Syk is required for INU1-induced thrombocytopenia. (A) Flow cytometric analysis of naive Wt and Syk^−/− platelets incubated with various agonists. Displayed are the FSC and SSC characteristics as well as integrin αIIbβ3 activation (JON/A-PE) and degranulation-dependent P-selectin exposure (RC, 0.12 µg/mL; Thr, 0.1 U/mL). (B) Wt and Syk^−/− 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). (C) Flow cytometric analysis of control and INU1-treated Syk^−/− platelets 1 hour after injection. Displayed are the FSC and SSC as well as the αIIbβ3 activation and degranulation-dependent P-selectin exposure. (D) Syk^−/− mice were bled 1 hour after INU1 injection and washed blood was incubated with an α-rat Ig-FITC antibody for 15 minutes and subsequently analyzed on a FACSCalibur. As in vitro control, untreated mice were bled, blood was incubated with 20 µg/mL INU1 for 15 minutes, washed, and incubated with an α-rat Ig-FITC antibody for 15 minutes. (E) Western blot analysis of CLEC-2 levels at the indicated time points post-INU1 injection in platelets lysates of Syk^−/− mice. GPIIIa served as a loading control. (F) Syk^−/− mice were injected with 100 µg of INU1-F488 antibody, 15 minutes after injection platelets were isolated, allowed to adhere to PLL-coated cover slips, fixed and stained with α-rat IgG-Cy3 under permeabilizing or nonpermeabilizing conditions, and stained subsequently with PhalF647 diluted in permeabilizing buffer. Samples were visualized using a Leica TCS SP5 confocal microscope equipped with a 100×/1.4 oil objective. Scale bar represents 1 µm. Results are representative of 3 individual experiments.