Distinct specificity and single-molecule kinetics characterize the interaction of pathogenic and non-pathogenic antibodies against platelet factor 4-heparin complexes with platelet factor 4

J Biol Chem. 2013 Nov 15;288(46):33060-70. doi: 10.1074/jbc.M113.481598. Epub 2013 Oct 4.

Abstract

Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans. However, only a fraction of patients with anti-PF4-heparin antibodies develop HIT, implying that only a subset of these antibodies is pathogenic. The basis for the pathogenic potential of anti-PF4-heparin antibodies remains unclear. To elucidate the intrinsic PF4-binding properties of HIT-like monoclonal antibody (KKO) versus non-pathogenic antibody (RTO) at the single-molecule level, we utilized optical trap-based force spectroscopy to measure the strength and probability of binding of surface-attached antibodies with oligomeric PF4 to simulate interactions on cells. To mimic the effect of heparin in bringing PF4 complexes into proximity, we chemically cross-linked PF4 tetramers using glutaraldehyde. Analysis of the force histograms revealed that KKO-PF4 interactions had ∼10-fold faster on-rates than RTO-PF4, and apparent equilibrium dissociation constants differed ∼10-fold with similar force-free off-rates (k(off) = 0.0031 and 0.0029 s(-1)). Qualitatively similar results were obtained for KKO and RTO interacting with PF4-heparin complexes. In contrast to WT PF4, KKO and RTO showed lower and similar binding probabilities to cross-linked PF4(K50E), which forms few if any oligomers. Thus, formation of stable PF4 polymers results in much stronger interactions with the pathogenic antibody without a significant effect on the binding of the non-pathogenic antibody. These results suggest a fundamental difference in the antigen-binding mechanisms between model pathogenic and non-pathogenic anti-PF4 antibodies that might underlie their distinct pathophysiological behaviors.

Keywords: Antibodies; Heparin-binding Protein; Platelets; Single Molecule Biophysics; Thrombosis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / immunology
  • Antibody Specificity*
  • Anticoagulants / adverse effects
  • Anticoagulants / chemistry*
  • Anticoagulants / immunology
  • Anticoagulants / therapeutic use
  • Autoantibodies / chemistry*
  • Autoantibodies / immunology
  • Binding Sites, Antibody
  • Heparin / adverse effects
  • Heparin / chemistry*
  • Heparin / immunology
  • Heparin / therapeutic use
  • Humans
  • Kinetics
  • Platelet Factor 4 / chemistry*
  • Platelet Factor 4 / immunology
  • Thrombocytopenia / chemically induced
  • Thrombocytopenia / immunology

Substances

  • Antibodies, Monoclonal
  • Anticoagulants
  • Autoantibodies
  • Platelet Factor 4
  • Heparin