Relationships between optical aggregometry (type born) and flow cytometry in evaluating ADP‐induced platelet activation

Sbrana, Silverio; Pina, Francesca Della; Rizza, Antonio; Buffa, Manuela; Filippis, Rossella De; Gianetti, Jacopo; Clerico, Aldo

doi:10.1002/cyto.b.20360

Cited by 24 publications

(18 citation statements)

References 26 publications

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“…A desirable cell response was obtained after incubation of whole blood with 10 µM ADP in the aggregation experiments and with 20 µM ADP when studying platelet activation. Such concentrations of ADP may appear high, but they are nevertheless used in studies of platelet function, particularly those concerning aggregation [41][42][43][44].…”

Section: Discussionmentioning

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: Discussionmentioning

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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“…Subsequently, the technique was improved and redefined as "continuous" (29) or "real-time flow cytometry" (30,31). In the platelet research field, kinetic flow cytometry was at first applied to investigations of calcium kinetics fluxes (24,(32)(33)(34)(35)(36) or activation markers (35,37).…”

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confidence: 99%

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

2020

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The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow‐cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real‐time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo‐3 (Ca2+), ION NaTRIUM Green‐2 (Na+), and ION Potassium Green‐2 (K+). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR‐1 agonist), AYPGKF (PAR‐4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry

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“…Microparticle formation reflects fragmentation of PLTs and release of cell membrane vesicles during exocytosis and impose a thrombotic burden to circulating blood [39,40,41,42]. Recently, this was found to correlate with atherosclerosis and the risk for major adverse cardiovascular events, such as heart infarct or stroke [43,44].…”

Section: Resultsmentioning

confidence: 99%

Hemocompatibility of Inorganic Physical Vapor Deposition (PVD) Coatings on Thermoplastic Polyurethane Polymers

Lackner

Waldhauser

Hartmann

et al. 2012

JFB

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Biocompatibility improvements for blood contacting materials are of increasing interest for implanted devices and interventional tools. The current study focuses on inorganic (titanium, titanium nitride, titanium oxide) as well as diamond-like carbon (DLC) coating materials on polymer surfaces (thermoplastic polyurethane), deposited by magnetron sputtering und pulsed laser deposition at room temperature. DLC was used pure (a-C:H) as well as doped with silicon, titanium, and nitrogen + titanium (a-C:H:Si, a-C:H:Ti, a-C:H:N:Ti). In-vitro testing of the hemocompatibility requires mandatory dynamic test conditions to simulate in-vivo conditions, e.g., realized by a cone-and-plate analyzer. In such tests, titanium- and nitrogen-doped DLC and titanium nitride were found to be optimally anti-thrombotic and better than state-of-the-art polyurethane polymers. This is mainly due to the low tendency to platelet microparticle formation, a high content of remaining platelets in the whole blood after testing and low concentration of platelet activation and aggregation markers. Comparing this result to shear-flow induced cell motility tests with e.g., Dictostelium discoideum cell model organism reveals similar tendencies for the investigated materials.

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Relationships between optical aggregometry (type born) and flow cytometry in evaluating ADP‐induced platelet activation

Cited by 24 publications

References 26 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

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

Hemocompatibility of Inorganic Physical Vapor Deposition (PVD) Coatings on Thermoplastic Polyurethane Polymers

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