INU1-induced thrombocytopenia depends on platelet CLEC-2 expression. (A) Wt and Clec2^−/− mice were IV injected with 100 µg of INU1 and platelet counts were determined on a FACSCalibur at the indicated time points. Results are mean ± SD in percentage of the initial platelet counts (n = 5 mice per group). (B) Western blot analysis of CLEC-2 levels, before and on day 5 post-INU1 injection in platelet lysates of Wt and Clec2^−/− mice. GPIIIa served as a loading control. (C) Flow cytometric analysis of αIIbβ3 activation (JON/A-PE) and degranulation-dependent P-selectin exposure on platelets on day 5 post-INU1 injection. Washed blood was incubated with the indicated agonist for 15 minutes and analyzed on a FACSCalibur (RC, 0.12 µg/mL; thrombin (Thr), 0.1 U/mL). Results are representative of 3 individual experiments. FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; PE, phycoerythrin; SD, standard deviation.