EXPERIMENTAL STUDY
The platelet-endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells
Luciano Cominacini, MD*,*,
Anna Fratta Pasini, MD*,
Ulisse Garbin, MD*,
Antonio Pastorino, BSc*,
Anna Rigoni, MD*,
Cristina Nava, MD*,
Anna Davoli, BSc*,
Vincenzo Lo Cascio, MD* and
Tatsuya Sawamura, MD
* Department of Biomedical and Surgical Sciences of Verona University, Verona, Italy
Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan
Manuscript received June 19, 2002;
revised manuscript received August 29, 2002,
accepted September 26, 2002.
* Reprint requests and correspondence: Dr. Luciano Cominacini, Dipartimento di Scienze Biomediche e Chirurgiche (Medicina D), Università di Verona, Ospedale Policlinico, 37134 Verona, Italy.
comina{at}medicinad.univr.it
OBJECTIVES: To address the potential role of the endothelial lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the thrombotic system, in this study we first examined whether platelet interaction with LOX-1 generated reactive oxygen species (ROS) and superoxide (O2·–) and then investigated the relationship between the intracellular production of O2·– and the availability of nitric oxide (NO).
BACKGROUND: Oxidative inactivation of NO is regarded as an important cause of its decreased biologic activity which may favor platelet-dependent arterial thrombosis.
METHODS: Bovine aortic endothelial cells (BAECs) and Chinese hamster ovary-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) were incubated at different times with human platelets. The ROS, O2·–, and NO were measured in cells by flow cytometry.
RESULTS: The incubation of BAECs and BLOX-1-CHO cells with human platelets induced a sharp and dose-dependent increase in intracellular concentration of ROS and O2·– (p from <0.01 to <0.001). The increase in intracellular concentration of O2·– was followed by a dose-dependent reduction in basal and bradykinin-induced intracellular NO concentration (p from <0.01 to <0.001). The increase in O2·– and the reduction of NO were inhibited by the presence of vitamin C and anti-LOX-1 monoclonal antibody (p < 0.001).
CONCLUSIONS: The results of this study show that one of the pathophysiologic consequences of platelet binding to LOX-1 may be the inactivation of NO through an increased cellular production of O2·–.
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Abbreviations and Acronyms
| | BAECs | | bovine aortic endothelial cells | | BLOX-1-CHO cells | | CHO-K1 cells stably expressing bovine LOX-1 | | CHO-K1 cells | | Chinese hamster ovary-K1 cells | | DAF-2 DA | | 4, 5 diaminofluorescein diacetate | | DCFH-DA | | 2',7'-dichlorofluorescin diacetate | | eNOS | | endothelial nitric oxide synthase | | HE | | hydroethidine | | LOX-1 | | lectin-like ox-LDL receptor-1 | | LOX-1 Ab | | anti-LOX-1 monoclonal antibody | | MFI | | mean fluorescence intensity | | NO | | nitric oxide | | O2·– | | superoxide | | ox-LDL | | oxidized low-density lipoprotein | | PEP | | phosphoenolpyruvate | | PK | | pyruvate kinase | | PS | | phosphatidylserine | | ROS | | reactive oxygen species |
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