Figure 2

Platelet isolation strategy yields samples >90% pure platelets by flow cytometry. (A) Purified platelets were double stained for allophycocyanin (APC)-conjugated, anti-CD45 (white blood cell marker, y-axis) and fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (platelet marker, x-axis). Double scatterplot demonstrates the majority of the sample stains positive for CD41, but not for CD45. (B) Histograms for CD41⁺ (91.36%) flow cytometry events consistent with platelet-specific enrichment, and (C), CD45⁺ (1.3%) events. (D) Differential centrifugation workflow for enrichment of the membrane proteome from platelets isolated from whole blood, prior to LC-MS/MS analysis. (E) Silver stain of fractions described in panel D. (F) Immunoblot demonstrating CD41 enrichment and actin depletion during the workflow to produce platelet membrane fraction (P3). LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry.