Serologic characterization of anti-protamine/heparin and anti-PF4/heparin antibodies

Anti-protamine (PRT)/heparin antibodies are a newly described class of heparin-dependent antibodies occurring in patients exposed to PRT and heparin during cardiac surgery. To understand the biologic significance of anti-PRT/heparin antibodies, we developed a murine monoclonal antibody (ADA) specific for PRT/heparin complexes and compared it to patient-derived anti-PRT/heparin antibodies, as well as comparing polyclonal and monoclonal antibodies with anti-platelet factor 4 (PF4)/heparin. Using monoclonal antibodies and polyclonal patient-derived antibodies, we show distinctive binding patterns of anti-PRT/heparin antibodies as compared with PF4/heparin antibodies. Whereas heparin-induced thrombocytopenia (HIT) antibody binding to PF4/heparin is inhibited by relatively low doses of heparin (0-1 U/mL), anti-PRT/heparin antibodies, including ADA, retain binding to PRT/heparin over a broad range of heparin concentrations (0-50 U/mL). Unlike PF4/heparin antibodies, which recognize PF4 complexed to purified or cell-associated glycosaminoglycans (GAGs), anti-PRT/heparin antibodies show variable binding to cell-associated GAGs. Further, binding of anti-PRT/heparin antibodies to PRT/dextran complexes correlates closely with the ability of antibodies to bind to cell-surface PRT. These findings suggest that antibody binding to PRT/dextran may identify a subset of clinically relevant anti-PRT/heparin antibodies that can bind to cell-surface GAGs. Together, these findings show important serologic differences between HIT and anti-PRT/heparin antibodies, which may account for the variability in disease expression of the two classes of heparin-dependent antibodies.