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

Przygodzki, Tomasz; Wolska, Nina; Talar, Marcin; Polak, Dawid; Gapińska, Magdalena; Watała, Cezary

doi:10.1016/j.vascn.2018.07.003

Cited by 4 publications

(3 citation statements)

References 18 publications

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“…In each field of a view 70 focal planes were acquired (logical size format X/Y/Z 512/512/70) [54]. For the analysis of images, FIJI software was used [55], according to our previously established protocol [56]. Briefly, the thresholding procedure was performed with the use of 'Auto Local Threshold' function (Bernsen method) with a radius value set at 5.…”

Section: Imaging and Image Analysismentioning

confidence: 99%

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

et al. 2019

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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 P2Y 12 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 P2Y 12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y 12 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 P2Y 12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y 12 antagonists were similar in high-and low-responders to P2Y 12 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.Molecules 2020, 25, 130 2 of 17 ADP is one of the key mediators of both physiological haemostasis and thrombosis, being not only a direct agonist of platelets, but also an important factor released from platelet intracellular structures, enhancing the platelet response initially induced by other activators. Platelets have two ADP receptors on their surface: the P2Y 1 receptor initiates platelet aggregation, while the P2Y 12 receptor enhances this process, eventually leading to the formation of a clot. Due to this fact, the P2Y 12 receptor is the main therapeutic target in anti-platelet therapy targeted at the ADP-dependent activation pathway [5]. Generally, the most commonly used clinically approved P2Y 12 inhibitors include the thienopyridine-class inhibitors (ticlopidine, clopidogrel and prasugrel), the ATP analogue cangrelor, and the cyclo-pentyl-triazolo-pyrimidine derivative ticagrelor [3,5]. Thienopyridines are prodrugs: their short-lived active metabolites irreversibly inactivate the receptor and consequently inhibit ADP-induced platelet activation. Cangrelor is the first (recently approved) intravenous P2Y 12 receptor inhibitor that reversibly and non-competitively blocks ADP signalling [6].Adenosine is an important purine metabolite, serving not only as a component of nucleic acids and ATP, the most important energy carrier in the cell, but also as a signalling molecule regulating many cell processes [7,8]. Adenosine receptors (AR) are a subfamily of highly conserved G protein-coupled receptors located in the membr...

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Section: Imaging and Image Analysismentioning

confidence: 99%

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

et al. 2019

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“…In this study, HE-NECA was found to increase cAMP levels with an EC 50 of 59 ± 3 nM, and inhibit ADP-induced human platelet aggregation (measured by turbidimetric method) with an IC 50 of 90 ± 6 nM [101,102]. Recently, HE-NECA was also used in work focused on a quantification of different imaging approaches to experiments carried out under flow conditions, where 10 µM HE-NECA was found to inhibit clot formation under flow in whole blood by 82%, based on the volumes of aggregates recorded by confocal microscopy [103].…”

Section: Adenosine Receptor Agonists—structure Chemical Propertiementioning

confidence: 99%

Blood Platelet Adenosine Receptors as Potential Targets for Anti-Platelet Therapy

Wolska

Różalski

2019

IJMS

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Adenosine receptors are a subfamily of highly-conserved G-protein coupled receptors. They are found in the membranes of various human cells and play many physiological functions. Blood platelets express two (A2A and A2B) of the four known adenosine receptor subtypes (A1, A2A, A2B, and A3). Agonization of these receptors results in an enhanced intracellular cAMP and the inhibition of platelet activation and aggregation. Therefore, adenosine receptors A2A and A2B could be targets for anti-platelet therapy, especially under circumstances when classic therapy based on antagonizing the purinergic receptor P2Y12 is insufficient or problematic. Apart from adenosine, there is a group of synthetic, selective, longer-lasting agonists of A2A and A2B receptors reported in the literature. This group includes agonists with good selectivity for A2A or A2B receptors, as well as non-selective compounds that activate more than one type of adenosine receptor. Chemically, most A2A and A2B adenosine receptor agonists are adenosine analogues, with either adenine or ribose substituted by single or multiple foreign substituents. However, a group of non-adenosine derivative agonists has also been described. This review aims to systematically describe known agonists of A2A and A2B receptors and review the available literature data on their effects on platelet function.

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“…A relatively novel approach uses quartz crystal microbalance system [4]. However, the most often used method of quantification is based on the evaluation of microscopic images of thrombi [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

Qualitative classification of thrombus images as a way to improve quantitative analysis of thrombus formation in flow chamber assays

Kamola,

Przygodzki

2024

PLoS ONE

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Background Thrombus formation in vitro under flow conditions is one of the most widely used methods to study haemostasis and to evaluate the activity of potential antithrombotic compounds. Assessment of the results of these experiments is often based on a quantification of microscopic images of thrombi. In a majority of reported analysis all thrombi visualised in an image are quantified as one homogenous class. In some protocols, qualitative assessment of thrombi morphology based on a visual comparison of evaluated images with representative images of predefined classes of thrombi are performed by experienced analysts. In presented paper we show how the quantitative analysis can be improved by classification of thrombi on the basis of defined morphological features prior to quantification and we suggest that machine learning-based approach can improve this way of analysis. Methods We tested the applicability of machine learning-based segmentation and classification of thrombi images to improve the outcome of quantification of the results of flow chamber assays. For this, we used the public domain machine learning software Ilastik for bioimage analysis developed at the European Molecular Biology Laboratory. A model was trained to distinguish two classes of thrombi based on certain morphological features which apparently correspond to the stage of thrombus development. Thrombi formed in the presence of a model antiplatelet compound—abciximab or in control conditions were quantified with the use of this model and the results were compared to quantification where all thrombi were quantified as a homogenous class. Results Machine learning-based analysis was capable of effective distinguishing of two classes of morphologically distinct platelet aggregates. The use of the model which segmented and quantified only the objects recognized as compacted structures provided results which better mirrored the actual effect of an antiplatelet treatment than quantification based on all structures. Conclusions Classification of thrombi enabled by machine learning increases the relevance of quantitative information and allows better evaluation of the results of in vitro thrombosis assays.

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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

Cited by 4 publications

References 18 publications

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

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

Blood Platelet Adenosine Receptors as Potential Targets for Anti-Platelet Therapy

Qualitative classification of thrombus images as a way to improve quantitative analysis of thrombus formation in flow chamber assays

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