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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

View larger version (30K): [in a new window]   Figure 1 (A) Effect of different amounts of non-activated (T–) and thrombin-activated (T+) platelets on intracellular concentration of reactive oxygen species (ROS) and superoxide in bovine aortic endothelial cells (BAECs). The BAECs were preincubated with 2',7'-dichlorofluorescein diacetate and hydroethidine at 37°C for 20 min. Different amounts of (T–) and (T+) platelets (from 0 to 4.5 x 107/ml) were added to the BAEC medium for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are the means ± SD of experiments performed in triplicate on six separate occasions. ¶p < 0.01, *p < 0.001 vs. no addition of platelets (Control). (B) Time-course of superoxide formation induced by platelets activated with different amounts of thrombin in BAECs. The BAECs were preincubated with hydroethidine for 20 min. Platelets (3 x 107/ml) activated with different doses of thrombin (from 0.03 to 1 U/l) were added to the BAEC medium for the indicated times at 37°C. The BAECs and platelets alone were used as controls. Results are expressed as MFI and are the means ± SD of experiments performed in triplicate on six separate occasions. By two-way analysis of variance there was a significant effect for the thrombin concentrations (p < 0.001) and for the times of incubation (p < 0.001) with a significant concentrations/times interaction (p < 0.001) on superoxide generation. Post-analysis of variance Tukey test for multiple comparisons: ¶p < 0.01, *p < 0.001 vs. time 0.

View larger version (20K): [in a new window]   Figure 2 Effect of vitamin C (Vit. C) and anti-LOX-1 monoclonal antibody (LOX-1 Ab) on platelet-induced variations of O2· concentration in bovine aortic endothelial cells (BAECs), Chinese hamster ovary-K1 (CHO) cells, and CHO-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) cells. Vitamin C (5 µmol/l) and LOX-1 Ab (30 µg/ml) were preincubated with BAECs, BLOX-1-CHO, and CHO cells at 37° for 30 min. Thrombin-activated platelets (3 x 107/ml) were then added to the cell medium for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are the means ± SD of experiments performed in triplicate on six separate occasions. *p < 0.001 vs. platelets alone.

View larger version (19K): [in a new window]   Figure 3 Effect of anti-LOX-1 monoclonal antibody (LOX-1 Ab), anti-CD41a antibody, annexin V, and immunoglobulin G (IgG) on platelet-induced variations of superoxide concentration in bovine aortic endothelial cells (BAECs). The LOX-1 Ab (30 µg/ml), anti-CD41a antibody (30 µg/ml) alone and mixed with LOX-1 Ab, annexin V (10 µmol/l), and IgG (30 µg/ml) were preincubated with BAECs for 30 min at 37°C. Thrombin-activated platelets (3 x 107/ml) were added to the cell medium for 10 min at 37°C. Results are expressed as percent variations of mean fluorescence intensity (MFI) and are the means ± SD of experiments performed in triplicate on six separate occasions. *p < 0.001 vs. platelets alone.

View larger version (29K): [in a new window]   Figure 4 Effect of different amounts of non-activated (T–) and thrombin-activated (T+) platelets on basal and bradykinin-stimulated intracellular concentration of nitric oxide (NO) in bovine aortic endothelial cells (BAECs). The BAECs were preincubated with 10 µmol/l 4, 5 diaminofluorescein diacetate for 10 min at 37°C with or without 100 nmol/l bradykinin. Different amounts of (T–) and (T+) (from 0 to 4.5 x 107/ml) platelets were then added to the BAEC medium for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are the means ± SD of experiments performed in triplicate on six separate occasions. *p < 0.001, ¶p < 0.01 vs. no addition of platelets (Control).

View larger version (33K): [in a new window]   Figure 5 Effect of vitamin C (Vit. C) and anti-LOX-1 monoclonal antibody (LOX-1 Ab) on platelet-induced variations of intracellular nitric oxide (NO) concentration in basal and bradykinin-stimulated bovine aortic endothelial cells (BAECs). The BAECs were preincubated with Vit. C (5 µmol/l) and anti-LOX-1 mAb (30 µg/ml) at 37°C for 30 min. Then the cells were incubated with 10 µmol/l DAF-2 DA at 37°C for 10 min with or without 100 nmol/l bradykinin. Thrombin-activated platelets (3.0 x 107/ml) were added to the BAEC medium for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are the means ± SD of experiments performed in triplicate on six separate occasions. ¶p < 0.01, *p < 0.001 vs. control; p < 0.01, p < 0.001 vs. platelets.