Activation of VWF by anti-NAIM nanobodies. (A-C) Platelet aggregation traces induced by addition of noted nanobodies. Human platelet-rich plasma was gently stirred, and, at 30 seconds, 6C4 (A), 6C11 (B), or 6D12 (C) was added to noted concentrations spanning from tens of nanomolar to 2 μM. Each concentration was tested in triplicate. Ristocetin was added in separate runs to 1.5 mg/mL, shown in black. Platelets stirred without addition of an agonist is shown in a gray trace. (D) Dose-aggregation curves were plotted for each concentration of each nanobody and fit to a 4-parameter dose-response curve. Percentage aggregation is the inverse of optical densities seen in panels A-C. Error bars are the standard deviation (SD) of the measurements. (E) ELISA binding isotherms of plasma VWF from normal pooled plasma (NPP) to immobilized GPIbα-LBD in the absence or presence of 500 nM noted nanobodies. Data are shown as mean ± SD (n = 3). Some error bars are not visible because they are smaller than the symbols.