Methylglyoxal potentiates thrombin and collagen‐induced human platelet aggregation.

Abstract

ObjectivesMethylglyoxal (MGO), a metabolite of the glycolysis pathway, is a precursor of advanced glycation end products (AGE). Levels of MGO are elevated in pre‐diabetic and diabetic patients (Dornadula 2015 et al. Chem Res Toxicol. 2015;28:1666–74). Because increased AGE formation has been proposed as one mechanism involved in vascular complications and platelet activation of diabetes (Hadas K et al, Plos One, 8:e74401; 2013), this study aimed to evaluate the effects of MGO on the human washed platelet aggregation induced by thrombin and collagen.MethodsThe study was approved by the local Ethics Committee (Protocol No. 66299817.4.0000.5137). Platelets were isolated from peripheral blood of human healthy volunteers (7 men and 10 women, aged 27.8±1.9 y.o.). Platelet samples (200 μL) were incubated with MGO (0.08–0.64 mM), and subsequently activated with either thrombin (0.01 to 1.0 U/ml) or collagen (0.2 to 20 μg/ml). Platelet aggregation was performed with an optical aggregometer (PAP‐8E Platelet Aggregation Profiler, PA, USA).ResultsThrombin produced concentration‐dependent platelet aggregation, achieving a maximum response at 0.3 and 1 U/ml (0.8 ± 0.5, 15.7±4.7, 27.8±5.5, 59.6±5.4 and 63.9±3.3% for 0.01, 0.03, 0.1, 0.3 and 1 U/ml, respectively). Pre‐incubation with MGO (0.08 mM) markedly increased (p<0.001) the thrombininduced platelet aggregation (14.0±13.5, 50.6±4.7, 72.4±4.2, 79.4±4.4 and 77.2±2.7%, respectively). Similar data were observed using higher concentrations of MGO (0.16 and 0.64 mM). In separate samples, addition of collagen induced concentration‐dependent platelet aggregation (0.2±0.2, 8.6±3.7, 14.4±4.4, 41.4±4.9 and 53.2±5.3 for 0.2, 1.0, 2.0, 10.0 and 20.0ug respectively). Pre‐incubation with MGO (0.08 mM) significantly increased (p<0.001) the collagen‐induced aggregation (0.5±0.3, 38.7±11.5, 38.3±6.8, 56.8±4.9 and 57.1±4.9 respectively). Higher concentrations of MGO (0.16 and 0.64 mM) also significantly potentiated collagen‐induced aggregation. MGO incubated had no effect in the absence of thrombin or collagen. Next, we evaluated if the potentiation of thrombin‐ and collagen‐induced platelet aggregation by MGO was due to inactivation of nitric oxide (NO) – cyclic GMP signaling Incubation of the NO donor sodium nitroprusside (3uM) inhibited by 32,7% a 60,9% (p<0.05) the thrombin‐ and collagen‐induced aggregation, respectively. However, MGO (0.08 e 0.16 mM) had no effect on the inhibitory effect of SNP on thrombin‐ and collagen‐induced aggregation. We also evaluated the protein expression by western blotting of both α1 and β1 subunits of soluble guanylate cyclase in the absence and presence of MGO. However, MGO did not significantly modify the expression of either subunits.ConclusionMGO enhances human platelet aggregation induced by thrombin and collagen by mechanisms independent of inactivation of NO‐cGMP signaling.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.