Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition.
Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin alpha IIb beta 3) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, cAMP-elevating agents [e.g. prostaglandin E1 and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of cAMP- and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet cAMP- or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.
Horstrup
K
K
Medizinische Universitätsklinik, Klinische Forschergruppe, Würzburg, Germany.
Jablonka
B
B
Hönig-Liedl
P
P
Just
M
M
Kochsiek
K
K
Walter
U
U
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D001798:Blood Proteins / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000097:blood*; D005576:Colforsin / Q000031:analogs & derivatives / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood*; D008840:Microfilament Proteins; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D010750:Phosphoproteins / Q000097:blood* / Q000737:chemistry; D010768:Phosphoserine / Q000032:analysis; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors*; D011494:Protein Kinases / Q000097:blood*; D011505:Protein-Tyrosine Kinases / Q000097:blood*; D012694:Serine; D014665:Vasodilator Agents / Q000494:pharmacology*; D000098662:Vasodilator-Stimulated Phosphoprotein
D001798:Blood Proteins; D015815:Cell Adhesion Molecules; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D010980:Platelet Membrane Glycoproteins; D014665:Vasodilator Agents; D000098662:Vasodilator-Stimulated Phosphoprotein; D009599:Nitroprusside; D010768:Phosphoserine; D005576:Colforsin; D012694:Serine; C002385:linsidomine; D000244:Adenosine Diphosphate; D008981:Molsidomine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D000527:Alprostadil