PLATELET RAP1B PHOSPHORYLATION IS A SENSITIVE MARKER FOR THE ACTION OF CYCLIC AMP-INCREASING AND CYCLIC GMP-INCREASING PLATELET INHIBITORS AND VASODILATORS

Rap1B, a ras-like protein expressed in high concentrations in human pl atelets, serves as a substrate for protein kinase A (PKA) and, eventua lly, protein kinase G (PKG). We measured rap1B phosphorylation by auto radiography of P-32-labeled proteins in platelets prelabeled with [P-3 2]-orthophosphate. Platelets coincubated with histamine-stimulated hum an umbilical vein endothelial cells (EC) showed increased phosphorylat ion of the 50-Kd vasodilator-stimulated phosphoprotein (VASP) of 2.6 /- 0.5-fold maximally and of rap 1B of 17.5 +/- 7.1-fold maximally (me an +/- SE, n = 4). Incubation of platelets with prostacyclin (PGI(2)), the PGI(2)-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 vap1B had a slow time course and was irreversible in contrast to that of VASP, wh ich was rapid and reversible. Phosphorylation of rap1B was dependent o n an increase of platelet cyclic AMP and/or cyclic GMP. Very small con centrations of ILO (50 pM), PGI(2) (1 nM), and SIN-1 (100 nM) increase d rap1B phosphorylation. Rap1B phosphorylation could also be detected by Western blot after incubation of platelet-rich plasma (PRP) with IL O or SIN-1. Measurement of platelet rap1B phosphorylation is a novel t ool that allows monitoring of the action of labile (PGI(2), NO) and mo re stable (ILO, SIN-1, SNP) platelet inhibitors and vasodilators that increase intracellular cyclic AMP and cyclic GMP. Determination of rap 1B phosphorylation by Western blot opens new possibilities of measurin g platelet-EC interactions in clinical studies and of monitoring the a ction of systemically applied PGI(2) analogues and nitrovasodilators i n pharmacologic studies.