Multiplexed phosphospecific flow cytometry enables large‐scale signaling profiling and drug screening in blood platelets

Abstract

To cite this article: Spurgeon BEJ, Aburima A, Oberprieler NG, Task en K, Naseem KM. Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets. J Thromb Haemost 2014; 12: 1733-43.Summary. Background: Dissecting the signaling events that contribute to platelet activation will increase our understanding of platelet function and aid in the development of new antiplatelet agents. However, high-throughput methodology for the quantitative analysis of platelet signaling events is still lacking. Objective: To develop a high-throughput assay for the analysis of platelet signaling events in whole blood. Methods and Results: We developed a fluorescent barcoding protocol to facilitate multiplexing and enable large-scale signaling profiling in platelets in whole blood. The methodology allowed simultaneous staining and acquisition of 24-96 samples in a single analysis tube with a standard flow cytometer. This approach significantly reduced experimental numbers, data acquisition time, and antibody consumption, while providing automated statistically rich quantitative data on signaling events. Using vasodilator-stimulated phosphoprotein (VASP), an established marker of platelet inhibition and antiplatelet drug therapy, we demonstrated that the assay could detect subtle changes in phosphoVASPSer157/239 in response to cAMP-elevating agents of varying potency and known modulators of the cAMP signaling cascade. The assay could be used with washed platelets or whole blood, analyzed immediately or frozen, without any significant change in assay performance. To demonstrate the usefulness of the assay as a drug discovery platform, we examined a prostaglandin screening library. Our screen of 70 prostaglandin derivatives revealed three previously uncharacterized lipids that stimulated phosphorylation of VASP-Ser157. Follow-up analyses demonstrated that these agents elevated intraplatelet cAMP and inhibited collagen-induced platelet aggregation. Conclusions: This novel method enables rapid, large-scale quantitative signaling profiling and compound screening in human platelets present in whole blood.

Phosphoflow cytometry and barcoding in blood platelets: Technical and analytical considerations

“…Permeabilization buffers dissolve/extract lipids from the platelet membrane so that antibodies can enter/leave the cell. The permeabilization protocol should be tailored to the phosphoprotein of interest as the choice of buffer can affect antigenicity . Platelets can be permeabilized with alcohols (e.g., methanol) or mild detergents (e.g., saponin and Triton X‐100).…”

“…Antibody titration is an important consideration in flow cytometry, and phosphospecific antibodies should be used at saturation. Platelets can be stained with monoclonal and/or polyclonal antibodies . Monoclonals can provide quantitative measures of phosphorylation (one antibody/one antigen), but their monospecificity can be limiting as binding can be inhibited by small changes in antigen conformation.…”

“…Samples can be deconvoluted with traditional software (e.g., FlowJo) or specialist applications (e.g., Cytobank) . FCB is relatively new in platelets—but it has already been effective in drug screens, clinical trials, and animal studies …”

“…Intraplatelet protein phosphorylation—coordinated by opposing kinase/phosphatase activity—regulates multiple aspects of platelet function (including glycoprotein expression) . Fast and flexible protocols have been developed for phosphoprotein analysis by flow cytometry . 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 ).…”

“…Phosphoflow can be used to dissect the signals that afflict platelet function, and it can be used to monitor antiplatelet efficacy, but routine protocols are ill‐suited to large‐scale application (e.g., batching/screening) as they are limited by low throughput. 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 . 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.…”