A2 blocked A1 exposed on activated VWF. (A,B) An anti-VWF antibody immunoprecipitated (ImP) A2, which was detected by an anti-His antibody in immunoblots (IB) (A) and an anti-His antibody immunoprecipitated VWF (B), which was detected by an anti-VWF antibody in TBI mice receiving A2. (C) A2 was incubated with plasma from TBI and sham mice and from TTP patients and healthy controls (NP, normal plasma) for 60 minutes at 37°C. Plasma VWF was then immunoprecipitated by an anti-His antibody and probed with a polyclonal VWF antibody through IB. As control, A2 was first treated with an equal molar concentration of the isolated A1-domain before incubation with plasma VWF from TBI mice (C, center; A-C, representatives from 6 individual experiments). (D) The binding kinetics of A2 to a recombinant human A1 measured by surface plasmon resonance after the nonspecific background binding was subtracted (n = 15). (E) Plasma VWF treated with 1 mg/mL of ristocetin blocked the anti-A2 antibody from binding to nickle-captured A2 in a dose-dependent manner; untreated plasma VWF did not do the same (n = 12, 1-way ANOVA, *P < .01 vs no ristocetin; #P < .001 vs the lowest concentration of VWF). (F,G) A2 blocked SIPA (F) and VWF:Rco (G) of human platelets in a dose-dependent manner. The GPIbα antibody AK2, which blocked VWF-induced platelet aggregation,⁵¹  was tested as a control for VWF:Rco (G, n = 9, 1-way ANOVA).