Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators.
Recently, we reported the purification of a 46-kDa membrane-associated platelet protein which is phosphorylated in intact platelets and platelet membranes by cGMP- and cAMP-dependent protein kinases (Halbrügge, M., and Walter, U. (1989) Eur. J. Biochem. 185, 41-50). Here we demonstrate that both cGMP- and cAMP-dependent protein kinases catalyze the rapid incorporation of up to 1.4 mol of phosphate/mol of this purified vasodilator-stimulated phosphoprotein (VASP). A specific rabbit antiserum was prepared which recognized both the 46-kDa dephospho form and the 50-kDa phospho form of VASP in Western blots. In untreated washed platelets, VASP was found to be present primarily as a 46-kDa dephosphoprotein. Sodium nitroprusside (100 microM) raised the intracellular platelet cGMP concentration from approximately 0.44 to 4.1 microM, without a significant effect on the cAMP level, and converted up to 50% of VASP to the 50-kDa phospho form. Prostaglandin E1 (10 microM) raised the platelet cAMP concentration from approximately 4.4 to 28.4 microM, without a significant effect on the cGMP level, and shifted up to 67% of VASP to the 50-kDa phospho form. Removal of the vasodilators sodium nitroprusside and prostaglandin E1 from the platelet suspension was followed by a return of the cyclic nucleotide concentration to basal levels and subsequent conversion of the 50-kDa phospho form of VASP to the 46-kDa dephospho form. The results support the hypothesis that VASP phosphorylation is an important component of the intracellular mechanism of action of these vasodilators in human platelets.
Halbrügge
M
M
Medizinische Universitätsklinik, Labor für Klinische Biochemie, Würzburg, Federal Republic of Germany.
Friedrich
C
C
Eigenthaler
M
M
Schanzenbächer
P
P
Walter
U
U
D000527:Alprostadil / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000097:blood; D006152:Cyclic GMP / Q000097:blood; D005292:Ferricyanides / Q000494:pharmacology*; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D008970:Molecular Weight; D009599:Nitroprusside / Q000494:pharmacology*; D010750:Phosphoproteins / Q000097:blood*; D010766:Phosphorylation; D010974:Platelet Aggregation
D001798:Blood Proteins; D005292:Ferricyanides; D007106:Immune Sera; D010750:Phosphoproteins; D009599:Nitroprusside; D000242:Cyclic AMP; D000527:Alprostadil; D006152:Cyclic GMP