Methylglyoxal induces platelet hyperaggregation and reduces thrombus stability by activating PKC and inhibiting PI3K/Akt pathway

Diabetes is characterized by a dysregulation of glucose homeostasis and platelets from patients with diabetes are known to be hyper-reactive and contribute to the accelerated development of vascular diseases. Since many of the deleterious effects of glucose have been attributed to its metabolite methylgyloxal (MG) rather than to hyperglycemia itself, the aim of the present study was to characterize the effects of MG on platelet function. Washed human platelets were pre-incubated for 15 min with MG and platelet aggregation, adhesion on matrix-coated slides and signaling (Western blot) were assessed ex vivo. In vivo, the effect of MG on thrombus formation was determined using the FeCl3-induced carotid artery injury model. MG potentiated thrombin-induced platelet aggregation and dense granule release, but inhibited platelet spreading on fibronectin and collagen. In vivo, MG accelerated thrombus formation but decreased thrombus stability. At the molecular level, MG increased intracellular Ca2+ and activated classical PKCs at the same time as inhibiting PI3K/Akt and the β3-integrin outside-in signaling. In conclusion, these findings indicate that the enhanced MG concentration measured in diabetic patients can directly contribute to the platelet dysfunction associated with diabetes characterized by hyperaggregability and reduced thrombus stability

Insulin Induces the Release of Vasodilator Compounds From Platelets by a Nitric Oxide–G Kinase–VAMP-3–dependent Pathway

Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis

Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions

MicroRNAs in platelet biogenesis and function : implications in vascular homeostasis and inflammation

Platelets are involved in vascular homeostasis and inflammation through interaction with circulating blood cells and vascular wall. MiRNAs are small, conserved and non-coding RNA molecules, which interact directly with specific mRNAs regions regulating gene expression. The purpose of this review is to gather all known platelet miRNAs and summarize their role in platelet biogenesis and function. Increasing evidence supports the role of miR-34a and miR-150 in megakaryocytopoiesis and platelet production. Although 284 miRNAs are described to be present in platelets, their role is mostly unknown. The most abundant miRNA in platelets is miR-223 followed by miR-126. The miR-96, miR-200b, miR- 495, miR-107 and miR-223 are critically involved in platelet reactivity, aggregation, secretion and adhesion. The presence of miRNAs known to regulate angiogenesis in platelets is also discussed. Furthermore, platelet-derived microvesicles and microparticles contain several miRNAs, which may facilitate the communication between platelets with other vascular cells, a mechanism that may play an important role in vascular homeostasis and inflammation. Further studies are needed to elucidate the exact roles of platelet miRNAs in platelet function and vascular biology

Effect of MG on the phosphorylation of β3 integrin and Akt.

<p>(a) Effect of MG (MG, 1 mmol/L, 15 minutes) on fibronectin (Fn) and collagen (coll)-induced tyrosine phosphorylation of β3 integrin (Tyr747). (b) Effect of MG on thrombin -induced tyrosine phosphorylation (Tyr747) of β3 integrin in washed human platelets. (c) Effect of MG on fibronectin (Fn) and collagen (coll)-induced phosphorylation of Akt (Ser 473). (d) Effect of wortmannin (Wt, 20 nmol/L, 30 minutes) on fibronectin (Fn) and collagen (coll)-induced phosphorylation of β3 integrin (Tyr747) and Akt (Ser 473). The graphs summarise the data from 6 different experiments; *P<0.05, ***P<0.001 versus sol or CTL and ^# P<0.05, ^{# # #} P<0.001 versus agonists.</p

Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPK&alpha;1 and &alpha;2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPK&alpha;&minus;/&minus;) or endothelial-specific deletion (AMPK&alpha;&Delta;EC) of the AMPK&alpha; subunits. In control and AMPK&alpha;1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPK&alpha;1 or &alpha;2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPK&alpha;1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPK&alpha;1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPK&alpha;1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction

Effect of MG on platelet [Ca²⁺]_i and degranulation.

<p>(a) Increase in [Ca²⁺]_i measured in washed human platelets treated with either solvent (CTL) or methylglyoxal (MG, 1 mmol/L, 15 minutes) prior to the stimulation with thrombin. (b) Effect of MG pre-treatment on the thrombin (0.03U/ml)-induced release of ATP and (c) on the TRAP-induced surface expression of P-selectin. The graphs summarise the data from at least 6 different individuals; *P<0.05, **P<0.01 versus CTL.</p