Testing of Nb binding and inhibition of GPVI signaling. (A) Surface binding assay of 1 Nb (100 nM) from each binding class to a GPVI-Fc–coated surface. All binding results have been normalized to Nb35, which gave the highest readout. Binding of each Nb to the Fc domain-coated surface was tested and subtracted from the GPVI-Fc readings. Binding was detected using HRP-conjugated anti-His antibody. The average binding of all the nanobodies to bovine serum albumin represents a nonspecific binding control. Data represent mean values of 3 experiments ± SD. (B) NFAT reporter assay of GPVI- and FcRγ-transfected DT40 cells stimulated by collagen (10 µg/mL) in the presence of the Nbs (100 nM). Results are plotted as a percentage of total signaling in the presence of collagen only. Dotted lines represent 100%, 50%, and 20% signaling levels, and nb2, 21, and 35 are colored in black, red, and orange, respectively. Data represent mean values of 3 experiments performed in triplicate ± SD.