hCLEC-2 can be immunodepleted using HEL1 or AYP1, with little effect on hemostasis. (A) Platelet count following intraperitoneal injection of INU1, AYP1, or HEL1 antibody (3 μg/g bodyweight). Transient thrombocytopenia lasting up to 4 days after injection can be seen for all antibody-treated groups compared with the untreated controls. Platelet count was determined by flow cytometry and is shown as the percentage of the baseline count. (B) hCLEC-2 surface expression determined by flow cytometry following depletion by either AYP1 or HEL1. For both antibodies, CLEC-2 could not be detected on the platelet surface for at least 11 days. (C) mCLEC-2 surface expression determined by flow cytometry following depletion by INU1 shows that CLEC-2 was absent for at least 7 days after injection. Measurements with anti-rat and anti-mouse IgG excluded the possibility that the abolished anti-CLEC-2-FITC binding was due to the presence of remaining anti-CLEC-2 antibodies on the platelets (supplemental Figure 8). (D) Depletion of CLEC-2 (using HEL1) had no effect on tail bleeding time. P > .05, Fisher exact test. Horizontal lines represent the median time to cessation of bleeding, with each circle representing 1 mouse. (E) Depletion of CLEC-2 had no effect on occlusive thrombus formation following mechanical injury of the abdominal aorta, and hCLEC-2^KI mice were comparable to WT. P > .05, Fisher exact test. Horizontal lines represent the median time to vessel occlusion, with each circle representing 1 mouse. For all experiments, a minimum of 5 mice were tested per group. FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; MFI, mean fluorescent intensity.