Comparison of three common whole blood platelet function tests for in vitro P2Y12 induced platelet inhibition.
Ontology highlight
ABSTRACT:
SUBMITTER: Dias JD
PROVIDER: S-EPMC7293977 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Patients treated with percutaneous coronary intervention receive aspirin and P2Y12 ADP receptor inhibitors to reduce thrombotic complications. The choice of methodology for monitoring the effects of treatment and assessing its efficacy is still a topic of debate. We evaluated how decreased P2Y12 function influences platelet aggregate (thrombus) size measured ex vivo.We used confocal videomicroscopy to measure in real time the volume of platelet thrombi forming upon blood perfusion over fibrillar collagen type I at a wall shear rate of 1500 s(-1). The average volume was significantly smaller in 31 patients receiving aspirin and clopidogrel (19) or ticlopidine (12) than in 21 controls, but individual values were above the lower limit of the normal distribution, albeit mostly within the lower quartile, in 61.3% of cases. Disaggregation of platelet thrombi at later perfusion times occurred frequently in the patients. Vasodilator-stimulated phosphoprotein phosphorylation, reflecting P2Y12 inhibition, was also decreased in the patient group, and only 22.6% of individual values were above the lower normal limit. We found no correlation between volume of thrombus formed on collagen fibrils and level of P2Y12 inhibition, suggesting that additional and individually variable factors can influence the inhibitory effect of treatment on platelet function.Measurements of platelet thrombus formation in flowing blood reflects the consequences of antiplatelet therapy in a manner that is not proportional to P2Y12 inhibition. Combining the results of the two assays may improve the assessment of thrombotic risk.
| S-EPMC3030676 | biostudies-literature
Project description:Platelet adhesion, activation and aggregation play a pivotal role in atherothrombosis. Intracoronary atherothrombosis is the most common cause of the development of acute coronary syndrome (ACS), and plays a central role in complications occurring around percutaneous coronary intervention (PCI) including recurrent ACS, procedure-related myocardial infarction or stent thrombosis. Inhibition of platelet aggregation by medical treatment impairs formation and progression of thrombotic processes and is therefore of great importance in the prevention of complications after an ACS or around PCI. An essential part in the platelet activation process is the interaction of adenosine diphosphate (ADP) with the platelet P2Y12 receptor. The P2Y12 receptor is the predominant receptor involved in the ADP-stimulated activation of the glycoprotein IIb/IIIa receptor. Activation of the glycoprotein IIb/IIIa receptor results in enhanced platelet degranulation and thromboxane production, and prolonged platelet aggregation. The objectives of this review are to discuss the pharmacological limitations of the P2Y12 inhibitor clopidogrel, and describe the novel alternative P2Y12 inhibitors prasugrel and ticagrelor and the clinical implications of the introduction of these new medicines.
| S-EPMC3269569 | biostudies-literature
Project description:A high-fidelity computational model using a 3D immersed boundary method is used to study platelet dynamics in whole blood. We focus on the 3D effects of the platelet-red blood cell (RBC) interaction on platelet margination and near-wall dynamics in a shear flow. We find that the RBC distribution in whole blood becomes naturally anisotropic and creates local clusters and cavities. A platelet can enter a cavity and use it as an express lane for a fast margination toward the wall. Once near the wall, the 3D nature of the platelet-RBC interaction results in a significant platelet movement in the transverse (vorticity) direction and leads to anisotropic platelet diffusion within the RBC-depleted zone or cell-free layer (CFL). We find that the anisotropy in platelet motion further leads to the formation of platelet clusters, even in the absence of any platelet-platelet adhesion. The transverse motion, and the size and number of the platelet clusters are observed to increase with decreasing CFL thickness. The 3D nature of the platelet-RBC collision also induces fluctuations in off-shear plane orientation and, hence, a rotational diffusion of the platelets. Although most marginated platelets are observed to tumble just outside the RBC-rich zone, platelets further inside the CFL are observed to flow with an intermittent dynamics that alters between sliding and tumbling, as a result of the off-shear plane rotational diffusion, bringing them even closer to the wall. To our knowledge, these new findings are based on the fundamentally 3D nature of the platelet-RBC interaction, and they underscore the importance of using cellular-scale 3D models of whole blood to understand platelet margination and near-wall platelet dynamics.
| S-EPMC4052243 | biostudies-other
Project description:OBJECTIVE:Commercial kits of column tests for pre-transfusion testing have progressively replaced conventional tube tests in most laboratories. Aim of this study was to compare three commercial test cell panels for the identification of irregular red blood cell (RBC) alloantibodies. Overall, 44 samples with a positive indirect antiglobulin test (IAT) by routine testing were used for comparison of following panels: Ortho RESOLVE® panelC (Ortho Clinical Diagnostics (OCD), Milan, Italy), ID-DiaPanel(-P) (Bio-Rad Laboratories, CA, USA) and Identisera Diana(P) (Grifols, Barcelona, Spain). Column agglutination techniques were used, with microtubes containing either microgel (Bio-Rad/Grifols) or glass bead microparticles (Ortho). RESULTS:Alloantibody identification was possible in 38 samples, of which identical identification was shown in 33 samples by all methods. The remaining samples showed differences between certain methods, with the gel card system being superior to the glass card system for analyzing stored samples Considering that not all samples were evaluated in all three methods, the concordance rate reached 100% between Bio-Rad and Grifols, 90.5% between Bio-Rad and OCD, 86.5% between OCD and Grifols and 90.5% between all methods. Although differences in sensitivities were seen for specific antibodies, the three methods showed comparable performance for the identification of RBC alloantibodies.
| S-EPMC7057655 | biostudies-literature
Project description:The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y(12) and P2Y(1) inhibition and P2Y(12) and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both G alpha(q) and Galpha (i) signalling. Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y(12) antagonist; MRS2179, a reversible P2Y(1) antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active alpha (IIb)beta(3) (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel. A synergistic effect regarding inhibition of ADP-induced platelet activation (10 microM) was obtained with different combinations of AR-C69931MX and MRS2179. Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect. To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described. Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies.
| S-EPMC1575192 | biostudies-other
Project description:Previous mouse studies have shown the increased presence of platelets in the myocardium during early stages of myocarditis and their selective detection by MRI. Here, we aimed to depict early myocarditis using molecular contrast-enhanced ultrasound of activated platelets, and to evaluate the impact of a P2Y12 receptor platelet inhibition. Experimental autoimmune myocarditis was induced in BALB/c mice by subcutaneous injection of porcine cardiac myosin and complete Freund adjuvant (CFA). Activated platelets were targeted with microbubbles (MB) coupled to a single-chain antibody that binds to the "ligand-induced binding sites" of the GPIIb/IIIa-receptor (=LIBS-MB). Alongside myocarditis induction, a group of mice received a daily dose of 100 g prasugrel for 1 month. Mice injected with myosin and CFA had a significantly deteriorated ejection fraction and histological inflammation on day 28 compared to mice only injected with myosin. Platelets infiltrated the myocardium before reduction in ejection fraction could be detected by echocardiography. No selective binding of the LIBS-MB contrast agent could be detected by either ultrasound or histology. Prasugrel therapy preserved ejection fraction and significantly reduced platelet aggregates in the myocardium compared to mice without prasugrel therapy. Therefore, P2Y12 inhibition could be a promising early therapeutic target in myocarditis, requiring further investigation.
| S-EPMC8699761 | biostudies-literature
Project description:Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a serious bleeding condition mostly caused by the reaction between maternal anti-HPA-1a antibodies and fetal platelets. This reaction leads to Fc-dependent platelet phagocytosis. Although several serological methods have been developed to identify maternal antibodies, a reliable laboratory parameter as a prognostic tool for FNAIT severity is still lacking. In this study, we developed whole blood platelet phagocytosis assay (WHOPPA), a flow cytometry-based phagocytosis assay that uses a pH-sensitive fluorescent dye (pHrodo-SE) to analyze anti-HPA-1a-dependent platelet phagocytosis in whole blood. WHOPPA revealed a high phagocytosis rate for the anti-HPA-1a opsonized platelets by monocytes but not by neutrophils. Analysis of different monocyte populations showed that all monocyte subsets, including classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes, were able to engulf opsonized platelets. A unique monocyte subset, termed shifted monocytes (CD14+CD16-), showed the highest phagocytosis rate and was detected after platelet engulfment. FcγR inhibition tests revealed that except for FcγRIIa, FcγRI and FcγRIII on monocytes were responsible for the phagocytosis of anti-HPA-1a opsonized platelets. Analysis of anti-HPA-1a antibodies from FNAIT cases (n = 7) showed the phagocytosis of HPA-1aa but not of HPA-1bb platelets by monocytes. The phagocytosis rate was highly correlated with bound antibodies measured by flow cytometry (p < 0001; r = 0.9214) and MAIPA assay (p < 0.001; r = 0.7692). The phagocytosis rates were equal for type I and II anti-HPA-1a antibodies recognizing the plexin-semaphoring-integrin (PSI) domain and PSI/epidermal growth factor 1 domain of β3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvβ3 integrin did not induce platelet phagocytosis. Furthermore, effector-silenced mAbs against HPA-1a inhibited the phagocytosis of anti-HPA-1a opsonized platelets. In conclusion, WHOPPA is a reliable in vitro platelet phagocytosis assay that mimics the phagocytosis of anti-HPA-1a opsonized platelets in whole blood. This assay allows to prove platelet phagocytosis ex vivo and evaluate the inhibitory capacity of different inhibitors as therapeutically strategies for the prevention of fetal thrombocytopenia in FNAIT in the future.
| S-EPMC10702767 | biostudies-literature
Project description:Standard bioaccumulation tests are commonly conducted using Macoma nasuta (clam), and Alitta virens (polychaete) for marine tests, and Lumbriculus variegatus (an oligochaete) for freshwater tests. Because the interlaboratory variability associated with these tests is unknown, four experienced laboratories conducted standard 28-day bioaccumulation tests with the above species using sediments contaminated with polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). Chemical analysis of tissue samples was performed by a single laboratory. The intralaboratory variance among replicates was relatively low for PCB tissue concentrations, with coefficients of variation (CVs) ranging from 9% to 28% for all laboratories and species, with the exception of one laboratory reporting higher variability for L. variegatus (CV = 51%). Intralaboratory variance for PCB tissue concentrations was higher than interlaboratory variance for A. virens and L. variegatus, and the magnitude of difference (MOD) for laboratory means ranged from 1.4 to 2.0 across species. Intralaboratory variability was also low for lipid content, and lipid normalization of PCB and PAH body residues generally had little impact on variability. In addition to variability across bioassay laboratories, analytical variability was evaluated by different laboratories measuring the concentration of PCBs and total lipids in a subsample of tissue homogenate of sediment-exposed test organisms. Variability associated with tissue analysis was higher than bioassay laboratory variability only in tests with L. variegatus. Statistical differences between samples may be observed due to the low intralaboratory variability; however, the biological significance of these differences may be limited because the MOD is low. Considering the MOD when comparing bioaccumulation across treatments accounts for uncertainty related to inherent variability of the test in the interpretation of statistically significant results. Environ Toxicol Chem 2022;41:1260-1275. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.
| S-EPMC9310598 | biostudies-literature
Project description:The P2Y(12) receptor (P2Y(12)R) is an ADP-activated G protein-coupled receptor (GPCR) that is an important target for antithrombotic drugs. Three homology models of P2Y(12)R were compared, based on different GPCR structural templates: bovine rhodopsin (bRHO), human A(2A) adenosine receptor (A(2A)AR), and human C-X-C chemokine receptor type 4 (CXCR4). By criteria of sequence analysis (25.6% identity in transmembrane region), deviation from helicity in the second transmembrane helix (TM2), docked poses of ligands highlighting the role of key residues, accessibility of a conserved disulfide bridge that is reactive toward irreversibly-binding antagonists, and the presence of a shared disulfide bridge between the third extracellular loop (EL3) and the N-terminus, the CXCR4-based model appeared to be the most consistent with known characteristics of P2Y(12)R. The docked poses of agonist 2MeSADP and charged anthraquinone antagonist PSB-0739 in the binding pocket of P2Y(12)R-CXC agree with previously published site-directed mutagenesis studies of Arg256 and Lys280. A sulfonate at position 2 of the anthraquinone core created a strong interaction with the Lys174(EL2) side chain. The docking poses of the irreversibly-binding, active metabolite (existing as two diastereoisomers in vivo) of the clinically utilized antagonist Clopidogrel were compared. The free thiol group of the 4S diastereoisomer, but not the 4R isomer, was found in close proximity (~4.7 Å) to the sulfur atom of a disulfide bridge involving Cys175, suggesting greater activity in covalent binding. Therefore, ligand docking to the CXCR4-based model of the P2Y(12)R predicted poses of both reversibly and irreversibly-binding small molecules, consistent with observed pharmacology and mutagenesis studies.
| S-EPMC3157290 | biostudies-literature
Project description:BackgroundImmature platelets (IP) are the youngest circulating platelets, released from megakaryocytes, and demonstrating increased dimensions, significant RNA content, and enhanced activity. Immature platelet research focuses on a differential diagnostic help in patients with thrombocytopenia. The objectives of this study were to compare the variability of IP in citrate and EDTA samples, and to determine stability over time.MethodsFifty-six patients were included for comparison between EDTA and citrate whole blood sample collection. Among the patients, 28 had thrombocytopenia (platelet count < 150G/L). Platelet measurement impedancemetry and fluorimetry were performed with Sysmex XN-9000. The immature platelet fraction (IPF) and absolute immature platelet count (A-IPC) were determined with a fluorescent method.ResultsThe mean value of platelet count with fluorescence was, in EDTA sample, 215 ± 171 and, in citrate sample, 153 ± 118 G/L. No significant difference was observed between IPF between EDTA and citrate (7.74 ± 6.68% vs. 8.45 ± 7.37%, p = 0.69), respectively. With the Bland-Altman analysis, the mean difference in the EDTA sample, between 1 and 24 h, was 8.06 ± 6.96% and 8.73 ± 7.12% for IPF, whereas in the citrate sample, between 1 and 6 h, it was 8.60 ± 7.29% and 7.54 ± 6.97%, for IPF. Comparing 1 h EDTA sample with 6 h citrate sample, the variance ratio was 0.974 (95% CI: 0.864-1.084) in IPF.ConclusionsWe confirmed the potential to conduct IP measurements up to 24 h in the EDTA sample and IPF measurements in the citrate sample for up to 6 h. These results may be useful for the use of IPF, which is a promising parameter whose interest in clinical practice and standardization is not yet well defined.
| S-EPMC10492457 | biostudies-literature