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

Background: Heparin-treated patients often develop antibodies, but only a subset cause heparin-induced thrombocytopenia. Results: In a single-molecule assay, a pathogenic monoclonal antibody bound more strongly to cross-linked platelet factor 4 than a non-pathogenic antibody. Conclusion: Oligomerization of platelet factor 4 may enhance binding of pathogenic antibodies. Significance: A molecular basis for specificity of pathogenic antibodies in heparin-induced thrombocytopenia is provided. 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 approximate to 10-fold faster on-rates than RTO-PF4, and apparent equilibrium dissociation constants differed approximate to 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.