Determinants of PF4/heparin immunogenicity

Abstract

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that occurs with variable frequency in patients exposed to heparin. HIT antibodies preferentially recognize large macromolecular complexes formed between PF4 and heparin over a narrow range of molar ratios, but the biophysical properties of complexes that initiate antibody production are unknown. To identify structural determinants underlying PF4/heparin immunogenicity, we characterized the in vitro interactions of murine PF4 (mPF4) and heparin with respect to light absorption, size, and surface charge (zeta potential). We show that PF4/heparin macromolecular assembly occurs through colloidal interactions, wherein heparin facilitates the growth of complexes through charge neutralization. The size of PF4/heparin macromolecules is governed by the molar ratios of the reactants. Maximal complex size occurs at molar ratios of PF4/heparin at which surface charge is neutral. When mice are immunized with complexes that differ in size and/or zeta potential, antibody formation varies inversely with heparin concentration and is most robust in animals immunized with complexes displaying a net positive zeta-potential. These studies suggest that the clinical heterogeneity in the HIT immune response may be due in part to requirements for specific biophysical parameters of the PF4/heparin complexes that occur in settings of intense platelet activation and PF4 release.

Figure 1

PF4 and heparin form visible aggregates in a heparin-dependent manner. Solutions containing mPF4 (10-200 μg/mL) were incubated with increasing concentrations of UFH (0-50 U/mL, shown on a log x-axis scale). Optical absorbance was measured at 280_nm (y-axis). Encircled UFH concentrations (0.5, 5, and 50 U/mL) and mPF4 at a concentration 200 μg/mL were chosen for further study in subsequent experiments. The inset shows changes in absorbance as a function of PF4 concentration.

Figure 2

Measurement of PF4/heparin particle size and particle stability over time. (A) PCS measurements were taken of solutions containing mPF4 (200 μg/mL) before and after addition of various concentrations of UFH (0.5, 5, and 50 U/mL). Particle size was largest upon addition of 5 U/mL UFH (PHR, 2.6:1) and smallest with 50 U/mL UFH (PHR, 1:5). (B) Evolution of PF4/heparin particle size over time. UFH (0.5, 5, or 50 U/mL) was added to 200 μg/mL PF4 at time 0, and particle size was measured every 8 minutes over the ensuing 2 to 3 hours until particle size stabilized. Serial measurements of particle sizes are shown.

Figure 3

Light microscopy of PF4/heparin particles. Panels showing solutions containing mPF4 (200 μg/mL) alone (A) or mixed with UFH at 0.5 U/mL (B), 5 U/mL (C), or 50 U/mL (D) and subjected to light microscopy using highly collimated oblique illumination. Microscopic images were obtained using a 10 × magnification with an Olympus microscope (model no. BX41; Olympus America). Images were captured using a Microfibre Optronics camera (Goleta, CA) and analyzed with Picture frame Optronics software.

Figure 4

Heparin-dependent changes in the zeta potential of PF4/heparin particles. The zeta potential of particles formed by solutions of mPF4 (10–200 μg/mL, individual graphs along y-axis) and increasing concentrations of heparin (0.1-50 U/mL along x-axis; log scale).

Figure 5

Effect of heparin on the development of anti-mPF4/heparin antibodies in vivo. BL6 mice were injected with mPF4 and UFH at various concentrations and antibodies to mPF4/heparin (mP + H) were assayed at baseline or D8 from the start of immunizations. (A) Cohorts (n = 10/cohort) were injected with mPF4 (200 μg/mL) alone or in combination with heparin at various PHRs (PHRs from left to right after PF4 alone, 26:1, 2.6:1, and 1:5). (B) Cohorts (n = 7/cohort for PF4 alone, and UFH 50 U/mL; n = 10/cohort for other conditions) were injected with mPF4 (100 μg/mL) alone or in combination with heparin at various PHRs (PHRs from left to right after PF4 alone, 20:1, 10:1, 1:1, and 1:5). Significant differences between cohorts are indicated at the top of the figures.

Figure 6

Zeta potential depiction of PF4 and heparin colloidal interactions. Y-axis represents positive or negative electrostatic potential as a function of increasing concentrations of UFH (on x-axis). (A) Positively charged PF4 molecules remain stably dispersed in the absence of heparin due to repulsive interactions. (B) With increasing amounts of heparin, PF4 charges are neutralized promoting floc or macromolecular assembly. (C) With heparin in molar excess, repulsive interactions once again predominate, leading to stable dispersion of smaller particles.