The platelet-activating properties of plasma containing multispecific HLA antibodies were studied in plasma from several persons stimulated by platelet or red blood cell transfusion or by pregnancy. Antibodies were characterized by their lymphocytotoxic effects and by a monoclonal antibody antigen--capture enzyme-linked immunoassay. Plasma from each patient induced dose-dependent aggregation and release of adenosine triphosphate with antigen-positive platelet-rich plasma. Plasma from three patients induced normal aggregation and adenosine triphosphate release with platelet-rich plasma from donors who had taken platelet inhibiting medications (e.g. aspirin, piroxicam). In contrast, these platelets failed to release adenosine triphosphate when stimulated with 5 mumol/L adenosine diphosphate. Platelets were fully activated when saturated with HLA antibodies from one patient, although little or no stimulation was observed at 50% saturation suggesting that additional plasma cofactors and/or a threshold of bound antibody were required for activation. With a murine monoclonal antibody specific for P-selectin and antimurine immunoglobulin G labeled with iodine 125, plasma from each patient was found to induce P-selectin expression that was approximately 50% of that induced by 0.2 U/ml thrombin. Expression on platelets of P-selectin induced by plasma from one patient was independent of whether the donor had taken aspirin. Purified immunoglobulin G from this same patient stimulated platelet activation, as did the patient's serum, but no activation was observed with F(ab')2 fragments prepared from the immunoglobulin G. These studies demonstrate that HLA antibodies (1) mediate expression of P-selectin on the platelet surface, (2) are as potent as thrombin in activating platelets previously exposed to antiinflammatory agents, and (3) require an intact Fc domain to activate platelets.