Expression and purification of individual VWF domains. (A) Mature VWF was purified from human plasma cryoprecipitate. This protein lacks VWFpp because the Arg ₇₆₃-Ser₇₆₄ bond is proteolytically cleaved. Propeptide VWFpp (D1-D2 domains) and other domains within the globular section of VWF (D′D3, A1, A2, and A3) were expressed as FLAG- and His-tagged proteins in CHO cells. Amino acid numbers provided are based on pre-pro-VWF. ΔPro-VWF is dimeric, full-length VWF. ΔD′D3-VWF is identical to ΔPro-VWF except it lacks the D′D3 domain. (B) Silver staining of purified VWF domains under reducing (with β-mercaptoethanol) and nonreducing (absence of β-mercaptoethanol) conditions. (C) Western blot of the domains using anti-His Ab under reducing conditions. (D) Western blot of ΔPro-VWF and ΔD′D3-VWF under reducing conditions detected using anti-VWF polyclonal Ab.