Effect of bivalent Nanobody format on VWF binding. (A) Surface plasmon resonance analysis with a Biacore sensor chip coupled with plasma-purified VWF. Binding and dissociation of 100nM PMP12A2h1 (gray dashed line) and ALX-0081 (black solid line) are shown. (B) Surface plasmon resonance analysis with a Biacore sensor chip coupled with recombinant VWF A1 domain. Binding and dissociation of 1nM PMP12A2h1 (gray dashed line), mock-PMP12A2h1 (gray solid line), PMP12A2h1-mock (black dashed line), and ALX-0081 (black solid line) are shown. Representative of 3 independent experiments. (C) Inhibition of VWF binding to human formalin-fixed platelets: formalin-fixed platelets were bound to poly-L-lysine–coated microtiter plates after which VWF was added in the presence of 1.5 mg/mL ristocetin and different amounts of ALX-0081 (●) or PMP12A2h1 (□). Residual bound VWF was detected colorimetrically. Optical density (OD) measured at 490 nm is shown in function of the Nanobody concentration (mean ± SEM, n = 21).