Grünberg B, Kruse H J, Negrescu E V, Siess W
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Germany.
J Cardiovasc Pharmacol. 1995 Apr;25(4):545-51. doi: 10.1097/00005344-199504000-00006.
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.
Rap1B是一种在人血小板中高浓度表达的类Ras蛋白，它是蛋白激酶A（PKA）以及最终蛋白激酶G（PKG）的底物。我们通过对用[32P]-正磷酸盐预标记的血小板中32P标记蛋白进行放射自显影来测量Rap1B的磷酸化。与组胺刺激的人脐静脉内皮细胞（EC）共孵育的血小板显示，50-Kd血管舒张刺激磷蛋白（VASP）的磷酸化最大增加2.6±0.5倍，Rap1B的磷酸化最大增加17.5±7.1倍（平均值±标准误，n = 4）。用前列环素（PGI2）、PGI2类似物伊洛前列素（ILO）、一氧化氮（NO）供体SIN-1或硝普钠（SNP）孵育血小板，结果显示Rap1B的浓度依赖性磷酸化比VASP更大。与VASP快速且可逆的磷酸化不同，Rap1B的磷酸化具有缓慢的时间进程且不可逆。Rap1B的磷酸化依赖于血小板环磷酸腺苷（cAMP）和/或环磷酸鸟苷（cGMP）的增加。非常低浓度的ILO（50 pM）、PGI2（1 nM）和SIN-1（100 nM）就能增加Rap1B的磷酸化。在用ILO或SIN-1孵育富血小板血浆（PRP）后，通过蛋白质印迹法也能检测到Rap1B的磷酸化。测量血小板Rap1B的磷酸化是一种新工具，可用于监测不稳定（PGI2、NO）和更稳定（ILO、SIN-1、SNP）的血小板抑制剂及血管舒张剂的作用，这些物质可增加细胞内cAMP和cGMP。通过蛋白质印迹法测定Rap1B的磷酸化，为临床研究中测量血小板与内皮细胞的相互作用以及在药理研究中监测全身应用的PGI2类似物和硝基血管舒张剂的作用开辟了新的可能性。
