title: Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets.
abstract: 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.
  
  
  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 phosphoVASP-Ser157/239 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.
authors: Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull, UK.
authors: B E J
authors: BE
authors: Spurgeon
authors: A
authors: A
authors: Aburima
authors: N G
authors: NG
authors: Oberprieler
authors: K
authors: K
authors: Taskén
authors: K M
authors: KM
authors: Naseem
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001792:Blood Platelets / Q000187:drug effects*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004586:Electrophoresis; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry; D011453:Prostaglandins / Q000737:chemistry; D015398:Signal Transduction; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D000906:Antibodies; D015815:Cell Adhesion Molecules; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D011453:Prostaglandins; D000098662:Vasodilator-Stimulated Phosphoprotein; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: flow cytometry; phosphorylation; platelets; protein kinase A; signal transduction