Platelet rap1B phosphorylation is a sensitive marker for the action of cyclic AMP- and cyclic GMP-increasing platelet inhibitors and vasodilators.

Abstract

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.