Abstract
AbstractAbstract 1415A recombinant human CHO-expressed von Willebrand factor (rVWF) consisting of ultra-high molecular weight (UHMW) multimers resembles the VWF stored in Weibel-Palade bodies of endothelial cells. Once secreted into plasma, UHMW multimers are rapidly cleaved by ADAMTS13 and are usually missing in plasma-derived VWF (pdVWF).Here we analyzed in vitro whether the kinetics of cleavage of rVWF by ADAMTS13 is similar to that of pdVWF. The kinetics of ADAMTS13-mediated proteolysis of rVWF were explored under denaturing conditions (1.5 M urea) or under shear stress so as to expose the ADAMTS13 cleavage site of VWF.Multiple assays showed that rVWF was efficiently cleaved by ADAMTS13. UHMW multimers disappeared within seconds at physiological concentrations of ADAMTS13. Using lower concentrations of ADAMTS13 (10-30 mU/ml, equivalent to 1–3% of normal human plasma), UHMW were cleaved within 30 minutes. The typical satellite bands appeared very early in an ADAMTS13 dose-dependent manner. Virtually the same results were obtained when human plasma was used as a source for ADAMTS13. Although pdVWF differs from rVWF in its multimeric structure, the decrease in activity was similar for rVWF and pdVWF. Finally, the extent of ADAMTS13 cleavage was similar for rVWF and pdVWF when exposed to shear stress using a cone-plate viscometer.Our data clearly indicate that rVWF is a good substrate for ADAMTS13. Ongoing phase 1 studies demonstrated that rVWF is indeed processed by the protease when administered in humans with severe VWD. Disclosures:Rottensteiner:Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Vejda:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.
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