Optimization of a murine immunization model for study of PF4/heparin antibodies

Abstract

Summary background: Heparin-induced thrombocytopenia (HIT) is a life-threatening thrombotic illness caused by drug-dependent antibodies recognizing complexes of platelet factor 4 (PF4) and heparin. Little is known about the immune pathogenesis of HIT, in particular factors influencing PF4/heparin antibody formation. To gain insight into the biologic basis of heparin sensitization, we have recently developed an animal model using wild-type (WT) mice in which murine PF4/heparin antibodies (anti-mPF4/H) arise de novo after antigen challenge.

Objectives and methods: This report describes technical refinements to the murine model and describes additional biologic features of the immune response to mPF4/heparin.

Results: Our studies indicate that antibody responses to mPF4/heparin are dependent on murine strain, injection routes and doses of mPF4 and heparin. C57BL/6 mice are more immunologically responsive to mPF4/heparin antigen than BALB/c mice and robust immunization can be achieved with intravenous, but not intraperitoneal, administration of antigen. We also observe a direct relationship between initial concentrations of mPF4 and antibody levels. Additionally, we demonstrate that mPF4/H immune response in mice decays with time, is not associated with thrombocytopenia and displays characteristics of immune recall on re-exposure to antigen.

Conclusions: These studies describe and characterize a murine model for studying the immunologic basis of PF4/heparin sensitization.

Figure 1. Strain dependent difference in the anti-mPF4/H response

BALB/c and C57BL/6 mice were injected with mPF4/H and monitored for anti-mPF4/H at D15. Individual measurements are shown as well as mean (bar). C57BL/6 not only showed higher rates of seroconversion, but also greater levels of anti-mPF4/H (mean anti-mPF4/H, p<0.0001).

Figure 2. Time course of anti-mPF4/H formation in BALB/c mice v C57BL/6

(A) BALB/c or (B) C57BL/6 mice were immunized with mPF4/H and anti-mPF4/H responses were followed over two to three months. Time course for individual mice within each cohort are shown by each line.

Figure 3. Effects of immunization route and time course of antibody responses

C57BL/6 mice were immunized intravenously {via retro-orbital (RO, n=10) or tail vein sites (TV, n=10)} or intraperitoneally (IP, n=10) with mPF4/H. Anti-mPF4/H levels were monitored at D15 for all mice (A). Antibody levels were followed over 2 ½ months for immunized mice injected by RO (B), TV (C) or IP routes (D) to document any delayed seroconversions.

Figure 4. Duration of antigen exposure

C57BL/6 mice were immunized intravenously (RO) with mPF4/H daily for 5, 4 or 1 day as shown. Anti-mPF4/H levels were monitored at D15 for all mice. Significant differences between cohorts are indicated at the top of the figure.

Figure 5. Effects of PF4:UFH dose on anti-mPF4/H responses

C57BL/6 mice were immunized intravenously (RO) with mPF4/H daily for 5 days with varying doses of PF4 and heparin adjusted to maintain a constant molar ratio of PF4:UFH of 2.6:1(n=5/cohort).

Figure 6. Effects of immunization on platelet counts

C57BL/6 mice were immunized intravenously with mPF4/H (□, n=10) or buffer (○, n=5). Platelet counts were checked at baseline (“Baseline”) or 8 days after immunization (“Day 8”). On D15, all mice (mPF4/H and buffer injected mice) were given one dose of heparin and platelet counts were checked 4 days after heparin administration on D19 (“4 days after heparin”). Mean platelet counts are indicated by line for each respective cohort and time point. Mice were also concurrently monitored for anti-mPF4/H development. Seropositive mice expressing antibody levels A_450nm ≥0.45 are indicated by solid symbols at D8 and D15.

Figure 7. Presence of immune recall in PF4/heparin sensitized mice

PF4/heparin sensitized C57BL/6 mice were immunized with mPF4/H and followed over time until antibody levels decreased to 2-3 fold over baseline values. Sensitized mice (n=5) were given one dose of mPF4/H on day 73 and seroconversion was monitored at day 8 and day 15 post re-immunization.