Degradation of VWF with rADAMTS13 was performed under conditions where VWF is partially denatured to make the cleavage motif accessible for proteolysis. The rADAMTS13 was pre-diluted to 10 and 100 IU/mL FRETS-VWF73 activity (final concentrations between 0.5 and 10 IU/mL) and activated with BaCl2 in the presence of 5 mM Tris and 1.5 M urea, pH 8.0, at 37 °C for 30 min. Activated rADAMTS13 was mixed 1 + 9 with patient plasma samples and further incubated at 37 °C for 2 and 5 h before proteolysis was stopped by the addition of Na2SO4 (8.25 mM final concentration). In control experiments to measure the effect of the endogenous ADAMTS13, the patient samples were mixed with buffer instead of rADAMTS13. Samples were centrifuged at 2500 ×g for 5 min and the VWF in the supernatants was analysed using the VWF:CB assay and by quantitative densitometry of semi-automated electrophoresis gels performed with the Sebia Phoresis rel. 9.2.0 software to calculate relative increase of VWF dimer levels relative to the total quantity of VWF determined from the areas under the densitometry curves. UHMW VWF multimers were quantified from the home case gels as described above.

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