Chung Ada W Y, Radomski Anna, Alonso-Escolano David, Jurasz Paul, Stewart Michael W, Malinski Tadeusz, Radomski Marek W
Department of Pharmacology, University of Alberta, Edmonton, Canada.
Br J Pharmacol. 2004 Dec;143(7):845-55. doi: 10.1038/sj.bjp.0705997. Epub 2004 Nov 8.
Platelet-leukocyte aggregation (PLA) links haemostasis to inflammation. The role of nitric oxide (NO) and matrix metalloproteinases (MMP-1, -2, -3, -9) in PLA regulation was studied. Homologous human platelet-leukocyte suspensions were stimulated with thrombin (0.1-3 nM) and other proteinase activated receptor-activating peptides (PAR-AP), including PAR1AP (0.5-10 microM), PAR4AP (10-70 microM), and thrombin receptor-activating peptide (1-35 microM). PLA was studied using light aggregometry with simultaneous measurement of oxygen-derived free radicals, dual colour flow cytometry, and phase-contrast microscopy. The release of NO was measured using a porphyrinic nanosensor, while MMPs were investigated by Western blot, substrate degradation assays, immunofluorescence microscopy, and flow cytometry. The levels of P-selectin and microparticles (MP) in PLA were measured by flow cytometry. PLA was also characterized using pharmacological agents: S-nitroso-glutathione (GSNO, 0.01-10 microM), 1H-Oxadiazole quinoxalin-1-one (ODQ, 1 microM), N(G)-L-nitro-L-arginine methyl ester (L-NAME, 100 microM) and compounds that modulate the actions of MMPs such as phenanthroline (100 microM), monoclonal anti-MMP antibodies, and purified MMPs. PAR agonists concentration-dependently induced PLA, an effect associated with the release of microparticles (MP) and the translocation of P-selectin to the platelet surface. NO and radicals were also released during PLA. Inhibition of NO bioactivity by the concomitant release of free radicals or by the treatment with L-NAME or ODQ stimulated PLA, while pharmacological administration of GSNO decreased PLA. PAR agonist-induced PLA resulted in the liberation of MMP-1, -2, -3, and -9. During PLA, MMPs were present on the cell surface, as shown by flow cytometry and immunofluorescence. PLA led to the activation of latent MMPs to active MMPs, as shown by Western blot and substrate degradation assays. Inhibition of MMPs actions by phenanthroline and by the antibodies attenuated PLA. In contrast, purified active, but not latent, MMPs amplified thrombin-induced PLA. It is concluded that NO and MMP-1, -2, -3, and -9 play an important role in regulation of PAR agonist-induced PLA.
血小板 - 白细胞聚集（PLA）将止血与炎症联系起来。研究了一氧化氮（NO）和基质金属蛋白酶（MMP - 1、-2、-3、-9）在PLA调节中的作用。用人同源血小板 - 白细胞悬浮液，用凝血酶（0.1 - 3 nM）和其他蛋白酶激活受体激活肽（PAR - AP）刺激，包括PAR1AP（0.5 - 10 microM）、PAR4AP（10 - 70 microM）和凝血酶受体激活肽（1 - 35 microM）。使用光聚集法同时测量氧衍生自由基、双色流式细胞术和相差显微镜来研究PLA。使用卟啉纳米传感器测量NO的释放，而通过蛋白质印迹、底物降解测定、免疫荧光显微镜和流式细胞术研究MMPs。通过流式细胞术测量PLA中P - 选择素和微粒（MP）的水平。还使用药物制剂对PLA进行表征：S - 亚硝基谷胱甘肽（GSNO，0.01 - 10 microM）、1H - 恶二唑喹喔啉 - 1 - 酮（ODQ，1 microM）、N（G）-L - 硝基 - L - 精氨酸甲酯（L - NAME，100 microM）以及调节MMPs作用的化合物，如菲咯啉（100 microM）、单克隆抗MMP抗体和纯化的MMPs。PAR激动剂浓度依赖性地诱导PLA，这种效应与微粒（MP）的释放以及P - 选择素向血小板表面的转位有关。PLA过程中也释放NO和自由基。自由基的伴随释放或用L - NAME或ODQ处理抑制NO生物活性会刺激PLA，而GSNO的药物给药会降低PLA。PAR激动剂诱导的PLA导致MMP - 1、-2、-3和-9的释放。如流式细胞术和免疫荧光所示，PLA过程中MMPs存在于细胞表面。如蛋白质印迹和底物降解测定所示，PLA导致潜伏MMPs激活为活性MMPs。菲咯啉和抗体抑制MMPs的作用会减弱PLA。相反，纯化的活性而非潜伏的MMPs会放大凝血酶诱导的PLA。结论是，NO和MMP - 1、-2、-3和-9在PAR激动剂诱导的PLA调节中起重要作用。
