The Y7A point mutation in the hemITAM motif of CLEC-2 abolishes platelet responses to CLEC-2 agonists and reproduces the lymphatic defects seen in CLEC-2–deficient mice. (A) Embryos from time-mated Y7A knockin (KI) heterozygous crosses at E12.5 and E14.5 show hemorrhages in the brain and edema and lymphatic blood-filling in the Y7A KI embryos, respectively. Representative pictures of wild-type and Y7A KI embryos from 5 different litters at each embryo stage. Original magnifications ×3.0 (upper panels [E12.5]) and ×1.8 (lower panels [E14.5]). (B) Adult mice transplanted with Y7A KI fetal liver cells after irradiation developed blood-filling of the lymphatics. Representative images of the mesenteric blood and lymphatic vessels are shown. Original magnification ×25. (C) CLEC-2 surface expression was maintained on Y7A KI platelets at levels comparable with wild-type platelets, as measured by flow cytometry. There was a small but significant reduction in the CLEC-2 surface levels in Y7A KI platelets (*P < .05), however, this was due to the anti–CLEC-2 antibody used (fluorescein isothiocyanate–conjugated INU1 immunoglobulin G) activating wild-type¹  but not Y7A KI platelets during the staining process, thus releasing a small intracellular pool of CLEC-2 to the platelet surface in wild-type platelets only. The results are presented as means with standard deviations, n = 20. (D) Western blotting of platelet lysates from wild-type and Y7A KI chimeric mice showed no difference in CLEC-2 protein levels or in the levels of the major hemITAM signaling proteins Syk, Lyn, Fyn, and PLCγ2. Blot represents 2 independent experiments. (E) Platelet integrin activation, measured as JON/A phycoerythrin antibody binding by flow cytometry, showed no differences in responses to the indicated agonists between wild-type and Y7A KI platelets from chimeric mice. (F) Y7A KI platelets showed no activation in response to rhodocytin, which could not be overcome by the exogenous addition of the secondary mediator agonist adenosine 5′-diphosphate (ADP) and the thromboxane mimetic U46619. Flow cytometry activation data are presented as means with error bars of standard deviations and are representative of 3 independent experiments, n = 4. (G) Washed platelet aggregometry experiments showed the complete loss of aggregation responses specifically to the CLEC-2 agonist rhodocytin in Y7A KI platelets (blue). Traces are representative of responses from n = 4 animals. (H) Pan-phosphotyrosine and phospho-specific western blots showed no induction of the hemITAM signaling cascade in CLEC-2 Y7A KI platelets after stimulation with 1 µg/ml rhodocytin. Representative western blots of 3 independent experiments. (I) There were no significant differences between the number of megakaryoctyes present per field of view (FOV) in chimeric wild-type and Y7A KI whole femora cyrosections; n = 3-6, minimum 5 fields of view per mouse. (J) There were no significant differences between chimeric wild-type and Y7A KI platelet counts as measured by flow cytometry. Counts were measured at 5 weeks posttransplantation, n = 12. CRP, collagen-related peptide; GMFI, geometric mean fluorescent intensity; Rhodo, rhodocytin.