SPR analysis of vWF interaction with rSPA-coupled CM5 sensor surfaces (solid lines) or uncoupled surfaces (dashed lines).

(A) After instrument equilibration, vWF (250 μg/mL in HEPES buffer) was injected (➁) followed by HEPES buffer (➀) and surface regeneration with glycine buffer (③) as described in “Materials and methods,” and RU was determined. (inset) Response measured as a function of time using different vWF concentrations (250/300/350/400/450/500 μg vWF/mL, lower through upper line) calculated by subtracting RU_vWF-CM5-RU_CM5. (B) Response curves after the injection of HSA (upper 2 lines) and BSA (lower 2 lines) followed by the injection of HEPES buffer. (C) Response after 4 injections of pooled human IgG (1060 nmol/L) (➁) followed by the injection of vWF (250 μg/mL) (➀). After regeneration of the surfaces, vWF (250 μg/mL) was injected (③), and surfaces were equilibrated and regenerated as in A. (D) Interaction of vWF and V8 protease-treated vWF with SPA. After immobilization of SPA (9300 RU), either vWF (100 μg/mL, 30 μL) (➁) or vWF digested with protease V8 (4 IU, 30 minutes at 37°C) (➀) was injected. After binding, surfaces were regenerated as described in A. Shown is the difference plot of signal obtained on [RU_vWF-CM5 − RU_CM5].