Rapid thrombocytopenia induced by TLR7 stimulation is caused by granulocyte (neutrophil) internalization of platelets and continued leukocyte aggregate formation. (A) Confocal microscopy of murine blood postinjection with Loxo. Mice were transfused with CFSE-labeled platelets; blood was drawn, fixed, and stained with 4,6 diamidino-2-phenylindole (DAPI). (B) Confocal microscopy of human blood prestained with CD41-FITC and CD45-CY5 (CD41-green stains are platelets and CD45-red stains are leukocytes) and stimulated with Loxo. DAPI (blue) stains the nucleus and was added postfixation. Cells are identified as neutrophils according to their lobularity and intensity of CD45. (C) Internalization coefficient measured by IDEAS-software (Amnis FlowSight Cytometry) in human blood. In these studies, CD45 was used to define the outside of the neutrophils, and CD41 is the internalization probe (n = 4). (D-E) Confocal images of human or murine blood showing large aggregates of platelets and WBCs. (D) Murine blood of mice transfused with CFSE-labeled platelets and injected with Loxo, fixed, and stained with DAPI. (E) Human blood treated ex vivo with Loxo (15 minutes) and stained with CD41 (green)-platelets, CD45 (red)-leukocytes, or DAPI (blue)-nucleus. In all cases, pictures are representative of: n = 3 (A), n = 5 (B); n = 4 (C), n = 3 (D), and n = 5 (E). Data in C are average ± SD and were analyzed by Student t test. Images in A-B and D-E were taken with spinning disk confocal microscope and Metamorph 7.4.2 software and merged by ImageJ (NIH) software. Pictures were taken with ×100 Plan Apo oil lens and the scale bar is 10 μm.