P-selectin expression and detection of activated GPIIb/IIIa by flow cytometry

Platelet reactivity was determined by assessing binding of the monoclonal antibody PAC-1 to activated GPIIb/IIIa and by the platelets’ surface expression of P-selectin, as previously published (19). In short, blood was diluted in phosphate-buffered saline (PBS) to obtain approximately 2×10⁴/μl platelets and incubated for 10 min without agonists or with suboptimal concentrations of the following three platelet agonists: PAR-1 agonist SFLLRN (Thrombin-receptor activating peptide [TRAP] –6; final concentration 14.25 μM; Bachem, Bubendorf, Switzerland), PAR-4 agonist AYPGKF (final concentration 0.714 mM; Verum Diagnostica, Munich, Germany) and the GPVI agonist collagen-related peptide (CRP; final concentration 0.04 μg/ml; a generous gift from Dr. R. W. Farndale, Department of Biochemistry, University of Cambridge, Cambridge, UK). The platelet population was identified by staining with anti-CD42b (clone HIP1, allophycocyanin labelled, Becton Dickinson, Immunocytometry Systems, San Jose, CA, USA), while P-selectin and activated GPIIb/IIIa expression were determined by the binding of the monoclonal antibodies anti-CD62P (P-selectin; clone CLB-Thromb6, phycoerythrin-labelled, Immunotech, Beckman Coulter Fullerton, CA, USA) and PAC-1 (fluorescein isothiocyanat-labelled, Becton Dickinson, Immunocytometry Systems). Isotype-matched control antibodies were used in separate vials for the determination of non-specific binding. After 15 min of incubation in the dark, their reaction was stopped by adding 500 μl cold PBS (4 °C) and samples were acquired after 10 min on a FACS Calibur flow cytometer (Becton Dickinson). Standard Becton Dickinson Calibrite beads were used for daily calibration of the cytometer.

A total number of 10,000 CD42b positive events were recorded. Positive analysis regions for P-selectin and activated GPIIb/IIIa were set with appropriate nonspecific controls. Percentage positive platelets and mean fluorescence intensity (MFI) of anti-CD62P and PAC-1 were evaluated.