Karolina Siewiera

Platelet activation patterns are different in mouse models of diabetes and chronic inhibition of nitric oxide synthesis

Currently, there are several animal models of diabetes mellitus and hypertension, but relatively little is known about blood platelet function in these models. The aim of this work was to characterise and compare platelet reactivity and activation in db/db mice (mouse model of diabetes) and mice receiving L-NAME (model of chronic inhibition of NO synthesis), using various platelet function assays. We found higher platelet activation (circulating resting platelets) in db/db mice than in db/+heterozygotes, as evidenced by elevated expressions of CD62P and CD40L and a lower expression of CD42b. The expression of COX-1 was significantly increased, and the phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser(157) significantly reduced in platelets from db/db mice. Similarly, we observed platelet hyperreactivity in db/db mice following the in vitro responses to 20μg/ml collagen (reflected by increased expressions of CD62P and CD40L, and reduced CD42b), 20μM ADP (reduced CD42b) and lower concentrations of thrombin (0.025 U/ml) (increased CD62P, JON/A, bound vWF, and bound fibrinogen). Otherwise, platelet hyporeactivity was revealed for higher thrombin (0.25 U/ml) (reduced CD62P and bound vWF), while hyperreactivity occurred for CD40L and bound Fg in db/db mice compared to non-diabetic control, db/+. Plasma levels of sCD40L, but not of sCD62P, were increased in db/db mice; also plasma TXB2 concentrations were over 3.5-fold higher in this group than in the heterozygous db/+mice (P<0.01). In contrast, in the mice administered with L-NAME, no statistical differences in expressions of platelet activation markers were found between mice supplemented with L-NAME and controls. Likewise, the TXB2 level did not differ between L-NAME mice and controls, but L-NAME mice had significantly higher plasma levels of sCD62P and sCD40L than controls. In conclusion, these two studied models differ in the overall picture of blood platelet activation and reactivity, as they demonstrated opposite time sequence patterns of platelet activation in circulating blood. More generally, our study provides another argument for the opinion that multiparametric analysis of platelet function offers a much better tool for investigation and minimizes the likelihood of artefacts.

Ambiguous effect of dendrimer PAMAM G3 on rat heart respiration in a model of an experimental diabetes - Objective causes of laboratory misfortune or unpredictable G3 activity?

Poly(amido)amine (PAMAM) dendrimer G3 was investigated for its ability to support the proper functioning of rat heart mitochondria exposed to hyperglycemia, in both the in vitro and in vivo experiments. The main aims of this study were to check whether PAMAM G3 dendrimer improves the efficiency of the impaired respiration of rat heart mitochondria. This study showed that mitochondria isolated from animals studied in different seasons respond to G3 (100 μM) exposure to a different extent. Probably, seasonal variations had the impact on rat metabolism and consequently on the received data. The used biological samples formed a heterogenous group and therefore the obtained results were not pooled together but treated separately. Nevertheless, the in vitro part of this study revealed that PAMAM G3 could be successfully used in the protection of heart mitochondria against MG-induced impaired respiratory activity. Despite these promising data, the protective effect of G3 was not confirmed in the in vivo experiment. This study revealed that dendrimer G3 (20 mg/kgbw) is toxic and very high mortality among the animals administered with G3 did not allow to perform a reliable data analysis.

Resorcylidene Aminoguanidine (RAG) Improves Cardiac Mitochondrial Bioenergetics Impaired by Hyperglycaemia in a Model of Experimental Diabetes

Diabetes is associated with a mitochondrial dysfunction. Hyperglycaemia is also clearly recognized as the primary culprit in the pathogenesis of cardiac complications. In response to glycation and oxidative stress, cardiac mitochondria undergo cumulative alterations, often leading to heart deterioration. There is a continuous search for innovative treatment strategies for protecting the heart mitochondria from the destructive impact of diabetes. Aminoguanidine derivatives have been successfully used in animal model studies on the treatment of experimental diabetes, as well as the diabetes-driven dysfunctions of peripheral tissues and cells. Considerable attention has been paid particularly to β-resorcylidene aminoguanidine (RAG), often shown as the efficient anti-glycation and anti-oxidant agent in both animal studies and in vitro experiments. The aim of the present study was to test the hypothesis that RAG improves oxidative phosphorylation and electron transport capacity in mitochondria impaired by hyperglycaemia. Diabetes mellitus was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (70 mg/kg body weight). Heart mitochondria were isolated from healthy rats and rats with streptozotocin-diabetes. Mitochondrial respiratory capacity was measured by high resolution respirometry with the OROBOROS Oxygraph-2k according to experimental protocol including respiratory substrates and inhibitors. The results revealed that RAG protects the heart against diabetes-associated injury by improving the mitochondrial bioenergetics, thus suggesting a possible novel pharmacological strategy for cardioprotection.

The Janus Face of PAMAM Dendrimers Used to Potentially Cure Nonenzymatic Modifications of Biomacromolecules in Metabolic Disorders—A Critical Review of the Pros and Cons

Diabetes mellitus, which is characterised by high blood glucose levels and the burden of various macrovascular and microvascular complications, is a cause of much human suffering across the globe. While the use of exogenous insulin and other medications can control and sometimes prevent various diabetes-associated sequelae, numerous diabetic complications are still commonly encountered in diabetic patients. Therefore, there is a strong need for safe and effective antihyperglycaemic agents that provide an alternative or compounding option for the treatment of diabetes. In recent years, amino-terminated poly(amido)amine (PAMAM) dendrimers (G2, G3 and G4) have attracted attention due to their protective value as anti-glycation and anti-carbonylation agents that can be used to limit the nonenzymatic modifications of biomacromolecules. The focus of this review is to present a detailed survey of our own data, as well as of the available literature regarding the toxicity, pharmacological properties and overall usefulness of PAMAM dendrimers. This presentation pays particular and primary attention to their therapeutic use in poorly controlled diabetes and its complications, but also in other conditions, such as Alzheimer’s disease, in which such nonenzymatic modifications may underlie the pathophysiological mechanisms. The impact of dendrimer administration on the overall survival of diabetic animals and on glycosylation, glycoxidation, the brain-blood barrier and cellular bioenergetics are demonstrated. Finally, we critically discuss the potential advantages and disadvantages accompanying the use of PAMAM dendrimers in the treatment of metabolic impairments that occur under conditions of chronic hyperglycaemia.

Long-term untreated streptozotocin-diabetes leads to increased expression and elevated activity of prostaglandin H2 synthase in blood platelets

Abstract In diabetes-related states of chronic hyperglycaemia elevated concentrations of glucose may alter the functioning of platelet enzymes involved in arachidonic acid metabolism, including prostaglandin H2 synthase (cyclooxygenase) (PGHS, COX). Therefore, the principal aim of this study was to assess the effects of experimental chronic hyperglycaemia on platelet PGHS-1 (COX-1) expression and activity. Blood platelet activation and reactivity were assessed in Sprague–Dawley rats with the 5-month streptozotocin (STZ) diabetes. The PGHS-1 abundance in platelets was evaluated with flow cytometry and Western blotting, while its activity monitored using a high resolution respirometry and the peroxidase fluorescent assay. The production of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in platelets were assayed immunoenzymatically. Circulating platelets from diabetic were characterised by increased size, elevated ‘priming’ and altered reactivity, compared to non-diabetic animals. Both Western blot analysis and flow cytometry revealed significantly elevated expressions of platelet PGHS-1 in STZ-diabetic rats (p < 0.05). We also observed significantly elevated platelet PGHS-1-related arachidonic acid metabolism in diabetic vs. non-diabetic animals, with the use of polarographic (p < 0.05) and total activity assay (p < 0.001). Such increases were accompanied by the elevated production of PGE2 (p < 0.001) and TXB2 (p < 0.05) in diabetic animals. The increased PGHS-1-dependent oxygen consumption and the total activity of PGHS-1 in diabetic animals remained very significant (p < 0.001) also upon adjusting for blood platelet PGHS-1 abundance. Therefore, our results further contribute to the explanation of the increased metabolism of arachidonic acid observed in diabetes.

Higher mitochondrial potential and elevated mitochondrial respiration are associated with excessive activation of blood platelets in diabetic rats

AIMS The high glucose concentration observed in diabetic patients is a recognized factor of mitochondrial damage in various cell types. Its impact on mitochondrial bioenergetics in blood platelets remains largely vague. The aim of the study was to determine how the metabolism of carbohydrates, which has been impaired by streptozotocin-induced diabetes may affect the functioning of platelet mitochondria. MATERIALS AND METHODS Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Platelet mitochondrial respiratory capacity was monitored as oxygen consumption (high-resolution respirometry). Mitochondrial membrane potential was assessed using a fluorescent probe, JC-1. Activation of circulating platelets was monitored by flow cytometry measuring of the expressions of CD61 and CD62P on a blood platelet surface. To determine mitochondrial protein density in platelets, Western Blot technique was used. KEY FINDINGS The results indicate significantly elevated mitochondria mass, increased mitochondrial membrane potential (ΔΨm) and enhanced respiration in STZ-diabetic animals, although the respiration control ratios appear to remain unchanged. Higher ΔΨm and elevated mitochondrial respiration were closely related to the excessive activation of circulating platelets in diabetic animals. SIGNIFICANCE Long-term diabetes can result in increased mitochondrial mass and may lead to hyperpolarization of blood platelet mitochondrial membrane. These alterations may be a potential underlying cause of abnormal platelet functioning in diabetes mellitus and hence, a potential target for antiplatelet therapies in diabetes.

Facts and Artifacts in the Evaluation of the Anti-diabetic Activity of Spent Hop Extract in Rat Hearts in the"Experimental Model of Diabetes"

The present study was undertaken to demonstrate the anti-diabetic mode of action of Humulus lupulus L. extract and its protective influence against changes in the respiratory capacity and coronary flow of the heart in experimental diabetes.

Effects of a single bout of strenuous exercise on platelet activation in female ApoE/LDLR−/− mice

Abstract Strenuous physical exercise leads to platelet activation that is normally counterbalanced by the production of endothelium-derived anti-platelet mediators, including prostacyclin (PGI2) and nitric oxide (NO). However, in the case of endothelial dysfunction, e.g. in atherosclerosis, there exists an increased risk for intravascular thrombosis during exercise that might be due to an impairment in endothelial anti-platelet mechanisms. In the present work, we evaluated platelet activation at rest and following a single bout of strenuous treadmill exercise in female ApoE/LDLR−/− mice with early (3-month-old) and advanced (7-month-old) atherosclerosis compared to female age-matched WT mice. In sedentary and post-exercise groups of animals, we analyzed TXB2 generation and the expression of platelet activation markers in the whole blood ex vivo assay. We also measured pre- and post-exercise plasma concentration of 6-keto-PGF1α, nitrite/nitrate, lipid profile, and blood cell count. Sedentary 3- and 7-month-old ApoE/LDLR−/− mice displayed significantly higher activation of platelets compared to age-matched wild-type (WT) mice, as evidenced by increased TXB2 production, expression of P-selectin, and activation of GPIIb/IIIa receptors, as well as increased fibrinogen and von Willebrand factor (vWf) binding. Interestingly, in ApoE/LDLR−/− but not in WT mice, strenuous exercise partially inhibited TXB2 production, the expression of activated GPIIb/IIIa receptors, and fibrinogen binding, with no effect on the P-selectin expression and vWf binding. Post-exercise down-regulation of the activated GPIIb/IIIa receptor expression and fibrinogen binding was not significantly different between 3- and 7-month-old ApoE/LDLR−/− mice; however, only 7-month-old ApoE/LDLR−/− mice showed lower TXB2 production after exercise. In female 4–6-month-old ApoE/LDLR−/− but not in WT mice, an elevated pre- and post-exercise plasma concentration of 6-keto-PGF1α was observed. In turn, the pre- and post-exercise plasma concentrations of nitrite (NO2−) and nitrate (NO3−) were decreased in ApoE/LDLR−/− as compared to that in age-matched WT mice. In conclusion, we demonstrated overactivation of platelets in ApoE/LDLR−/− as compared to WT mice. However, platelet activation in ApoE/LDLR−/− mice was not further increased by strenuous exercise, but was instead attenuated, a phenomenon not observed in WT mice. This phenomenon could be linked to compensatory up-regulation of PGI2-dependent anti-platelet mechanisms in ApoE/LDLR−/− mice.

Flow cytometry analysis reveals different activation profiles of thrombin- or TRAP-stimulated platelets in db/db mice. The regulatory role of PAR-3

INTRODUCTION Recent studies have shown that it may be the concentration of thrombin, which is discriminative in determining of the mechanism of platelet activation via protease activated receptors (PARs). Whether the observed phenomenon of differentiated responses of mouse platelets to various thrombin concentrations in non-diabetic db/+ and diabetic db/db mice depends upon the concerted action of various PARs, remains to be established. RESULTS We found elevated reactivity of platelets, as well as the enhanced PAR-3 expression in response to both the used concentrations of AYPGKF in db/db mice, as compared to db/+ heterozygotes. At low concentration of thrombin platelets from diabetic mice demonstrated hyperreactivity, reflected by higher expression of PAR-3. For higher thrombin concentration, blood platelets from db/db mice appeared hyporeactive, compared to db/+ animals, while no significant differences in PAR-3 expression were observed between diabetic and non-diabetic mice. CONCLUSIONS The novel and previously unreported finding resulting from our study is that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low thrombin (when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to higher reactivity of platelets in db/db mice.

Only the Truth Would Enlighten Us — The Advantages and Disadvantages of Flow Cytometry as a Method of Choice in the Study of Mouse and Rat Platelets

Increasing number of transgenic and knockout strains of laboratory rodents has been developed to provide reliable models of human cardiovascular diseases. Due to apparent differences in platelet physiology, morphology, biochemistry, etc. between rodents and men, methods employed to study blood platelets in rodents should always consider these differences in a reasonably critical way. Flow cytometry is a conven‐ ient tool that enables to easily cope with the minute amounts of the available biologi‐ cal material and providing an extremely versatile information. This review focuses on the practical and methodological aspects of flow cytometry, pointing to the key elements of the commonly used protocols for determining of multiple parameters of blood platelet (patho)physiology in mice and rats. We summarized and critically reviewed the available procedures, as well as figured out how to overcome possible obstacles, shortcomings, drawbacks or artefacts that a researcher may encounter when monitor‐ ing various phenomena intimately associated with blood platelet biology. Flow cytometry assays have been also collated with some alternative techniques (intravital fluorescence microscopy, in vitro platelet adhesion under flow conditions). We hope that our paper may further facilitate other researchers to study mouse and rat platelets with the use of the most optimal and the least artefact-prone procedures.