Characterization of a murine immunization model of anti-PF4/heparin. (A) Antibody formation. Euthymic BALB/c mice were injected intravenously with antigens (mP+H, mPF4, heparin, or buffer) as described in “Study design.” Antibodies to the immunizing antigen were measured by ELISA 14 days later. Horizontal bars indicate the mean. (B) Temporal course of antibody formation. Mice injected as with mP+H (by intravenous or intraperitoneal route) or buffer (controls) were followed for about 90 days from the onset of immunization. Error bars indicate standard deviation. (C) Antigen specificity of anti-mP+H. Mice developing high-titer antibody responses after intravenous or intraperitoneal injection (IV P+H no. 2, IV P+H no. 0, and IP P+H no. 2) were tested for antigen specificity toward mPF4, mPF4/heparin, mPF4/heparin in the presence of excess heparin and to rat albumin. (D) Heparin-dependent platelet activation by anti-mPF4/heparin antibody. Murine plasma containing PF4/heparin autoantibody (anti-mP+H) or control plasma (con) in the presence of low-dose (0.02 U/mL; Lo Hep) or high-dose heparin (20 U/mL; Hi Hep) was incubated with WT platelets lacking FcγRIIA or platelets from transgenic mice expressing human FcγRIIA (FcγRIIA⁺). Phorbol 12-myristate 13-acetate (PMA) was used as a positive control. Platelet activation was measured by expression of annexin V binding as described in “Study design.”