Platelet rap1B phosphorylation is a sensitive marker for the action of cyclic AMP- and cyclic GMP-increasing platelet inhibitors and vasodilators.
Rap1B, a ras-like protein expressed in high concentrations in human platelets, serves as a substrate for protein kinase A (PKA) and, eventually, protein kinase G (PKG). We measured rap1B phosphorylation by autoradiography of 32P-labeled proteins in platelets prelabeled with [32P]-orthophosphate. Platelets coincubated with histamine-stimulated human umbilical vein endothelial cells (EC) showed increased phosphorylation of the 50-Kd vasodilator-stimulated phosphoprotein (VASP) of 2.6 +/- 0.5-fold maximally and of rap1B of 17.5 +/- 7.1-fold maximally (mean +/- SE, n = 4). Incubation of platelets with prostacyclin (PGI2), the PGI2-analogue iloprost (ILO), the nitric oxide (NO) donors SIN-1 or sodium nitroprusside (SNP) showed greater concentration-dependent phosphorylation of rap1B than of VASP. Phosphorylation of rap1B had a slow time course and was irreversible in contrast to that of VASP, which was rapid and reversible. Phosphorylation of rap1B was dependent on an increase of platelet cyclic AMP and/or cyclic GMP. Very small concentrations of ILO (50 pM), PGI2 (1 nM), and SIN-1 (100 nM) increased rap1B phosphorylation. Rap1B phosphorylation could also be detected by Western blot after incubation of platelet-rich plasma (PRP) with ILO or SIN-1. Measurement of platelet rap1B phosphorylation is a novel tool that allows monitoring of the action of labile (PGI2, NO) and more stable (ILO, SIN-1, SNP) platelet inhibitors and vasodilators that increase intracellular cyclic AMP and cyclic GMP. Determination of rap1B phosphorylation by Western blot opens new possibilities of measuring platelet-EC interactions in clinical studies and of monitoring the action of systemically applied PGI2 analogues and nitrovasodilators in pharmacologic studies.
Grünberg
B
B
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Germany.
Kruse
H J
HJ
Negrescu
E V
EV
Siess
W
W
D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001345:Autoradiography; D015415:Biomarkers; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001797:Blood Protein Electrophoresis; D015153:Blotting, Western; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000097:blood*; D006152:Cyclic GMP / Q000097:blood*; D011464:Epoprostenol / Q000097:blood; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000097:blood; D010766:Phosphorylation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology*; D019323:omega-N-Methylarginine; D020668:rap GTP-Binding Proteins
D015415:Biomarkers; D010975:Platelet Aggregation Inhibitors; D014665:Vasodilator Agents; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D001120:Arginine; D011464:Epoprostenol; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D006152:Cyclic GMP