Comprehensive VWF Functional Assays

VWF:Ag levels were measured using immunoturbidometric assay (HemosIL von Willebrand Factor Antigen, Instrumentation Laboratory, Bedford, MA). Platelet-dependent VWF activity was measured using VWF:GPIbR (HemosIL von Willebrand Factor Ristocetin Cofactor Activity, Instrumentation Laboratory, Bedford, MA). FVIII clotting activity (FVIII:C) was assessed using a 1-stage assay and VWF collagen binding (VWF:CB) with collagen type I, III, or a combination of thereof using an enzyme-linked immunosorbent assay method.¹² (link) VWF multimer analysis was performed using a semiautomatic Hydrasys 2 scan (Sebia, Lisses, France), and we used the HYDRAGEL 5 von WILLEBRAND MULTIMERS kit.¹³ (link) A commercially available enzyme-linked immunosorbent assay kit (Sanquin, Amsterdam, The Netherlands) was used to measure the VWF propeptide (VWFpp) antigen.¹⁴ (link) To evaluate the synthesis and secretion of VWF we used the FVIII:C/VWF:Ag ratio and VWFpp.¹⁴ (link) VWF clearance was assessed using the VWFpp/VWF:Ag ratio with a ratio exceeding 1.6 considered an indicator of accelerated clearance.¹⁵ (link)