Evaluation of von Willebrand Factor cleaving protease independent of von Willebrand Factor (vWF)

KEMPFER, ANA C; FARÍAS, CRISTINA; AMARAL, MARÍA M; SILAF MARÍA R; CARBALLO GONZALO A; LAZZARI MARÍA A

Paris

Congreso; XVIII CONGRESS. THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS; 2001

THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS

Different methods to measure vWF cleaving protease activity on unfolded vWF were developed. In the immunoblotting method described by Furlan et al exogenous vWF was added to the samples and to diluted reference plasma. It has been proposed to dilute plasma between 1/20 and 1/640, but we have found no proteolytic effect at so high dilutions. Other workers partially digest endogenous vWF with urea. Samples are then diluted between 1/6 and 1/320 and exogenous vWF is added to detect the proteasic effect. The aim of this work was to choose a sample treatment to avoid endogenous vWF interference, because if the protease is normal and endogenous vWF is abnormal, it will remain in the sample and only exogenous vWF will be digested, carrying to erroneous results. So, we have ultrasonicated plasma in order to systematically digest endogenous vWF (normal or abnormal), without disturbing the protease. Besides, we dialyzed the samples against urea in cellulose membranes (dialysis tubing) to unfold vWF with urea. The plasma samples were ultrasonicated at 20000 Hz, during 20 min to eliminate large and intermediate endogenous vWF multimers. Then were first diluted 1/5, 1/10, 1/20, 1/40 with 5 mM Tris, pH 8.0 and the sample (aliquots of 50 µl) vWF cleaving protease was activated with 9,3 mM BaCl2 were added. After mixing with 100 µl purified vWF purified (final concentration of 3mM BaCl2), the samples were incubated against 1.5 M urea, 5 mM Tris, pH 8.0. in a dialysis tubing in a magnetic stirrer for 4 h at 37°C. After stopping the digestion by dialysis with 5 mM Tris, pH 8.0 during 90 min and addition of 2mM EDTA the samples were analyzed by SDSelectrophoresis on 1% agarose and inmunoenzymatic staining of digested purified vWF with biotinylated antivWF, avidin-biotin-peroxidase and 4-Cl-1-naphtol. Aliquots of 50 µl normal human plasma pool ultrasonicated (NHP) dilutions 1/5, 1/10, 1/20, 1/40, 1/80, 1/160 and 1/320 in 5 mM Tris, pH 8.0 were used for the calibration curve. The stained gels were analyzed by densitometrical scanning. The 100 % of vWf multimers and 0% of protease activity is defined as the distance between densitometric tracing starting point and final point for NHP. A patient with thrombotic thrombocytopenic purpura (TTP) tested by the immunoblotting method was used to compare the results. The protease activity of TTP patient (25%) was similar in both methods was similar. The method described have three advantages 1) we avoid the interference of the endogenous vWF (normal and abnormal), 2) an easier form to recovery the sample because in our hands the immunoblotting method caused the loss of the majority of the samples and 3) to eliminate directly urea to avoid its interference in the vWF detection.