The platelet-endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells

Methods

Results

In our experimental conditions, the incubation of BAECs with increasing amounts of platelets for 10 min induced a sharp and dose-dependent increase in intracellular concentration of ROS and O2·− (p from <0.01 to <0.001) (Fig. 1A). We also performed some time-course experiments of O2·− formation in BAECs incubated with platelets activated with different amounts of thrombin. By two-way ANOVA with repeated measures, 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 O2·− generation. As shown in Figure 1B, from the evaluation of the time-course of O2·− formation in BAECs using post-ANOVA Tukey test for multiple comparisons, it was clear that the effect of platelets was already present after 2 min of incubation even with submaximal doses of thrombin.

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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 × 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 × 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.

To exclude the interference of biologically highly active compounds secreted by thrombin-activated platelets on ROS and O2·− generated by BAECs, the effects of thrombin-activated platelet releasate and of specific antagonists were analyzed. The incubation of thrombin-activated platelet releasate with BAECs did not significantly affect intracellular concentration of ROS and O2·−. Similarly the preincubation of BAECs with SQ29548, ketanserin, yohimbine, and with the ADP scavengers apyrase and PEP-PK did not modify intracellular ROS and O2·− concentration induced by activated platelets in BAECs.

To test the response specificity and to verify if O2·− increase was dependent on platelets binding to LOX-1, vitamin C and LOX-1 Ab were incubated with BAECs, CHO-K1, and BLOX-1-CHO cells. As shown in Figure 2, the O2·− concentration was markedly reduced in BAECs and BLOX-1-CHO cells preincubated with vitamin C (p < 0.001) and LOX-1 Ab (p < 0.001), whereas control CHO-K1 cells were not affected. Besides LOX-1 Ab, anti-CD41a slightly inhibited the production of O2·−, and the simultaneous application of anti-LOX-1 and anti-CD41a showed an additive effect (p < 0.001). Finally, annexin V significantly reduced the production of O2·− induced by thrombin-activated platelets (p < 0.001) (Fig. 3). Annexin V also decreased the generation of O2·− induced by non-stimulated platelets (3 × 10⁷/ml) by 14 ± 1.3% (p < 0.05).

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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 × 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.

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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 × 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.

The PS liposomes (at concentrations of 10 and 20 μmol/l total lipids/10⁶cells) determined a dose-dependent increase in O2·− production in BAECs (mean fluorescence intensity [MFI] from 0.3 ± 0.029 to 1.5 ± 0.13 and to 2.7 ± 0.23, p < 0.001, n = 10) and BLOX-1-CHO cells (MFI from 0.24 ± 0.020 to 1.8 ± 0.17 and to 3.1 ± 0.29, p < 0.001, n = 10). The PS liposomes produced no effect on CHO-K1 cells.

Anti-CD40L, antiCD11a, anti-CD62, anti-CD31, anti-VWF, sialyl Lewis X oligosaccharide, and control immunoglobulin G (IgG) did not affect O2·− generation.

The incubation of BAECs with increasing amounts of platelets for 10 min in the presence of DAF-2 DA dose-dependently reduced basal and bradykinin-induced intracellular NO concentration (p from <0.01 to <0.001) (Fig. 4). Similarly, concentrations of nitrite in the supernatant of stimulated BAECs dropped from 635 ± 25 pmol/well to 211 ± 12 pmol/well (p < 0.001, n = 12) after the addition of activated platelets (4.5 × 10⁷/ml), the contribution of activated platelets alone being 124 ± 8 pmol/4.5 × 10⁷platelets.

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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 × 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).

The incubation of platelet releasate with BAECs did not significantly affect basal and stimulated concentration of intracellular NO and supernatant nitrite. Similarly, the preincubation of BAECs with SQ29548, ketanserin, yohimbine, and with the ADP scavengers apyrase and PEP-PK did not interfere with the modifications of basal and stimulated intracellular NO and supernatant nitrite induced by activated platelets.

To test if the reduction of intracellular NO concentration induced by platelets was dependent on O2·− generation, we preincubated BAECs with vitamin C and LOX-1 Ab. Figure 5shows that the preincubation of BAECs with vitamin C and LOX-1 Ab significantly counteracted the effect of activated platelets on basal and stimulated generation of NO (p from <0.01 to <0.001).

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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 × 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.

The effect of platelets on endothelial cell eNOS activity was examined by the ³H citrulline assay. Activated platelets (4.5 × 10⁷/ml) did not significantly modify the ability of eNOS to metabolize L-arginine to L-citrulline (BAECs = 76.6 ± 10.4 pmol citrulline/mg protein/min, BAECs + platelets = 72 ± 9.9 pmol citrulline/mg protein/min, p = NS).

As for the oxidative system involved, we found that aspirin, L-NAME, L-NMMA, D-NAME, D-NMMA, oxypurinol, and rotenone did not modify O2·− concentration in endothelial cells after platelet incubation, whereas DPI drastically reduced the intracellular amount of O2·− (MFI from 8.20 ± 0.88 to 0.34 ± 0.04, p < 0.001, n = 10).

Discussion

Lectin-like ox-LDL receptor-1 is a type II membrane protein with a C-type lectin-like structure at the C terminus (15). The expression of LOX-1 in endothelial cells in vitro and in vivo is highly regulated (22). Besides ox-LDL, LOX-1 binds aged/apoptotic cells (19)and platelets (18). The binding of platelets to LOX-1 has recently been shown to increase the release of endothelin from endothelial cells supporting the induction of endothelial dysfunction (18).

In this study, we demonstrated for the first time that the binding of platelets to LOX-1 reduces the intracellular concentration of NO in cultured BAECs. This decrease was associated with a contemporary fall of nitrite in the supernatant, indicating that the reduction in intracellular NO concentrations also reflects the extracellular NO concentration. Taken together, these results suggest a role of platelets in the selective impairment of endothelium-derived relaxation which is characteristic of the early stages of atherosclerosis (6,7).

The rapid decrease in NO induced by platelets was paralleled by a specular fast increase in ROS and O2·− formation. The increased cellular production of ROS and O2·− was prevented by preincubating BAECs with vitamin C, an antioxidant known to work as a radical scavenger and with LOX-1 Ab. Furthermore, the incubation of platelets with BLOX-1 CHO cells determined a time- and dose-dependent significant increase in ROS and O2·− formation, which once again were abolished by vitamin C and LOX-1 Ab. As for the effect of LOX-1 Ab, our results agree with those of Kakutani et al. (18), who demonstrated that the preincubation of BAECs or BLOX-1 CHO cells with LOX-1 Ab greatly reduced the binding of platelets to LOX-1. To compare with other molecules possibly involved in platelet-endothelium interaction, antibodies or antagonists for these molecules were introduced into the medium. Besides LOX-1 Ab, anti-CD41a inhibited the O2·− formation approximately 25%, indicating that glycoprotein IIb/IIIa-mediated binding is also involved in the platelet–endothelium interaction. The simultaneous addition of LOX-1 Ab and anti-CD41 antibody showed an additive effect on O2·−, indicating, as previously shown (18), that LOX-1 and glycoprotein IIb/IIIa work independently on platelet-endothelium interaction. Anti-CD40L, anti-CD11a, anti-CD62, anti-CD31, anti-VWF, sialyl Lewis X oligosaccharide, and control IgG did not affect the O2·− production induced by platelets.

As for the mechanism of platelet-endothelium interaction, the results we obtained with annexin V, a PS binding protein (28), are consistent with the hypothesis that PS may be one of the major determinants of platelet binding to LOX-1. Phosphatidylserine in fact is an efficient ligand for LOX-1 (19), and although PS binding sites have also been demonstrated in non-activated platelets (20), they are mainly exposed on the surface of activated platelets (19,20). In addition, the results of this study show that PS dose-dependently increased O2·− production both in BAECs and BLOX-1-CHO cells. Even if these results should be considered preliminary, another conclusion of this study is that PS not only may be one of the major determinants of platelet binding to LOX-1, but may also trigger a train of events leading to O2·− generation.

Taken together, these data show that the incubation of platelets with BAECs is associated with an increased intracellular production of ROS and O2·− and that the platelet ligation to LOX-1 plays a crucial role in intracellular ROS and O2·− generation. The results of this study also demonstrate that the decrease of intracellular NO concentration was prevented by preincubating BAECs with vitamin C and with LOX-1 Ab. The data support the conclusion that the incubation of platelets with BAECs is associated with a receptor-dependent, abnormally increased intracellular production of ROS, and in particular of O2·−. The NO decrease therefore may be secondary to O2·− formation, which is known to inactivate NO in a chemical reaction during which the cytotoxic radical peroxynitrite is formed (12). In our experimental conditions, platelets did not significantly alter the ability of eNOS to metabolize L-arginine to L-citrulline. Because the conversion of ³H arginine into ³H citrulline, under apparent maximum velocity conditions (14,27), is a measure of eNOS levels, the results of this study show that the binding of platelets to LOX-1 did not alter, at least quantitatively, the ability to produce NO.

As for the oxidative system involved in ROS and O2·− generation, the results of this study clearly show that only DPI, a non-selective inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (29), reduced O2·− generation in BAECs stimulated by platelets. Because specific inhibitors of cyclooxygenase, eNOS, xanthine oxidase, and mitochondrial nicotinamide adenine dinucleotide dehydrogenase did not reduce O2·− generation in BAECs stimulated by platelets, another conclusion of this study is that the increase of O2·− release after platelet exposure may be related to the increased activity of endothelial NADPH oxidase.

The decrease in intracellular NO concentration as a result of O2·− generation may also have implications in vivo. For example, the impaired endothelium-dependent vasodilation is associated with an augmented susceptibility of atherosclerotic blood vessels to the development of vasospasm in vivo (6), the inappropriate constriction leading to ischemic manifestations (30). Increased oxidative stress within the vascular wall may facilitate platelet activation (31,32). The O2·− produced by the ligation of activated platelets to LOX-1 may finally inactivate NO in a chemical reaction during which peroxynitrite is formed. Furthermore, a deficiency of NO has been shown to be associated with arterial thrombosis in animal models (33)and in individuals with acute coronary syndromes (34). The decrease in NO availability induced by the binding of platelets to LOX-1 may, therefore, in turn further increase platelet activation, induce adhesion and aggregation, and finally favor platelet-dependent arterial thrombosis.

In conclusion, the results of this study show that one of the pathophysiologic consequences of platelets binding to LOX-1 may be the inactivation of NO through an increased cellular production of O2·−.