Blood Journal
Leading the way in experimental and clinical research in hematology

Dynamic antibody binding properties in the pathogenesis of HIT

  1. Bruce S. Sachais1,*,
  2. Rustem I. Litvinov2,
  3. Serge V. Yarovoi1,
  4. Lubica Rauova3,
  5. Jillian L. Hinds1,
  6. Ann H. Rux1,
  7. Gowthami M. Arepally4,
  8. Mortimer Poncz3,
  9. Adam Cuker5,
  10. John W. Weisel2, and
  11. Douglas B. Cines1
  1. 1 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States;
  2. 2 Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States;
  3. 3 Department of Pediatrics, Children's Hospital of Philadelphia and Perelman School of Medicine, Philadelphia, Pennsylvania, United States;
  4. 4 Division of Hematology, Duke University Medical Center, Durham, North Carolina, United States;
  5. 5 Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
  1. * Corresponding author; email: sachais{at}mail.med.upenn.edu

Abstract

Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect anti-platelet factor 4 (PF4)/heparin antibodies is hindered by identification of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, while RTO does not. KKO binding to PF4/heparin is specifically inhibited by human HIT-antibodies that activate platelets, while inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but did not increase total-binding or binding to non-tetrameric PF4K50E. Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive towards PF4 tetramers and formed stronger complexes than RTO, but not to PF4K50E dimers. KKO, but not RTO, promoted oligomerization of PF4, but not PF4K50E. These studies reveal differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT.

  • Submitted January 27, 2012.
  • Accepted April 23, 2012.