scholarly journals The Role of Human Platelet Preparation for Toll-Like Receptors 2 and 4 Related Platelet Responsiveness

TH Open ◽  
2019 ◽  
Vol 03 (02) ◽  
pp. e94-e102 ◽  
Author(s):  
Juergen Koessler ◽  
Marius Niklaus ◽  
Katja Weber ◽  
Angela Koessler ◽  
Sabine Kuhn ◽  
...  
Keyword(s):  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Toll Like Receptors ◽  
Preparation Methods ◽  
Expression Levels ◽  
Basal Expression ◽  
Healthy Donors ◽  
Light Transmission Aggregometry ◽  
Bacterial Patterns ◽  
Study Designs

Background Like immune cells, platelets express the repertoire of toll-like receptors (TLR), among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. Receptor-mediated functional effects in platelets have been investigated, but reliable conclusions are tampered due to heterogeneous study designs with variable platelet preparation methods. This study compares TLR2- and TLR4-dependent platelet responsiveness in platelet-rich plasma (PRP) and in washed platelets (WPs). Material and Methods Fresh peripheral blood samples from healthy donors served for the preparation of PRP and WP. Basal and agonist-stimulated TLR2 and TLR4 expression levels were evaluated by flow cytometry. Light transmission aggregometry was used to investigate functional effects of TLR2 and TLR4 stimulation with Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli) as ligands. The capacity of chemokine release was determined by immunoassays. Results Pam3CSK4 and LPS (in combination with thrombin) were able to induce aggregation in WP, but not in PRP, with threshold concentrations of 15 µg/mL. Basal expression levels of TLR2 and TLR4 were higher in WP than in PRP, increasing several-fold rapidly and persistently upon platelet activation with potent agonists. Pam3CSK4 (15 µg/mL) or LPS led to the submaximal release of RANTES, PF4, PDGF, NAP-2, and sCD40L from WP. In PRP, secretory effects are less pronounced for RANTES, PDGF, or PF4, and not detectable for NAP-2 or sCD40L. Conclusion The effects mediated by TLR2 and TLR4 stimulation are dependent on platelet preparation, an important issue for experimental designs and for manufacturing of platelet concentrates in transfusion medicine.

Standardization of Light Transmission Aggregometry for Diagnosis of Platelet Disorders: An Inter-Laboratory External Quality Assessment

2019 ◽  
Vol 119 (07) ◽  
pp. 1154-1161 ◽  
Author(s):  
Karina Althaus ◽  
Barbara Zieger ◽  
Tamam Bakchoul ◽  
Kerstin Jurk ◽  
Keyword(s):  
Platelet Function ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Reference Ranges ◽  
Technical Problems ◽  
Platelet Function Disorders ◽  
Light Transmission Aggregometry ◽  
Laboratory Survey ◽  
Definition Of

AbstractSeveral in vitro platelet function tests are available for the diagnosis of inherited platelet function disorders. Currently, the light transmission aggregometry (LTA) is recommended as one of the first-step tests. LTA is available in most specialized hemostasis laboratories. Although the LTA is accepted as a ‘gold standard’ assay for the evaluation of platelet function, its standardization in the clinical practice is still challenging. The GTH-based THROMKID-Plus Study Group has performed an inter-laboratory trial in Germany and Austria. Five different agonists were selected according to the Scientific and Standardization Committee/International Society on Thrombosis and Haemostasis recommendations and shipped in 3 different sets (one should represent a healthy control and two should simulate platelet function disorders) to 15 specialized laboratories in Germany and Austria. Agonists were analyzed by APACT or PAP4/8 aggregometer using platelet-rich plasma from healthy donors. In addition, laboratory-internal platelet agonists were tested in platelet-rich plasma from a healthy donor. All laboratories (9 used APACT, 6 used PAP4/PAP8) showed very consistent data regarding the maximum percentage of aggregation induced by the tested agonists and identified the differential diagnosis of the simulated platelet function disorders with one exception, which was due to technical problems. In contrast, there was a high variability of the laboratory-internal inductors regarding reagent type, concentrations and pathological cut-off values. Our study showed that the shipment of agonists is suitable for an inter-laboratory survey of LTA. However, there is still a remarkable need for standardization of agonist reagents and their concentration as well as for definition of reference ranges.

Assessment of ADP-induced platelet aggregation with light transmission aggregometry and multiple electrode platelet aggregometry before and after clopidogrel treatment

2008 ◽  
Vol 99 (01) ◽  
pp. 121-126 ◽  
Author(s):  
Siegmund Braun ◽  
Stefan Jawansky ◽  
Wolfgang Vogt ◽  
Julinda Mehilli ◽  
Albert Schömig ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Whole Blood ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Platelet Aggregometry ◽  
Light Transmission Aggregometry ◽  
Before And After ◽  
Standardized Method ◽  
Multiple Electrode

SummaryThe level of platelet aggregation, measured with light transmission aggregometry (LTA) in platelet rich plasma (PRP), has been shown to predict outcomes after percutaneous coronary intervention (PCI). However, measuring parameters of platelet function with LTA is time consuming and weakly standardized. Thus, a fast and standardized method to assess platelet function after clopidogrel treatment would be of great value for clinical practice. A new method, multiple electrode platelet aggregometry (MEA), to rapidly measure platelet aggregation in whole blood has recently been developed. The aim of this study was to assess parameters of platelet function with MEA and LTA before and after administration of 600 mg clopidogrel. Blood samples from 149 patients scheduled for coronary angiography were taken after clopidogrel treatment; in addition, in 60 of the patients samples were available before clopidogrel treatment. ADP-induced platelet aggregation was measured with LTA and simultaneously in whole blood with MEA on the Multiplate analyzer. Platelet aggregation measured with MEA decreased significantly after clopidogrel treatment (P<0.0001). ADP-induced platelet aggregation assessed with MEA and LTA correlated significantly (Spearman rank correlation coefficient=0.71; P<0.0001).The results of MEA, a fast and standardized method to assess the platelet response to ADP prior to and after clopidogrel treatment, correlate well with LTA.

Platelet Aggregation Induced By Formaldehyde

1981 ◽  
Author(s):  
J A Zeller ◽  
K Eurenius ◽  
R E Dayhoff ◽  
R S Ledley ◽  
L S Rotolo
Keyword(s):  
Platelet Aggregation ◽  
Small Volume ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Microscopic Analysis ◽  
Formaldehyde Concentration ◽  
Microscopic Method ◽  
Light Transmission Aggregometry ◽  
Quantitative Image ◽  
Aggregation Analysis

Formaldehyde causes platelet aggregation (or agglutination) which varies with dosage. Aggregation was studied in ten blood samples drawn from normal volunteers. A small volume of formaldehyde was added to platelet rich plasma in a light transmission aggregcmeter to produce final formaldehyde concentrations between 0.1% and 8%. Measurements were made by three methods: (1) light transmission aggregometry, (2) visual semi-quantitative microscopic analysis, and (3) quantitative image analysis (Computerized Platelet Aggregation Analysis) . Using the visual semi-quantitative microscopic method, the dose response curve (expressed as the percentage of platelets involved in aggregates) increased from 11% at a formaldehyde concentration of 0.1% to 41% at a 0.5% formaldehyde concentration; it then decreased to 11% at a formaldehyde concentration of 8%. This curve may reflect the influence of two different formaldehyde effects: an aggregating effect which increases until about 0.5% concentration, whereafter a fixative effect may predominate. Light transmission aggregometry recordings did not provide a reliable indicator of the presence and degree of aggregation. A comparison of the visual semi-quantitative method and CPAA show similar detection of aggregating effects. In sunmary, formaldehyde has an aggregating effect on normal platelets. The physiologic significance of this effect is unknown; however, because formaldehyde is used to process platelets in studies of platelet aggregation, this effect may be of importance.

Abstract 19437: Elevated Platelet Turnover in Patients Receiving Dual Antiplatlet Therapy Could Permit the Formation of Uninhibited Platelet Clusters That Act as Seeds for Thrombus Formation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Thomas Hoefer ◽  
Timothy D Warner
Keyword(s):  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Imaging Techniques ◽  
Once Daily ◽  
Increased Risk ◽  
Light Transmission Aggregometry ◽  
Platelet Aggregates ◽  
Dynamic Relationships ◽  
Drug Free

Introduction: ACS patients who also have conditions associated with increased platelet turnover, such as diabetes or chronic kidney disease, are at increased risk of atherothrombotic events despite receiving dual-antiplatelet therapy (DAPT). It may well be that aspirin and thienopyridines, such as clopidogrel or prasugrel, prescribed once daily as pharmacologically short-lived but irreversibly acting agents, are less effective in these patients because of the daily emergence of a significant subpopulation of uninhibited platelets. Hypothesis: Here we investigated the potentially crucial roles of subpopulations of uninhibited platelets in overcoming DAPT and driving aggregation. Methods: Aliquots of platelet rich plasma were incubated with aspirin, prasugrel active metabolite (PAM) plus aspirin, or vehicle and platelets were differently labelled with membrane dyes. Aliquots were then combined in various proportions and platelet responses measured in standard light transmission aggregometry. Aggregates formed in response to platelet agonists were then fixed and detailed structural analyses made by advanced imaging techniques, including confocal microscopy and ImageStream flow cytometry, to determine the interactions of different platelet populations. Results: Platelet aggregation responses and images of platelet aggregates demonstrated complex, dynamic relationships between platelet subpopulations. Summary analysis indicated that aggregates containing aspirin-treated (90-60%) and drug-free (10-40%) platelets had random distributions of drug-free platelets in response to ADP or AA. In contrast, aggregates containing DAPT-inhibited (90-60%) platelets and drug-free (10-40%) platelets showed marked clustering of drug-free platelets at aggregate cores surrounded by recruited DAPT-inhibited platelets. Conclusions: We show that a subpopulation of drug-free platelets can provide seeds for the formation of aggregates that then recruit DAPT-inhibited platelets. Such interaction between platelet subpopulations are of direct relevance to ACS patients with underlying conditions associated with elevated platelet turnover and may well provide an explanation for the failure of DAPT with aspirin and thienopyridines.

Platelet aggregation in response to ADP is highly variable in normal donors and patients on anti-platelet medication

2016 ◽  
Vol 54 (7) ◽  
Author(s):  
Eimear Dunne ◽  
Karl Egan ◽  
Siobhán McFadden ◽  
David Foley ◽  
Dermot Kenny
Keyword(s):  
Platelet Aggregation ◽  
Therapeutic Response ◽  
Light Transmission ◽  
Platelet Reactivity ◽  
Coronary Intervention ◽  
P2y12 Inhibitors ◽  
Aggregation Response ◽  
Healthy Donors ◽  
Percutaneous Coronary ◽  
Light Transmission Aggregometry

AbstractP2Y12 inhibitors are indicated in patients following percutaneous coronary intervention. Several studies have demonstrated that high on treatment platelet reactivity is correlated with outcomes yet prospective studies of guided therapy have failed to show benefit. There is a paucity of studies on the platelet aggregation response to ADP before P2Y12 therapy is started. The aim of this study was to characterize platelet responses to 20 μM ADP by light transmission aggregometry (LTA) in a homogenous population.Platelet aggregation was assessed in 201 patients on dual antiplatelet therapy, 98 patients on aspirin alone and 47 normal, healthy volunteers free from anti-platelet medication.Consensus guidelines suggest that a platelet aggregation response in response to the agonist ADP of <57% is an adequate therapeutic response to P2Y12 inhibition. Seven healthy donors and 38 patients taking aspirin only had aggregation responses below 57%.The results of our study demonstrate that 15% of normal donors and 38% of patients taking aspirin only would be classified as having a therapeutic response to P2Y12 inhibition using current guidelines.

Evaluation of Platelet Dysfunction In Children: Improved Detection and Efficiency

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1446-1446
Author(s):  
Diane J. Nugent ◽  
Ryan Roberts ◽  
Peggy Nakagawa
Keyword(s):  
Platelet Function ◽  
Mutational Analysis ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Clot Formation ◽  
Activity Levels ◽  
Food History ◽  
Platelet Dysfunction ◽  
Light Transmission Aggregometry ◽  
Arachadonic Acid

Abstract Abstract 1446 Currently, there is no single assay that will detect platelet function abnormalities in all individuals. We prospectively studied 369 patients with a strong history of bruising and mucosal membrane bleeding for possible platelet dysfunction following documentation of normal Von Willebrand antigen and activity levels. In an effort to evaluate platelet function under more diverse conditions we chose to simultaneously evaluate 1) aggregation using platelet rich plasma and light transmission aggregometry (LTA), 2) adhesion under high shear using the Platelet Function Analyzer (PFA-100, ADP-collagen, and Epinephrine-collagen cartridges) and 3) platelet initiated clot formation using heparinized whole blood and the two standard mapping agonists, arachadonic acid (AA) and ADP (Hemascope Thromboelastograph Analyzer). Of the 369 platelet evaluations performed, 87 patients (24%) were found to be normal in all three assays with all agonists. On repeated assays with increased attention to medication and food history, an additional 152 patients were felt to have a transient or acquired dysfunction which improved or normalized on further testing. Leaving 130 patients with persistent bleeding and documented abnormalities on one or more of the assays used. There were no patients with only Collagen (CN) or arachadonic acid (AA) aggregation alone on LTA. Using LTA, there was one patient with combined CN and ADP aggregation defect only, another with AA and EPI absent aggregation only, and one with only AA and Thrombin receptor agonist peptide (TRAP) aggregation abnormalities. A summary of the remaining 127 patients are displayed in the table below:Abnormal AssayLTA EPILTA ADPLTA ADP + EPILTA ADP, EPI CNLTA ADP, EPI, AALTA ADP, EPI CN, AAPFA and PLT Mapping ONLYNumber201240841825M/F3M/17F6M/6F16M/24F1M/7F1M/3F8M/10F12M/13FPFA1M/2F4M7M/4F01M01M/3FPlt Mapping2M/2F1M/2F3F1F009M/7FBoth PFA and Mapping1M0001F2M/9F2M/3F Summary: By using a combination of three assays, we were able to identify 25 additional patients with significant platelet dysfunction detected with abnormal platelet mapping or PFA-100 despite normal light transmission aggregometry. Patients with the most abnormalities on aggregation, also demonstrated abnormal adhesion and platelet initiated clot formation. However, the use of PFA-100 and/or platelet mapping alone would miss the majority of patients with aggregation defects. In the future, the unique combination of platelet function defects as measured by these assays, and future technologies, will not only improve detection, but also facilitate phenotype to genotype associations and expedite mutational analysis. Disclosures: Off Label Use: Rituximab to treat ITP.

In Vitro Reversal of the Anti-Aggregant Effect of Ticagrelor Using Untreated Platelets

2018 ◽  
Vol 118 (11) ◽  
pp. 1895-1901 ◽  
Author(s):  
Paul Kruger ◽  
Jack Hirsh ◽  
Vinai Bhagirath ◽  
Ke Xu ◽  
Brian Dale ◽  
...  
Keyword(s):  
Platelet Transfusion ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Coronary Syndrome ◽  
Coronary Artery Stent ◽  
Anti Platelet Agent ◽  
Light Transmission Aggregometry ◽  
Optimal Quantity

Background Ticagrelor is an anti-platelet agent that is indicated for prevention of thrombosis after acute coronary syndrome or intra-coronary artery stent implantation, but it increases the risk of bleeding. Platelet transfusion has the potential to treat or prevent bleeding in patients taking ticagrelor, but the optimal quantity of platelets and timing of administration have not been fully defined. Methods and Results Ten healthy subjects took ticagrelor in combination with acetylsalicylic acid for 5 days, and had blood collected prior to treatment and at 2, 10, 24, 48, 72 and 96 hours after the last doses. The potential of platelet transfusion to prevent or reverse bleeding was evaluated by mixing subject and donor platelet-rich plasma in vitro in nine different proportions, and measuring adenosine diphosphate-mediated aggregation by light transmission aggregometry. Spontaneous offset of the anti-aggregant effect of ticagrelor occurred gradually and was complete at 72 hours after the last dose. The addition of donor platelets enhanced the recovery. The addition of the equivalent of six apheresis platelet units produced a 50% relative reversal at 10 hours, and > 90% reversal at 24 hours. Conclusion Donor platelets enhance reversal of the anti-aggregant effect of ticagrelor in vitro. Donor platelets given in clinically relevant amounts partially reversed ticagrelor at 10 hours after the last dose, and almost fully reversed ticagrelor at 24 hours. The results inform on the potential to reverse ticagrelor in patients who develop bleeding or require emergency surgery.

Comparison of platelet aggregation parameters and expression of platelet granule markers

2019 ◽  
Author(s):  
В.В. Малышева ◽  
О.А. Шустова ◽  
С.Г. Хаспекова ◽  
Я.А. Наймушин ◽  
А.В. Мазуров
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Thrombin Receptor ◽  
Platelet Surface ◽  
Dense Granules ◽  
Turbidimetric Method ◽  
Healthy Donors ◽  
Aggregation Parameters

Введение. Активация тромбоцитов стимулирует их агрегацию и ассоциированный с секрецией экзоцитоз внутриклеточных гранул. Агрегацию чаще всего изучают турбидиметрическим методом, а экзоцитоз гранул методом проточной цитофлуориметрии, определяя экспрессию их маркеров на поверхности тромбоцитов. Цель исследования: сравнение чувствительности двух методов оценки активации тромбоцитов, исследования их агрегации и экспрессии маркеров внутриклеточных гранул. Материалы и методы. Тромбоциты здоровых доноров активировали АДФ и пептидом, активирующим рецептор тромбина (thrombin receptor activating peptide, TRAP). Агрегацию тромбоцитов изучали турбидиметрическим методом в обогащенной тромбоцитами плазме, регистрируя максимальный уровень светопропускания (Т макс). Экспрессию маркеров гранул изучали в цельной крови с помощью проточной цитофлуориметрии, регистрируя процент тромбоцитов, окрашенных антителами против маркеров альфагранул (CD62P) и плотных гранул (CD63). Результаты. У всех доноров 20 мкМ АДФ и 10 мкМ TRAP стимулировали мощную, необратимую агрегацию тромбоцитов (62,1 10,3 и 65,1 5,1 T макс, соответственно). Средние уровни агрегации были ниже при ее стимуляции 2,5 мкМ АДФ (30,9 23,2 T макс) и 1 мкМ TRAP (24,4 29,1 T макс). Экспрессия маркеров гранул была максимальной при активации тромбоцитов 10 мкМ TRAP (77,6 13,7 CD62P и 73,9 13,3 CD63), ниже при активации 1 мкМ TRAP (46,3 25,1 CD62P и 38,3 23,8 CD63), еще ниже при активации 20 мкМ АДФ (19,8 8,5 CD62P и 13,6 5,2 CD63) и минимальной при активации 2,5 мкМ АДФ (8,0 4,2 CD62P и 8,3 3,3 CD63). Адреналин (20 мкМ) не стимулировал экспрессию маркеров гранул, но при совместном добавлении с 20 мкМ АДФ повышал ее в 1,9 раза для обоих маркеров CD62P и CD63. Заключение. При активации тромбоцитов АДФ исследование агрегации является более чувствительным методом, чем определение экспрессии маркеров гранул. При активации тромбоцитов TRAP чувствительность обоих методов приблизительно одинакова. Экспрессия маркеров гранул увеличивается при совместном добавлении к тромбоцитам АДФ и адреналина, хотя адреналин сам по себе не стимулирует их экспрессию. Introduction. Platelet activation stimulates their aggregation and secretion associated exocytosis of intracellular granules. Aggregation is usually studied by turbidimetric method and granule exocytosis by detecting expression of their markers on platelet surface using flow cytofluorimetry. Aim: сomparison of the sensitivity of two methods for evaluation of platelet activation, studies of their aggregation and expression of intracellular granule markers. Materials and methods. Healthy donors platelets were activated by ADP and thrombin receptor activating peptide (TRAP). Platelet aggregation was investigated in platelet rich plasma by turbidimetric method, assessed by the maximal level of light transmission (T max). Expression of granule markers was detected in whole blood by fl ow cytofl uorimetry registering the percent of platelets stained with antibodies against the markers of alphagranules (CD62P) and dense granules (CD63). Results. In all donors 20 M ADP and 10 M TRAP stimulated strong irreversible platelet aggregation (62.1 10.3 and 65.1 5.1 T max). Mean aggregation levels were lower when it was stimulated by 2.5 M ADP (30.9 23.2 T max) and 1 M TRAP (24.4 29.1 T max). Expression of granule markers was maximal at platelet activation by 10 M TRAP (77.6 13.7 CD62P and 73.9 13.3 CD63), lower at activation by 1 M TRAP (46.3 25.1 CD62P and 38.3 23.8 CD63), more lower at activation by 20 M ADP (19.8 8.5 CD62P and 13.6 5.4 CD63), and minimal at activation by 2.5 M ADP (8.0 4.2 CD62P and 8.3 3.3 CD63). Epinephrine (20 M) did not stimulate expression of granule markers but at combined addition with 20 M ADP increased its level by 1.9 for both markers, CD62P and CD63. Conclusion. Platelet aggregation is more sensitive method than detection of expression of granule markers when platelets are activated by ADP. Both methods demonstrate about the same sensitivity when platelets are activated by TRAP. Expression of granule markers is increased at combined addition of ADP and epinephrine although epinephrine by itself fails to stimulate their expression.

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