Potential Role of Mitochondria as Modulators of Blood Platelet Activation and Reactivity in Diabetes and Effect of Metformin on Blood Platelet Bioenergetics and Platelet Activation.

Blood platelet dysfunctions are strongly involved in the development of the micro- and macrovascular complications in diabetes mellitus (DM). However, the molecular causes of abnormal platelet activation in DM remain unclear. Experimental data suggests that platelet mitochondria can regulate the prothrombotic phenotype of platelets, and changes in these organelles may influence platelet activation and modify platelet responses to stimulation. The present study evaluates the impact of DM on mitochondrial respiratory parameters and blood platelet activation/reactivity in a rat model of experimental diabetes following 1, 2.5 and 5 months of streptozotocin (STZ)-induced diabetes. Moreover, a mild inhibition of the mitochondrial respiratory chain with the use of metformin under in vitro and in vivo conditions was tested as a method to reduce platelet activation and reactivity. The platelets were studied with a combination of flow cytometry and advanced respirometry. Our results indicate that prolonged exposure of blood platelets to high concentrations of glucose, as in diabetes, can result in elevated blood platelet mitochondrial respiration; this may be an effect of cell adaptation to the high availability of energy substrates. However, as these alterations occur later than the changes in platelet activation/reactivity, they may not constitute the major reason for abnormal platelet functioning in DM. Moreover, metformin was not able to inhibit platelet activation and reactivity under in vitro conditions despite causing a decrease in mitochondrial respiration. This indicates that the beneficial effect of metformin on the coagulation system observed in vivo can be related to other mechanisms than via the inhibition of platelet activation.

Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel in vivo by Decreasing of Fibrinogen Density in Thrombus.

Blood platelets' adenosine receptors (AR) are considered to be a new target for the anti-platelet therapy. This idea is based on in vitro studies which show that signaling mediated by these receptors leads to a decreased platelet response to activating stimuli. In vivo evidence for the antithrombotic activity of AR agonists published to date were limited, however, to the usage of relatively high doses given in bolus. The present study was aimed at verifying if these substances used in lower doses in combination with inhibitors of P2Y12 could serve as components of dual anti-platelet therapy. We have found that a selective A2A agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HE-NECA) improved the anti-thrombotic properties of either cangrelor or prasugrel in the model of ferric chloride-induced experimental thrombosis in mice. Importantly, HE-NECA was effective not only when applied in bolus as other AR agonists in the up-to-date published studies, but also when given chronically. In vitro thrombus formation under flow conditions revealed that HE-NECA enhanced the ability of P2Y12 inhibitors to decrease fibrinogen content in thrombi, possibly resulting in their lower stability. Adenosine receptor agonists possess a certain hypotensive effect and an ability to increase the blood-brain barrier permeability. Therefore, the effects of anti-thrombotic doses of HE-NECA on blood pressure and the blood-brain barrier permeability in mice were tested. HE-NECA applied in bolus caused a significant hypotension in mice, but the effect was much lower when the substance was given in doses corresponding to that obtained by chronic administration. At the same time, no significant effect of HE-NECA was observed on the blood-brain barrier. We conclude that chronic administration of the A2A agonist can be considered a potential component of a dual antithrombotic therapy. However, due to the hypotensive effect of the substances, dosage and administration must be elaborated to minimize the side-effects. The total number of animals used in the experiments was 146.

Intravital Assessment of Blood Platelet Function. A Review of the Methodological Approaches with Examples of Studies of Selected Aspects of Blood Platelet Function.

Platelet biology owes to intravital studies not only a better understanding of platelets' role in primary hemostasis but also findings that platelets are important factors in inflammation and atherosclerosis. Researchers who enter the field of intravital platelet studies may be confused by the heterogeneity of experimental protocols utilized. On the one hand, there are a variety of stimuli used to activate platelet response, and on the other hand there are several approaches to measure the outcome of the activation. A number of possible combinations of activation factors with measurement approaches result in the aforementioned heterogeneity. The aim of this review is to present the most often used protocols in a systematic way depending on the stimulus used to activate platelets. By providing examples of studies performed with each of the protocols, we attempt to explain why a particular combination of stimuli and measurement method was applied to study a given aspect of platelet biology.

Adenosine Receptor Agonists Exhibit Anti-Platelet Effects and the Potential to Overcome Resistance to P2Y12 Receptor Antagonists.

Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y12 antagonists were similar in high- and low-responders to P2Y12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.

Adenosine receptor agonists deepen the inhibition of platelet aggregation by P2Y12 antagonists.

Several adenosine receptor (AR) agonists have been shown in the past to possess anti-platelet potential; however, the adjunctive role of AR agonists in anti-platelet therapy with the use of P2Y12 receptor inhibitors has not been elucidated so far. This in vitro aggregation-based study investigates whether the inhibition of platelet function mediated by cangrelor or prasugrel metabolite can be potentiated by AR agonists. It evaluates the effect of non-selective (2-chloroadenosine), A2A-selective (UK 432097, MRE 0094, PSB 0777) and A2B-selective AR agonists (BAY 60-6583) on platelet function in relation to their toxicity, specificity towards adenosine receptor subtypes, structure and solubility. UK 432097, 2-chloroadenosine, MRE 0094 and PSB 0777 were found to be more or less potent inhibitors of ADP-induced platelet aggregation when acting alone, and that they remained non-cytotoxic to the cells. These AR agonists were also effective in the potentiation of the effects exerted by P2Y12 antagonists. Considering the estimated IC50 value, UK 432097, showing a relatively high binding affinity to the A2A adenosine receptor, has been identified as the most potent anti-aggregatory agent. This compound diminished platelet aggregation at nanomolar concentrations and further augmented platelet inhibition by P2Y12 antagonists by approx. 60% (P < .01). Our results indicate the importance of adenosine receptors as therapeutic targets and point out challenges and potential benefits of therapeutic use of a combined therapy of P2Y12 antagonist and AR agonist in cardioprotection. Our comparative analysis of the effects of AR agonists on platelet response in plasma and whole blood may indirectly suggest that other blood morphology elements contribute little to the inhibition of platelet function by AR agonists.

Comparison of different microscopy approaches to quantification of inhibitory effect on thrombus formation under flow conditions by the example of adenosine receptor agonist HE-NECA.

INTRODUCTION: Thrombus formation in vitro in flow conditions and its visualization and quantification with the use of microscopy are widely utilized to evaluate activity of compounds with a potential antithrombotic activity. Visualization and quantification of thrombi can be performed with the use of wide-field or confocal microscopy. Acquiring reliable numerical data from wide-field microscopy images of objects which have a complex three-dimensional structure is strongly influenced by the methods used for image analysis. This can be a possible source of inaccuracy in assessment of antithrombotic activity of a tested substance. We aimed to verify how different approaches to the quantification of wide-field images can affect the evaluation of an antiplatelet effect of a tested substance. METHODS: We compared three algorithms of image analysis to evaluate an effect of 2-hexynyl-5'-ethylcarboxamidoadenosine (HE-NECA), a compound of a moderate antiplatelet activity on thrombus formation, and of abciximab - a potent antiplatelet compound. Also, we studied how the results obtained in a wide-field imaging correspond to those obtained by means of confocal imaging. RESULTS: Three algorithms for analysis of wide-field images showed antiplatelet effect of HE-NECA or abciximab. Absolute values of thrombus area and outcomes of the evaluation of inhibition efficacy of HE-NECA were significantly different between the algorithms. Analysis of volumes and heights of thrombi obtained by confocal imaging confirmed inhibitory effect of HE-NECA, but the evaluated levels of inhibition were significantly different from that obtained by wide-field imaging. DISCUSSION: We conclude that wide-field imaging provides reliable qualitative data on an inhibitory effect on thrombus formation, despite differences which can emerge from various approaches to image analysis. However, quantitative evaluation and comparison of the efficacy of inhibitors on the basis of total area occupied by thrombi obtained by wide-field microscopy should be made with caution. To obtain a reliable quantitative assessment of the effect of a tested compound on thrombus structure the use of confocal microscopy is inevitable.

The impact of 1MeTIQ on the dopaminergic system function in the 6-OHDA model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder which is caused by degeneration of dopaminergic neurons of the nigrostriatal pathway. As a model of PD we used 6-hydroxydopamine (6-OHDA) which exerts toxic effects on catecholaminergic neurons and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) as neuroprotective compound. The aim of the present study, was to investigate the potential neuroprotective properties of 1MeTIQ against 6-OHDA-induced neurotoxic effects in the rat. METHODS: In the behavioral study, we measured locomotor activity and catalepsy. In the biochemical studies using HPLC methodology, we analyzed the concentration of dopamine and its metabolites in rat brain. RESULTS: Behavioral tests showed that 6-OHDA decreased rat locomotor activity and produced an increase of catalepsy. These effects did not blocked by 1MeTIQ injections. Biochemical studies indicated that 6-OHDA lesion significantly reduced the concentration of dopamine and its metabolites in the nigro-striatal pathway in the lesioned (ipsilateral) side. Moreover, 6-OHDA induced an increase in the rate of dopamine oxidation. Both acute and chronic administration of 1MeTIQ did not reverse the effects of 6-OHDA lesion on the ipsilateral side, however, it produced a significant elevation of the dopamine concentration in the contralateral side. It is evident that multiple treatments with 1MeTIQ stimulate undamaged neurons to increased activity. CONCLUSION: 1MeTIQ was shown to possess neuroprotective potential to the dopaminergic neurons damaged by 6-OHDA lesion. This compound has a protective effect but does not have neurorestorative capacity. It does not reverse damage already caused but will maintain the function and activity of undamaged dopamine neurons at physiological level.