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Tutorials : Feb 1, 2011 ( )
In Vitro Platelet-Function Assay Kits
VASP Assay Detects Responsiveness to Clopidogrel and Other Anti-P2Y12 Drugs
Managing drug dosage is critical to the overall success of pharmaceutical intervention, not only in the clinic but also in clinical trials. Inadequate dose management of certain drugs can have fatal consequences; therefore, optimizing the dose of a drug has become a necessary component of successful clinical trial management.
One example of the necessity of dose management is with a class of drugs that is administered to inhibit blood clotting. The most popular thienopyridine, clopidogrel (commercially known as Plavix), irreversibly inhibits clotting by targeting the platelet ADP/P2Y12 receptor. Adenosine diphosphate (ADP) is a major in vivo platelet agonist and binds to the platelet ADP/P2Y12 receptor. Clopidogrel competes with ADP binding to this receptor.
Cardiologists reported in several clinical trials that patients exhibit a variable response to clopidogrel dosage, resulting in the need to modulate the dosage in order to increase the antithrombotic effect of the drug and potentially the bleeding risk during various surgical procedures.
There are a number of assays designed to assess platelet responsiveness to clopidogrel, one of which is the vasodilator-stimulated phosphoprotein (VASP) flow cytometry assay developed by BioCytex/Diagnostica Stago. The use of such assays has demonstrated interpatient variability in drug responsiveness not only to clopidogrel but also to prasugrel
The ADP/P2Y12 receptor is a seven-transmembrane domain receptor and is biophysically linked to an inhibitory G-protein (Gi). Signaling through this receptor leads to lower cyclic adenosine monophosphate (cAMP) levels and inactivation of protein kinase A (PKA), one of the substrates of which is VASP. Also feeding into this pathway is P2Y1 receptor-mediated activation of platelets. In contrast, the inhibiting prostanoid prostaglandin E1 (PGE1) leads to higher levels of cAMP and activation of PKA.
The mechanism of signaling through this pathway is as follows: when platelets that are not treated with an anti-P2Y12 drug are incubated in vitro with PGE1, VASP is phosphorylated and platelets are inhibited. In contrast, in platelets left untreated, but incubated with PGE1 plus ADP, VASP is dephosphorylated and the platelets are activated. However, in platelets treated with an anti-P2Y12 drug and then incubated with PGE1 alone or in combination with ADP, VASP is constitutively phosphorylated and the platelets are inactivated (Figure).
This occurs due to the fact that the anti-P2Y12 drug irreversibly or reversibly binds to the ADP/P2Y12 receptor, thus preventing ADP from binding to its receptor and inhibiting the dephosphorylation of VASP.
In other words, the binding of clopidogrel, or any other P2Y12-targeting drug, to the platelet P2Y12 receptor results in direct inactivation of the intracellular signaling cascade that ultimately inactivates the platelet’s ability to form a clot. VASP is a reliable reporter of the platelet’s ability to be activated by ADP, and the degree to which VASP is phosphorylated can be measured in a direct, cost-effective manner.
Principle, Protocol, Instrumentation
The assay works by a basic, dual-color flow cytometric principle. In terms of sample type, the specimen is uncoagulated whole blood collected from patients who are taking anti-P2Y12 drug; the specimen is collected in a Vacutainer (BD) tube containing the anticoagulant sodium citrate. The specimen is stable at room temperature for up to 48 hours prior to testing. Only 30 microliters of specimen is needed to run the assay.
The sample is incubated with PGE1 in the presence and absence of ADP. Following incubation, the sample is fixed in formaldehyde. The fixed sample is then permeabilized and labeled with a primary antibody against the phosphorylated Ser-239 residue of VASP (mouse monoclonal anti-VASP-P). This is followed by staining with fluorescein isothiocyanate (FITC)-conjugated goat antimouse polyclonal antibody and counterstaining with antibody against platelet surface marker CD61. A negative isotype control is also run simultaneously to assess background reactivity of platelets with primary antibody.
All steps are performed at room temperature. All of these reagents are contained within the kit, and the protocol takes about 30 minutes to complete.
The fluorescence in each sample is measured by flow cytometry, which results in a statistic known as the platelet reactivity index (PRI). The following is the equation for calculating PRI:
PRI = [MFIPGE1 - MFIPGE1 + ADP/ MFIPGE1] X 100
MFI stands for mean fluorescence intensity. Based on these calculations: the higher the PRI percentage, the lower the responsiveness to anti-P2Y12 drug. In terms of instrumentation required for the assay, any flow cytometer can be used to measure the fluorescence, even benchtop models.
Use of VASP Assay in Clinical Trials
Like other platelet-function assays, the VASP assay has been incorporated in clinical trials to assess patient responsiveness to anti-P2Y12 drugs. In all trials, the mechanism of cardiovascular pathology was enhanced platelet reactivity, which was measured by in vitro platelet function assays such as the VASP assay.
For example, one small clinical trial has shown that, overall, the VASP assay is comparable to light transmission-based platelet aggregation assays, but has found that the VASP assay is better at identifying low responders to treatment with Cangrelor. In addition, several clinical trials have shown that clopidogrel, both alone and in combination with aspirin, reduced risk of atherosclerosis in patients with diabetes mellitus. In a more recent clinical trial, the VASP assay was used to determine responsiveness to Ticagrelor combination therapy.
Other potential clinical applications for the VASP assay include assessment of response to clopidogrel prior to angioplasty, assessment of treatment response prior to discharge following angioplasty, long-term assessment of treatment effectiveness, assessment of novel P2Y12 antagonists in preclinical and clinical development, and assessment of potential hemorrhaging following treatment with clopidogrel. Moreover, with its increasing use in clinical trials as a way of assessing responsiveness to clopidogrel (and other anti-P2Y12 compounds), there are discussions about using the VASP assay as a companion diagnostic.
In summary, traditional platelet aggregation tests are often not sensitive and specific enough to detect resistance or potential bleeding problems caused by anti-P2Y12 drug treatment. These limitations led to the development of the VASP assay kit, which is labeled for research use only and not for use in diagnostic procedures in the U.S. and Canada.
Paul Riley, Ph.D. (Paul.Riley@stago-us.com), is product manager at Diagnostica Stago.
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