D′D3 is the only domain in multimeric VWF that binds VWFpp. (A) Eight hundred response unit (RU) VWFpp was covalently coupled onto the SPR biosensor. Each of the VWF domains (D′D3, A1, A2, and A3) at a concentration of 250nM were then injected over this surface in MES buffer (pH 6.2) containing 10mM CaCl2. Inset shows the inverse experiment in which binding of the 250nM VWFpp to 800 RU immobilized D′D3 was measured. Only D′D3 binds VWFpp. (B) Binding of 10 μg/mL of VWF isolated from human plasma cryoprecipitate (multimeric, pVWF), recombinant dimeric ΔPro-VWF, and ΔD′D3-VWF to immobilized VWFpp was measured using ELISA. Binding observed in the case of pVWF and ΔPro-VWF was eliminated when either the D′D3 domain was deleted (ΔD′D3-VWF) or when anti-D′D3 mAb DD3.1 was applied. *P < .05 for pVWF and ΔPro-VWF in the absence of any mAb.
