Glycine reduces platelet aggregation

Abstract

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl(-) influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1-10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.

Figures

Fig. 1

The effect of glycine on bleeding time in rats. Rats were fed 5 % dietary glycine for 5 days while controls received a diet containing 5 % isonitrogenous valine. Bleeding time was measured as described in “Materials and methods”. Values are mean ± SEM, n = 6. *p < 0.05 compared to valine treated controls using Student’s t test

Fig. 2

Dietary glycine inhibits rat platelet aggregation in vivo in a dose-dependent manner. Blood was drawn from rats fed diets containing 0.6, 1.3, 2.5 or 5 % glycine for 5 days. Heparinized whole blood was used to measure the amplitude of platelet aggregation after a ADP (0.3 µM) or b collagen (8 µg/ml) from changes of impedance at 37 °C with an aggregometer as described in “Materials and methods”. Values are mean ± SEM, n = 6, p < 0.05 for comparison to platelet aggregation without glycine (control) by one-way ANOVA with Student–Newman–Keul’s post hoc test

Fig. 3

Glycine inhibits rat platelet aggregation in vitro in a dose-dependent manner. Heparinized blood was incubated at 37 °C for 10 min with glycine at a final concentration of 1 to 10 mM. Subsequently, platelet aggregation was induced with either a ADP (0.3 µM) or b collagen (8 µg/ml), and changes of impedance were measured as described in “Materials and methods”. Values are mean ± SEM, n = 6, p < 0.05 for comparison to platelet aggregation without glycine (control) by one-way ANOVA with Student–Newman–Keul’s post hoc test

Fig. 4

Strychnine blunts inhibitory effects of glycine on rat platelet aggregation. Heparinized whole blood was incubated at 37 °C for 10 min with glycine at a final concentration of 3 mM. Subsequently, platelet aggregation was induced with collagen (8 µg/ml) and changes of impedance were measured as described in “Materials and methods”. Strychnine was added at a final concentration of 1 µM. Values are mean ± SEM, n = 8. *p < 0.05 for comparison to the control group; #p < 0.05 for comparison to the glycine group by one-way ANOVA with Student–Newman–Keul’s post hoc test

Fig. 5

Western blot of glycine receptor in spinal cord and rat platelets. Western blotting was performed as described in “Materials and methods”. Western blot of 3 µg spinal cord membrane extract (SC) and 30 µg of platelet membrane extract (RP) using anti-GlyR4a monoclonal antibody is shown

Fig. 6

Western blot of glycine receptor in erythrocytes and human platelets. Western blotting was performed as described in “Materials and methods”. 10 µg of human platelet membrane extract (HP) and erythrocyte cell extract (E) was labeled with anti-glycine receptor alpha 1- + alpha 2 antibody. Loading control was done using anti-β-actin antibody