Fig. 5. Divalent and tetravalent nanobodies activate human CLEC-2 in mouse platelets.

Humanised CLEC-2 mouse (hCLEC1b^KI) platelet aggregation was monitored by light transmission aggregometry at 37°C with constant stirring at 1200 rpm for 10 min. Representative traces of washed hCLEC-2^KI platelet aggregation (2 × 10⁸ platelets/ml) induced by (a) LUAS-2 (1-100 nM) and (b) LUAS-4 (1-10 nM). c Percentage of maximal platelet aggregation induced by LUAS-2 and LUAS-4 (n = 4–6 biologically independent experiments). Representative traces of washed hCLEC-2^KI platelet aggregation (2 × 10⁸ platelets/ml) induced by (d) LUAS-2 (100 nM) and (e) LUAS-4 (10 nM) in the absence (vehicle) or presence of Syk inhibitor BI1002494 (BI) (500 nM) and AYP1 Fab (208 nM). Platelets were preincubated with inhibitors for 5 min at 37 °C prior to stimulation. f Percentage of platelet maximal aggregation induced by LUAS-2 and LUAS-4 in the presence of BI or AYP1 Fab (n = 5–6 biologically independent experiments). Significance was measured with a one-way ANOVA with Bonferroni post-hoc for where P ≤ 0.05. Data are presented as mean ± SD.