41 Reduced thrombin generation and/or decreased platelet response in women with HMB minutes, within 3 hours from withdrawal, to obtain platelet-poor plasma (PPP). For the normal pooled plasma (NPP), blood from 24 volunteers was prepared by double centrifugation at 2630 g during 10 minutes at room temperature. Plasmas were pooled, followed by ultracentrifugation (100 000 g, 70 minutes). Aliquots of 1 mL were stored at −80°C. Platelet function Platelet function assays were done within three hours from collection. Agonist-induced platelet P-selectin expression and αIIbβ3 activations were measured using phycoerythrin (PE)-conjugated monoclonal mouse antihuman-P-selectin antibody (BD Bioscience) and fluores- cein phycoerythrin (FITC)-conjugated polyclonal sheep antihuman fibrinogen (Dako).14 Whole blood was 10-fold diluted (5 μL: 45 μL) in HEPES-buffered saline (HBS; 10 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L MgSO4, 5 mmol/L KCl, pH 7.4), containing 20 μg/mL FITC- labelled antifibrinogen, 20 μg/mL PE-labelled anti-P-selectin, with either 30 μmol/L ADP, 625 ng/mL XL-CRP and 10 μmol/L PAR-1 agonist TRAP (SFLLRN) to study agonist specific responses.19 The assay kits were prepared in large badges and stored at −20°C for a maximum of 2 months in nonsterile tubes (Brand GMBH, Wertheim, Germany). Assays were stopped after 20-minute incubation with 500 μL fixation buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 1.12 mmol/L NaH2PO4, 1.15 mmol/L KH2PO4, 10.2 mmol/L Na2HPO4, 4 mmol/L EDTA and 0.5% formalin (37% formaldehyde)), and all samples were analyzed using flow cytometry on the same day of processing. Single platelets were gated based on forward and side scatter properties, and their median fluorescence intensity (MFI) was measured. Dose-response graphs and optimal agonist concentrations were determined as the shoulder of the dosedependent curves. Measurements were normalized to measurements of P-selectin expression and αIIbβ3 activation in 26 healthy control subjects, whose blood was collected at the same time and location as patient blood collection to guarantee identical pre-analytical treatment of the blood. The control samples were set at 100%, and the corresponding patients were presented as percentage of these controls. All samples were analyzed on a FACS Canto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The CVs of platelet function are below 10% for all platelet activation measurements, except ADP- induced P-selection expression, which has a CV below 20% (22). Analysis of VWF function and antigen levels Von Willebrand factor function was measured, using ristocetin sulphate-induced endogenous VWF binding to the patient platelets. 5 μL whole blood was supplied to 45 μL HBS with 20 μg/ mL FITC- labelled polyclonal sheep antihuman VWF and ristocetin suphate (0.4 μg/mL). The assay kits were prepared in large batches and stored at −20°C for a maximum of 2 months in nonsterile tubes (Brand GMBH, Wertheim, Germany). Assays were stopped after 20-minute incubation with 500 μL fixation buffer, and all samples were analysed by flow cytometry on the same day of processing. Single platelets were gated based on forward and side scatter properties, and their MFI was measured. Dose-response graphs and optimal ristocetin sulphate con- centration were determined as the shoulder of the dose-dependent curve in the unprocessed blood. Measurements were normalized to measurements of VWF binding in controls. All MFI 3
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