Comparison of a new ELISA assay with the flow cytometric assay for platelet vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation in whole blood to assess P2Y12 inhibition

2012 ◽  
Vol 107 (02) ◽  
pp. 388-395 ◽  
Author(s):  
Nicolas Bourguet ◽  
Danièle Boulay-Moine ◽  
Atsuhiro Sugidachi ◽  
Shinji Yamaguchi ◽  
Paul Barragan ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Platelet Reactivity ◽  
P2y12 Receptor ◽  
Reactivity Index ◽  
Blood Collection ◽  
Variable Response ◽  
Vasp Phosphorylation ◽  
P2y12 Antagonists ◽  
Wide Range

SummaryThienopyridines and other agents target the platelet P2Y12 receptor and inhibit several platelet activities mediated by adenosine diphosphate (ADP). The measurement of vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation, expressed as platelet reactivity index (PRI), mirrors the degree of P2Y12 receptor inhibition and can detect the well-known variable response to clopidogrel. The commercially available VASP assay uses flow cytometry (FC) and requires that the test be run within 48 hours of blood collection. A new ELISA VASP assay offers the advantages of using more widely available technology and the potential to freeze and store samples before analysis. The objectives of the present study were to compare the performance of the ELISA and FC methods and to describe the relative flexibility of the ELISA-based assay. Human blood samples encompassing a wide range of levels of P2Y12 blockade achieved in vitro by preincubation with P2Y12 antagonists or in vivo from patients treated with clopidogrel were included, reflecting the wide spread of values reported in clinical studies. The correlation between the PRI measured by ELISA and FC was highly significant (r=0.95, p<0.001), (n=80). After the initial activation, samples were stable for at least four weeks when frozen (−20°C) prior to analysis by ELISA. Frozen samples from patients treated with clopidogrel appeared stable for up to nine weeks. Based on these results, the ELISA-based assay appears to provide a reliable and more flexible alternative to the FC method to determine P2Y12 receptor blockade and may enable more extensive utilisation of the VASP assay in clinical studies.Note: All work done at BioCytex, Marseille, France and Daiichi Sankyo, Tokyo, Japan.

Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y12 receptor

2006 ◽  
Vol 96 (12) ◽  
pp. 767-773 ◽  
Author(s):  
Agnieszka Pampuch ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Light Transmission ◽  
Platelet Reactivity ◽  
Flow Cytometric Analysis ◽  
Individual Variability ◽  
P2y12 Receptor ◽  
Reactivity Index ◽  
Drug Induced ◽  
Light Transmission Aggregometry ◽  
Adp Receptor

SummaryThere is need to improve platelet function testing to monitor the response to antiplatelet drugs. We compared flow-cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) to light-transmission aggregometry for the detection of drug-induced in-vitro inhibition of the platelet P2Y12 ADP receptor on 22 healthy subjects (10 males, 12 females, 28.5 ± 6.6 years). The platelet reactivity index (PRI) of VASP was calculated both from mean fluorescence intensity (MFI) and percent of fluorescence-positive platelets in the presence of PGE1 with or without ADP (10 µM). Platelet aggregation was induced by ADP (1.25, 2.5 and 5 µM). Cangrelor, a competitive inhibitor of the P2Y12 receptor, preincubated 5 minutes, induced a concentration-dependent inhibition of platelet ADP-receptor function in both tests. Indeed PRI (%) based on either MFI or percent platelets gated were highly correlated with each other (r = 0.97, p<0.0001) and with aggregation in- duced by ADP. The IC50 of cangrelor against each of the three ADP concentrations used in aggregometry increased from 5.8 ± 3.4 nM to 23.1 ± 4.0 nM and to 98 ± 25 nM, respectively. The IC50 of cangrelor based on VASP-P was within the same range (25.5 ± 7.7 nM). No correlation was observed between IC50 values of cangrelor and ADP concentrations giving 50% effect (EC50) in the absence of the drug. However, at 10 nM cangrelor seven subjects could be identified by the VASP-P assay as “low responders” to the drug (PRI> 50%), and six of them also had an aggregation response to 5 µM ADP > 50%. These six subjects showed the lowest ADP EC50 values in the absence of the drug, possibly reflecting high sensitivity of their platelet P2Y12 receptors to ADP. In conclusion, both the VASP-P assay and light-transmission aggregometry detect in a comparable way in-vitro pharmacological inhibition of the platelet P2Y12 ADP receptor and its individual variability.

Impact of aspirin dose on adenosine diphosphate-mediated platelet activities

2013 ◽  
Vol 110 (10) ◽  
pp. 777-784 ◽  
Author(s):  
Antonio Tello-Montoliu ◽  
Estela Thano ◽  
Fabiana Rollini ◽  
Ronakkumar Patel ◽  
Ryan E. Wilson ◽  
...  
Keyword(s):  
Platelet Reactivity ◽  
Stable Coronary Artery Disease ◽  
Adenosine Diphosphate ◽  
P2y12 Receptor ◽  
Reactivity Index ◽  
Receptor Blockade ◽  
Aspirin Dose ◽  
Dosing Regimens ◽  
The Impact

SummaryDifferent aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 μM) of prasugrel’s active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (p<0.001). However, there were no differences according to aspirin dosing regimen at any P-AM concentration (vehicle: p=0.899; 1 ïM: p=0.888; 3 ïM: p=0.524; 10 ïM: p=0.548). Similar findings were observed in purinergic markers assessed by MEA (ADP and ADP+PGE1). P-AM addition significantly reduced AA and collagen induced platelet aggregation (p<0.001 for all measures), irrespective of aspirin dose. In conclusion, aspirin dosing does not appear to affect PD measures of ADP-mediated platelet reactivity irrespective of the degree of P2Y12 receptor blockade. P2Y12 receptor blockade modulates platelet reactivity mediated by alternative activators.

scholarly journals Head-to-Head Comparison of Consensus-Recommended Platelet Function Tests to Assess P2Y12 Inhibition—Insights for Multi-Center Trials

2020 ◽  
Vol 9 (2) ◽  
pp. 332
Author(s):  
Jean-Christophe Bélanger ◽  
Fabio Luiz Bandeira Ferreira ◽  
Mélanie Welman ◽  
Rahma Boulahya ◽  
Jean-François Tanguay ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Reactivity ◽  
Receptor Activation ◽  
P2y12 Receptor ◽  
Reactivity Index ◽  
Specific Method ◽  
Vasp Phosphorylation ◽  
Receptor Inhibitor

The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary objective of this study was to compare the performance of VASP assessment by ELISA and flow cytometry in relation to functional platelet aggregation testing by Multiplate® whole-blood aggregometry. Blood from 24 healthy volunteers was incubated with increasing concentration of a P2Y12 receptor inhibitor (AR-C 66096). Platelet function testing was carried out simultaneously by Multiplate® aggregometry and by VASP assessment through ELISA and flow cytometry. As expected, increasing concentrations of the P2Y12 receptor inhibitor induced a proportional inhibition of platelet aggregation and P2Y12 receptor activation across the modalities. Platelet reactivity index values of both ELISA- and flow cytometry-based VASP assessment methods correlated strongly (r = 0.87, p < 0.0001) and showed minimal bias (1.05%). Correlation with Multiplate® was slightly higher for the flow cytometry-based VASP assay (r = 0.79, p < 0.0001) than for the ELISA-based assay (r = 0.69, p < 0.0001). Intraclass correlation (ICC) was moderate for all the assays tested (ICC between 0.62 and 0.84). However, categorization into low, optimal, or high platelet reactivity based on these assays was strongly concordant (κ between 0.86 and 0.92). In conclusion, the consensus-recommended assays with their standardized cut-offs should not be used interchangeably in multi-center clinical studies but, rather, they should be standardized throughout sites.

A comparison of the antiplatelet effects of prasugrel and high-dose clopidogrel as assessed by VASP-phosphorylation and light transmission aggregometry

2008 ◽  
Vol 99 (01) ◽  
pp. 215-222 ◽  
Author(s):  
Christopher D Payne ◽  
Ying G Li ◽  
Nagy A Farid ◽  
John T Brandt ◽  
David S Small ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Light Transmission ◽  
Platelet Reactivity ◽  
Platelet Inhibition ◽  
P2y12 Receptor ◽  
Reactivity Index ◽  
High Dose ◽  
Receptor Blockade ◽  
Vasp Phosphorylation ◽  
Light Transmission Aggregometry

SummaryPlatelet inhibition as measured by vasodilator-stimulated phosphoprotein (VASP) and light transmission aggregometry (LTA) have shown concordance following dosing of clopidogrel. No reports have directly compared theVASP assay and LTA at the levels of P2Y12 blockade after loading doses (LDs) of prasugrel or high dose clopidogrel (600 and 900 mg).The aim was to compare theVASP assay and LTA during the loading dose phase of a comparative study of prasugrel and clopidogrel. Prasugrel 60 mg LD/10 mg maintenance dose (MD) and clopidogrel 300 mg/75 mg and 600 mg/75 mg LD/MD regimens were compared in a 3-way crossover study in 41 healthy, aspirin-free subjects. Each LD was followed by seven daily MDs and a 14-day washout period. P2Y12 receptor blockade was estimated using theVASP assay, expressed as platelet reactivity index (VASP-PRI). Platelet aggregation was assessed by light transmission aggregometry (20 and 5 μM ADP).Twenty-four hoursafter prasgurel 60 mg or clopidogrel 300 mg and 600 mg, respectively, VASP-PRI decreased from ∼80% to 8.9%, 54.7%, and 39.0 %, and maximal platelet aggregation (MPA) decreased from ∼79% to 10.8%, 42.7%, and 31.2%, with an overall VASP:MPA correlation of 0.88 (p<0.01). VASP assay responses after the clopidogrel LDs showed a wider range of values (300 mg: 0–93%; 600 mg: 0–80%) than prasugrel (0–13%); MPA responses followed a similar trend. Pearson’s correlation suggested a strong agreement between VASP and LTA (20 μM ADP) for MPA (r=0.86, p<0.0001).VASP and LTA demonstrated concordance across the response range of P2Y12 receptor blockade following thienopyridine LDs.

‘VASPFix’ for measurement of VASP phosphorylation in platelets and for monitoring effects of P2Y12 antagonists

2014 ◽  
Vol 111 (03) ◽  
pp. 539-548 ◽  
Author(s):  
Natalia Dovlatova ◽  
Ann E. White ◽  
Kiren Dhillon ◽  
Stan Heptinstall ◽  
Susan C. Fox ◽  
...  
Keyword(s):  
Platelet Reactivity ◽  
Platelet Rich Plasma ◽  
Indirect Measurement ◽  
Single Step ◽  
P2y12 Receptor ◽  
Distinct Advantage ◽  
Clinical Investigations ◽  
Step Procedure ◽  
Vasp Phosphorylation ◽  
P2y12 Antagonists

SummaryVasodilator-stimulated phosphoprotein (VASP) is phosphorylated and dephosphorylated consequent to increases and decreases in cyclic nucleotide levels. Monitoring changes in VASP phosphorylation is an established method for indirect measurement of cyclic nucleotides. Here we describe the use of an innovative cocktail, VASPFix, which allows sensitive and reproducible measurement of phosphorylated VASP (VASP-P) in a simple, single-step procedure using cytometric bead technology. Frozen VASPFix-treated samples are stable for at least six months prior to analysis. We successfully used VASPFix to measure VASP-P in platelets in both platelet-rich plasma and blood in response to compounds that increase (dibutyryl cAMP, adenosine, iloprost, PGE1) and decrease (ADP, PGE1) cAMP, and to determine the effects of certain receptor antagonists on the results obtained. The change in VASP-P brought about by adding ADP to PGE1-stimulated platelets is a combination of the effect of ADP at the P2Y12 receptor and of PGE1 at both IP and EP3 receptors. For iloprost-stimulated platelets EP3 receptors are not involved. A procedure in which iloprost, ADP and VASPFix were used to determine effectiveness of clopidogrel and prasugrel in patients was compared with an established commercial procedure that uses PGE1 and ADP; the latter produced higher platelet reactivity values that were the result of PGE1 interacting with platelet EP3 receptors. We conclude that VASPFix can be used both as a research tool and for clinical investigations and provides better specificity for P2Y12 receptor inhibition. The latter confers a distinct advantage over existing methods used to monitor effects of P2Y12 antagonists on platelet function.

scholarly journals Advances in bacterial specific imaging

2005 ◽  
Vol 48 (spe2) ◽  
pp. 145-152 ◽  
Author(s):  
David Wareham ◽  
J. Michael ◽  
Satya Das
Keyword(s):  
Nuclear Medicine ◽  
Clinical Studies ◽  
Human Disease ◽  
Diagnostic Technique ◽  
Sterile Inflammation ◽  
Microbial Infection ◽  
Course Of Disease ◽  
Wide Range ◽  
Diagnosis Of Infection

Nuclear medicine is a powerful diagnostic technique able to detect inflammatory foci in human disease. A wide range of agents have been evaluated for their ability to distinguish lesions due to microbial infection from those due to sterile inflammation. Advances continue to be made on the use of radiolabelled antibiotics which as well as being highly specific in the diagnosis of infection may be useful in monitoring the treatment and course of disease. Here we provide an update on in-vitro and clinical studies with a number of established and novel radiopharmaceuticals

scholarly journals Role of VASP Phosphorylation Assay in Monitoring the Antiplatelet Therapy

2013 ◽  
Vol 13 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Marian Fedor ◽  
R. Simonova ◽  
J. Fedorova ◽  
I. Skornova ◽  
L. Duraj ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Stent Thrombosis ◽  
Cardiac Death ◽  
P2y12 Receptor ◽  
Response To Treatment ◽  
Reactivity Index ◽  
Inadequate Response ◽  
Vasp Phosphorylation ◽  
Platelet Receptor ◽  
Increased Risk

Abstract Dual antiplatelet treatment with clopidogrel and aspirin represents standard regimen in prevention of thromboembolic events in patients with ischemic heart disease undergoing percutaneous coronary intervention (PCI). One of the greatest pitfalls of clopidogrel treatment is large inter-individual variability in response. Large amount of patients does not respond adequately and therefore are not „protected“ even in spite of receiving the therapy. Poor responders are exposed to three-fold increased risk of myocardial infarction, stent thrombosis and cardiac death. Clopidogrel is an antiplatelet prodrug, whose active metabolite inhibits platelet function by irreversible binding to the (adenosine diphosphate) platelet receptor P2Y12. Receptor P2Y12 plays primal role in ADP-mediated platelet activation, and also in mechanism of action of ADP inhibitors such as clopidogrel, prasugrel etc. Reasons stated above, raised the necessity for implementing reliable laboratory test in order to identify the unprotected patients. In an ideal scenario, such test would serve to adjust the dose and guide the individual tailored treatment. Vasodilator Stimulated Phosphoprotein (VASP) is an intracellular platelet protein which is non phosphorylated at basal state. Since its relation in cascade with P2Y12 receptor, VASP phosphorylation corerlates with inhibition of P2Y12 which is the receptor of prime importance in ADP mediated activation of platelets and as is primary target of ADP inhibitors action. Outcome of the assay is represented as the value of platelet reactivity index (PRI), where PRI values above 50% are considered inadequate response to treatment and signal exposure to increased risk of myocardial infarction, post-PCI stent thrombosis and cardiac death. VASP-P flow cytometric assay is emerging into the spotlight as the promising method, mostly for its specificity for ADP inhibitors, better outlook for standardising results and lesser sample manipulation compared to multiple electrode aggregometry.

scholarly journals Lactiplantibacillus plantarum 299v (LP299V®): three decades of research

2021 ◽  
pp. 1-26
Author(s):  
E. Arvidsson Nordström ◽  
C. Teixeira ◽  
C. Montelius ◽  
B. Jeppsson ◽  
N. Larsson
Keyword(s):  
Clinical Studies ◽  
Iron Status ◽  
Human Consumption ◽  
Comprehensive Overview ◽  
Scientific Publications ◽  
Healthy Human ◽  
Positive Effects ◽  
Freeze Dried ◽  
Wide Range

This review aims to provide a comprehensive overview of the in vitro, animal, and clinical studies with the bacterial strain Lactiplantibacillus plantarum 299v (L. plantarum 299v; formerly named Lactobacillus plantarum 299v) published up until June 30, 2020. L. plantarum 299v is the most documented L. plantarum strain in the world, described in over 170 scientific publications out of which more than 60 are human clinical studies. The genome sequence of L. plantarum 299v has been determined and is available in the public domain (GenBank Accession number: NZ_LEAV01000004). The probiotic strain L. plantarum 299v was isolated from healthy human intestinal mucosa three decades ago by scientists at Lund University, Sweden. Thirty years later, a wealth of data coming from in vitro, animal, and clinical studies exist, showing benefits primarily for gastrointestinal health, such as reduced flatulence and abdominal pain in patients with irritable bowel syndrome (IBS). Moreover, several clinical studies have shown positive effects of L. plantarum 299v on iron absorption and more recently also on iron status. L. plantarum 299v is safe for human consumption and does not confer antibiotic resistance. It survives the harsh conditions of the human gastrointestinal tract, adheres to mannose residues on the intestinal epithelial cells and has in some cases been re-isolated more than ten days after administration ceased. Besides studying health benefits, research groups around the globe have investigated L. plantarum 299v in a range of applications and processes. L. plantarum 299v is used in many different food applications as well as in various dietary supplements. In a freeze-dried format, L. plantarum 299v is robust and stable at room temperature, enabling long shelf-lives of consumer healthcare products such as capsules, tablets, or powder sachets. The strain is patent protected for a wide range of indications and applications worldwide as well as trademarked as LP299V®.

scholarly journals Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
G. J. Schmidt ◽  
C. M. Reumiller ◽  
H. Ercan ◽  
U. Resch ◽  
E. Butt ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Fold Increase ◽  
Surface Expression ◽  
Discrimination Power ◽  
Vasp Phosphorylation ◽  
Platelet Proteome ◽  
Quantitative Changes

AbstractThere is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and –inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen’s d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.

Relationship between degree of P2Y12 receptor blockade and inhibition of P2Y12-mediated platelet function

2010 ◽  
Vol 103 (06) ◽  
pp. 1210-1217 ◽  
Author(s):  
Robert Buckland ◽  
Atsuhiro Sugidachi ◽  
Joseph Jakubowski ◽  
Robert Storey ◽  
Heather Judge
Keyword(s):  
Platelet Function ◽  
Annexin V ◽  
Coronary Intervention ◽  
P2y12 Receptor ◽  
Receptor Blockade ◽  
Strong Inhibition ◽  
Receptor Antagonists ◽  
Vasp Phosphorylation ◽  
Microparticle Formation

SummaryThe thienopyridine P2Y12 receptor antagonists clopidogrel and prasugrel prevent arterial thrombosis and are routinely used following percutaneous coronary intervention. However, the optimal level of P2Y12 blockade to effectively inhibit platelet function is unknown. These studies utilised the active metabolite of prasugrel (R-138727) to achieve a range of P2Y12 blockade in vitro and assessed several aspects of platelet function. Blood from healthy volunteers was incubated with R-138727 (0–10 μM). P2Y12 receptor number was assessed using a 33P-2MeSADP binding assay. Platelet aggregation (PA) was measured by optical aggregometry with ADP 2–20 μM. VASP phosphorylation, annexin V binding, microparticle formation and P-selectin expression were assessed by flow cytometry. Increasing numbers of unblocked receptors were required for a sustained aggregation response with decreasing concentrations of ADP. A P2Y12 receptor blockade of 60–80% resulted in strong inhibition of final PA response, P-selectin expression, microparticle formation and vasodilator-stimulated phosphoprotein (VASP). PA induced by ADP 2 μM and P-selectin expression were particularly sensitive to low levels of receptor blockade whereas the VASP phosphorylation assay was relatively insensitive, requiring 60% receptor blockade to achieve substantial inhibition. Different assays varied in their ability to discriminate particular ranges of P2Y12 blockade and 80% or greater P2Y12 receptor blockade is required for consistently strong inhibition of several aspects of platelet function. These data guide the interpretation of results from different assays used to monitor the effects of P2Y12 receptor antagonists.

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