Phosphorylation is reversible, so platelets must be fixed with paraformaldehyde (PFA) solution. 14 Results should be interpreted with caution if blood cannot be processed immediately as prolonged storage can cause artifactual activation.
14 Platelets are highly responsive in freshly drawn blood (<5 min), but they can be kept at room temperature for 1 hr before signaling starts to wane. 13 Inhibitory signaling (e.g., pVASP) is seemingly impervious to the choice of anticoagulant, but activatory signaling (e.g., pErk1/2) is blunted by EDTA. 12 Albeit, heparin should be used with caution as it can inhibit thrombin-induced platelet activation. 7 Assays are often performed on citrated blood, but comparable results can be obtained with heparin. 11 In any case, blood should be drawn into an anticoagulant tube (e.g., BD Vacutainer), and sample preparation should begin as soon as possible. Phosphoflow is commonly performed on whole blood, but it can be performed on platelet-rich plasma (PRP), washed platelets, and murine samples with comparable ease. 2 TECHNICAL CONSIDERATIONS 2.1 Blood collection Accordingly, we review the technical considerations for successful phosphoflow and barcoding in blood platelets. Widespread application will foster calls for standardized protocols and prompt laboratories to learn its capabilities and limitations. 6, 10 As FCB allows large-scale experiments to be performed with standard equipment and consumables, it provides an attractive and accessible platform for platelet function testing. The advent of an innovative multiplexing technique called fluorescent cell barcoding (FCB)-adapted by our laboratory-amplifies analytical throughput by allowing the rapid acquisition of ≤96 samples in one test tube. Phosphoflow can be used to dissect the signals that afflict platelet function, 6, 8 and it can be used to monitor antiplatelet efficacy, 9 but routine protocols are ill-suited to large-scale application (e.g., batching/screening) as they are limited by low throughput. 6, 7 These phosphoflow protocols provide significant advantages over traditional approaches that rely on cell lysis and protein extraction (e.g., immunoassays, immunoblotting, and mass spectrometry) as they allow multiple molecular measurements on individual (intact) cells in the (patho)physiologic milieu of whole blood (Table 1).
4, 5 Fast and flexible protocols have been developed for phosphoprotein analysis by flow cytometry. 3 Intraplatelet protein phosphorylation-coordinated by opposing kinase/phosphatase activity-regulates multiple aspects of platelet function (including glycoprotein expression). 2 Naturally, platelet function rests on dynamic interplay between activatory and inhibitory signaling. 1 Common tests are performed on inducible glycoproteins (e.g., CD62P), but-while useful in some respects-they are merely endpoint markers that provide an incomplete snapshot of the platelet activation process. Flow cytometry is a powerful tool in platelet function testing it can provide critical information on disease progression and drug responsiveness in cardiovascular medicine.
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