close to the exocytotic site. However, not all the data are consistent with this postulate.