VWFpp and VWF interact in plasma. (A) Anti-VWF Ab (30 μg/mL) or control (antimyosin) was added to immunoprecipitate VWF from 1 mL of heparinized human PPP in the presence or absence of 30 μg/mL of DD3.1 overnight at 4°C. The protein complex was isolated using protein-A/G beads and VWFpp was subsequently released using HEPES buffer containing 5mM EDTA. Released VWFpp was detected with a sandwich ELISA using mAb 242.2 for capture and HRP-conjugated 239.3 for detection. *P < .05 with respect to all other treatments. (B) Studies similar to those in panel A were conducted, only PPP was incubated with 0-20 μg/mL of FLAG-tagged VWFpp for 10 minutes before addition of the mixture to wells bearing anti-FLAG mAb (clone M2). Bound plasma VWF was measured using HRP-conjugated polyclonal anti-VWF Ab. Wells without mAb M2 serve as a negative control. VWF-VWFpp binding in this assay could be blocked by 50 μg/mL of DD3.1, but not by control mAb DD3.3 (panel A, panel B inset).