CLEC-2 internalization depends on SFK activity. (A) Wt and FcRγ^−/− mice were pretreated with Dasatinib (5 mg/kg) for 1 h followed by IV injection of 100 µg of INU1. Platelet counts were determined 2 hours after injection on a FACSCalibur. Results are mean ± SD in percentage of starting values (n = 5 mice per group). (B) Flow cytometric analysis of INU1-injected Dasatinib-treated Wt and FcRγ^−/− platelets 2 hours after injection. Displayed are the FSC and SSC as well as the αIIbβ3 activation and degranulation-dependent P-selectin exposure (Thr, 0.1 U/mL). (C) Two hours after INU1 injection, washed blood of Dasatinib-treated Wt and FcRγ^−/− mice was incubated for 15 minutes with an α-rat Ig-FITC antibody 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. (D) Wt and (E) Syk^−/− platelets were in vitro incubated for 15 minutes with 25 µM PP3 (negative control) or PP2 followed by an incubation with 20 µg/mL INU1-F488 for 15 minutes. Thereafter, platelets were stained and imaged as described in Figure 3F. (F) Wt platelets were in vitro incubated for 5 minutes with eptifibatide (40 µg/mL) and 20 µg/mL INU1 antibody. Subsequently, platelets were lysed at the indicated time points. Western blot analysis was performed with an antibody recognizing the intracellular N-terminal region of CLEC-2. β-actin served as a loading control. Results are representative of 3 individual experiments.