title: Platelet-leukocyte aggregation induced by PAR agonists: regulation by nitric oxide and matrix metalloproteinases.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Alberta, Edmonton, Canada.
authors.forename: Ada W Y
authors.initials: AW
authors.lastname: Chung
authors.forename: Anna
authors.initials: A
authors.lastname: Radomski
authors.forename: David
authors.initials: D
authors.lastname: Alonso-Escolano
authors.forename: Paul
authors.initials: P
authors.lastname: Jurasz
authors.forename: Michael W
authors.initials: MW
authors.lastname: Stewart
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Malinski
authors.forename: Marek W
authors.initials: MW
authors.lastname: Radomski
mesh_terms: D015153:Blotting, Western; D002449:Cell Aggregation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000187:drug effects*; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D009569:Nitric Oxide / Q000502:physiology*; D010974:Platelet Aggregation / Q000187:drug effects*; D011166:Porphyrins / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D011166:Porphyrins; D009569:Nitric Oxide; D013917:Thrombin; D020782:Matrix Metalloproteinases