title: Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP.
abstract: 8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.
authors.affiliation: Medizinische Universitätsklinik, Klinische Forschergruppe, Würzburg, Federal Republic of Germany.
authors.forename: E
authors.initials: E
authors.lastname: Butt
authors.forename: C
authors.initials: C
authors.lastname: Nolte
authors.forename: S
authors.initials: S
authors.lastname: Schulz
authors.forename: J
authors.initials: J
authors.lastname: Beltman
authors.forename: J A
authors.initials: JA
authors.lastname: Beavo
authors.forename: B
authors.initials: B
authors.lastname: Jastorff
authors.forename: U
authors.initials: U
authors.lastname: Walter
mesh_terms: D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000032:analysis / Q000037:antagonists & inhibitors; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000032:analysis*; D013873:Thionucleotides / Q000494:pharmacology*
chemical_list: D047428:Protein Kinase Inhibitors; D013873:Thionucleotides; C016276:8-bromocyclic GMP; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D011494:Protein Kinases; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D006152:Cyclic GMP