Characterization of platelet activation induced by HLA antibodies associated with alloimmune thrombocytopenia.

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.

Human platelet activating antibodies.

Platelet antibodies identified in the plasma of three multiply transfused patients and a woman who had delivered a baby with neonatal alloimmune thrombocytopenia were investigated for their platelet activating properties. Three patients possessed multispecific HLA antibodies reactive with 90 to 100% of the cells on a lymphocytotoxic panel. These antibodies were also detected using the MAIPA assay and MAb w6/32, which recognizes an epitope common to all HLA class I molecules. In addition to HLA antibodies, three of the patients possessed platelet-specific antibodies that were identified by the MAIPA assay as anti-HPA-1a and anti-HPA-3a (one patient) and anti-HPA-1b (two patients). Each of the HLA antibodies when reacted with platelets expressing the corresponding HLA antigens, potently induced aggregation and release of ATP from dense granules. In contrast, the HPA-1b antibodies induced platelet agglutination, but failed to trigger ATP release. However, platelets coated with these latter antibodies were now refractory to subsequent stimulation by ADP. Similarly, when HLA antibodies were reacted with platelets to produce suboptimal activation, the platelets could now be stimulated only poorly or not at all by either epinephrine or thrombin. This was also true for anti-HPA-1b, which, although not inducing aggregation or ATP release by itself, was capable of almost completely blocking thrombin-induced platelet activation. The thrombin-inhibiting activity of these antibodies could partially be reversed by pretreating antibody-coated platelets with epinephrine immediately followed by stimulation with thrombin. These findings suggest that transfused platelets may either be activated or inhibited by reaction with various platelet antibodies. Therefore it is conceivable that the presence of platelet reactive antibodies in multiply transfused recipients may contribute to the increased thrombotic and hemorrhagic symptoms often observed among these patients.

Cefotetan-induced immunologic thrombocytopenia.

Profound thrombocytopenia accompanied by a severe coagulopathy developed in an elderly female patient being treated with cefotetan while undergoing surgery for closure of a perforated gastric ulcer. During the acute phase of the bleeding diathesis, the patient had a platelet count of 12 x 10(9)/l, a prothrombin time of 88 s (normal 10.0-11.8 s) and a PTT of 105 s (normal 23.0-37.0 s). Potent IgG cefotetan-dependent anti-platelet antibodies, which also were weakly reactive with ampicillin, were detected in the patient's serum using immunofluorescence and a recently developed protein A-agarose rosette forming assay. Unlike typical cephalosporin- and penicillin-induced antibodies that react with cells pretreated with drug, this antibody only reacted with platelets in the presence of exogenous drug. Failure of the antibody to react with drug-coated platelets suggests the possibility that, in this patient, sensitization to cefotetan involved mechanisms other than formation of typical hapten-carrier complexes normally described for members of the cephalosporin family of antibiotics. This appears to be the first definitive report that cefotetan, or any other cephalosporin derivative, can induce immunologic thrombocytopenia.

Antigenic differences among bovine and porcine Factor VIII concentrates.

Three anti-Factor-VIII antibodies from hemophiliacs were reacted with samples of batches of Maws commerical bovine and porcine Factor VIII concentrates manufactured over a 12-year period. The apparent antibody concentrations varied widely with the different batches of concentrates. The variations are probably due to intrinsic differences in the antigenic nature of the Factor VIII in the different preparations. With the older porcine concentrates, the low apparent concentrations may be in part due to the reaction of antibody with inactive Factor VIII. When animal Factor VIII concentrates are used in treating hemophiliac patients who have anti-Factor-VIII antibodies, the least reactive batch should be chosen. Random batches of animal concentrates are not suited as a standard for measuring anti-Factor-VIII antibody concentration.