PF4/heparin complexes are T cell-dependent antigens

Abstract

Heparin-induced thrombocytopenia (HIT) is a life-threatening, thrombotic disorder associated with development of anti-platelet factor 4 (anti-PF4)/heparin autoantibodies. Little is known about the antigenic and cellular requirements that initiate the immune response to these complexes. To begin to delineate mechanisms of autoantibody formation in HIT, we studied the immunizing effects of murine PF4 (mPF4)/heparin in mice with and without thymic function. Euthymic mice were injected with mPF4/heparin complexes, mPF4, heparin, or buffer. Mice injected with mPF4/heparin, but not mPF4 or heparin alone, developed heparin-dependent autoantibodies that shared serologic and functional characteristics of human HIT antibodies, including preferential binding to mPF4/heparin complexes and causing heparin- and FcRgammaIIA-dependent platelet activation. In contrast, athymic mice did not develop HIT-like antibodies. Taken together, these studies establish that PF4/heparin complexes are highly immunogenic and elicit self-reacting anti-PF4/heparin antibodies in a T cell-dependent manner.

Figure 1.

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.”

Figure 2.

T-cell requirement for anti-mPF4/heparin antibody development. (A) ELISA measurements (A_450nm) of antibody development 14 days after inoculation of either mPF4/heparin (mP+H) or DNP-Ficoll into either euthymic BALB/c WT mice or BALB/c athymic (nu/nu) mice. Individual measurements are shown as well as mean (bar). Statistically different values are indicated at the top. (B) Antibody measurements of nu/nu and WT mice injected with mP+H 42 days after inoculation.