Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells.
Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA- and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 microM cAMP and 3.1 microM catalytic subunit of PKA, which is equivalent to 6.2 microM cAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 microM cGMP and 7.3 microM PKG monomer, equivalent to 14.6 microM cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 microM. Treatment of washed human platelets with 10 microM (or 10 mM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 microM (10 microM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein). Treatment of washed human platelets with 100 microM (or 1 microM) sodium nitroprusside elevated the platelet cGMP level to 4 microM (0.9 microM with 1 microM sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1 microM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increase in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA- and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation.(ABSTRACT TRUNCATED AT 400 WORDS)
Eigenthaler
M
M
Medizinische Universitätsklinik, Labor für Klinische Biochemie, Würzburg, Federal Republic of Germany.
Nolte
C
C
Halbrügge
M
M
Walter
U
U
D000328:Adult; D000527:Alprostadil / Q000494:pharmacology; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000242:Cyclic AMP / Q000097:blood*; D006152:Cyclic GMP / Q000097:blood*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D009599:Nitroprusside / Q000494:pharmacology; D010750:Phosphoproteins / Q000097:blood* / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood* / Q000302:isolation & purification
D046911:Macromolecular Substances; D010750:Phosphoproteins; D009599:Nitroprusside; D000242:Cyclic AMP; D011494:Protein Kinases; D000527:Alprostadil; D006152:Cyclic GMP