Further testing of top inhibitory Nbs of GPVI. (A) Nbs 2, 21, and 35 (5 µM) binding to washed platelets in the presence or absence of 200 μM PAR1 by flow cytometry. Gray histograms show unstained washed platelets, black histograms show nonspecific staining of anti-His alexafluor647 secondary, red histograms show nanobody binding to resting platelets, and green histograms show nanobody binding to PAR1 peptide–activated platelets. (B) Platelet aggregation in response to (i-ii) 5 µg/mL collagen and (iii-iv) 10 µg/mL CRP in the presence of Nb2, 21, and 35. (i, iii) Representative aggregation curves for Nb2. (ii, iv) Maximum aggregation values for all Nbs. IC₅₀ values of 172, 85, and 115 nM for collagen and 1, 22, and 1 nM for CRP were determined for Nb2, 21, and 35, respectively. The effect of different concentrations of the nanobody compared with the vehicle (PBS) was determined using 2-way analysis of variance with Dunnett’s correction for multiple comparisons. (C) Solid-phase binding assay showing GPVI-Fc (100 nM) displacement from a collagen surface in the presence of increasing concentration Nb2, 21, and 35, with IC₅₀ values of 18, 62, and 39 nM, respectively. Data represent mean values of 3 ± SD. (D-E) SPR data showing Nb2 at a range of concentrations binding to (D) GPVI-Fc and (E) GPVI immobilized on a surface. The binding affinity was determined by kinetic analysis with calculated K_D values of 0.7 ± 0.03 nM for GPVI-Fc and 0.58 ± 0.06 nM for GPVI.