Sera of 34 patients with heparin-associated thrombocytopenia (HAT), giving a positive result in the serotonin release assay (SRA), were assessed in a platelet factor 4 (PF4)/heparin ELISA. Three sera revealing indeterminate results in the SRA and 10 control sera were also investigated. Both tests correlated closely (Kappa 0.742; p = 2.67 x 10(-7)), but one positive serum in the SRA was negative in the pF4/heparin ELISA. We have isolated the HAT antibodies by absorbtion and elution of HAT sera using endothelial cells (HUVEC). Eluates gave similar results as the sera in the PF4/heparin ELISA (Kappa 0.837, p = 9.26 x 10(-9)), and they also correlated very closely with the SRA (Kappa 0.888; p = 8.89 x 10(-10)). This demonstrates that HAT antibodies bind to the same epitope on platelets and on endothelial cells. High heparin concentrations released PF4 in a dose dependent manner from microtiter plates if PF4/heparin, but not if PF4 alone, was covalently linked. Concomitant to the release of PF4, binding of HAT antibodies to PF4/heparin decreased, as indicated by the median optical density (OD) values of OD 0.88 in the presence of buffer compared to OD 0.181 in the presence of 100 IU/ml heparin. The latter values were similar to those obtained when plates were coated with PF4 alone (median OD 0.203). Binding of three eluates was not inhibited by high heparin concentrations and they reacted also with PF4 alone. We conclude that multimolecular PF4/heparin complexes represent the major antigen in HAT. These multimolecular complexes might present several epitopes and form immune complexes after HAT antibody binding which activate platelets via the FcRII. In a few cases, PF4 alone can be recognized by the antibody. However, there is also evidence that other molecules might be involved in some patients.