The epitope of the antibody used in the REAADS VWF activity assay is quaternary

The REAADS VWF activity assay is often assumed to be specific for the A1 domain, the portion of VWF that binds platelet GPIbα. We tested this assay on the A1A2A3 region of VWF with each domain expressed independently of one another and together in combination as a tri-domain. The monoclonal antibody used in this assay is found to be insensitive to the single A domains and does not recognize free A1 domains as it is often assumed. Rather, we find the assay to effectively recognize A1A2A3 with the domains together in their natural glycosylated sequence context. Furthermore, type 2M and 2B Von Willebrand Disease mutations differentially disrupt the sensitivity of the assay, indicating that mutational effects on the structure of A1 in the A1A2A3 context concomitantly disrupt the epitope of the antibody. The REAADS VWF activity assay therefore is conformationally sensitive to the native quaternary association of the A domains together and it is not specific to freely exposed A1 domains.