Delayed onset heparin-associated thrombocytopenia (HAT) is thought to be a result of formation of antiplatelet antibodies which cause platelet aggregation in the presence of heparin. Platelet aggregation in response to serum from patients with HAT has been studied in platelet-rich plasma (PRP) from a panel of normal blood donors. Heparin-dependent aggregation with any HAT serum occurred in PRP from only some donors. PRP from the non-responding donors did, however, aggregate in the presence of heparin with other HAT sera. The same patterns of aggregation or lack of response to HAT sera were seen in washed platelet suspensions. Heparin (0.06-2 U/ml) did not cause aggregation in the presence of normal serum with PRP from these donors. However, in PRP from four of the 17 individuals studied, heparin (0.25-1 U/ml) alone caused rapid platelet aggregation and some HAT sera heated at 56 degrees C caused platelet aggregation without added heparin. Sub-aggregating concentrations of adrenaline could replace heparin in promoting aggregation by heated HAT sera in PRP of the other donors. HAT IgG showed the same platelet specificities as the serum in causing either heparin- or adrenaline-dependent aggregation. Thus in HAT, antibodies are directed towards different platelet antigens which are expressed differently in different individuals. Platelet activation by heparin and adrenaline either exposes these antigens or causes aggregation of antibody-coated platelets.
Previously described platelet-aggregating antibodies associated with thrombosis and thrombocytopenia required heparin for their in vivo and in vitro expression. We have observed a patient with thrombosis who became thrombocytopenic during heparin treatment, but who suffered further thrombotic events and continued thrombocytopenia for 3 months after heparin withdrawal. The patient's plasma contained a potent platelet aggregating factor reactive with both his own and normal platelets in the absence of heparin. It also caused [14C]serotonin secretion from labelled platelets from normal donors and patients with either Glanzmann's thrombasthenia or Bernard-Soulier syndrome. This factor was an IgG and was neutralized by antibody specific for IgG lambda light chains. While the patient was thrombocytopenic an IgG paraprotein with lambda light chains was detected by isoelectrofocussing. After corticosteroid treatment it disappeared and the patient recovered. The active, but not the recovery serum contained IgG which immunoprecipitated a glycoprotein with characteristics of Glycoprotein IV from platelets labelled with Na[3H]BH4/periodate. Thus platelet-aggregating IgG antibodies with direct specificity for platelet surface glycoproteins may be associated with thrombosis/thrombocytopenia.
The relationship between platelet-associated IgG (PA-IgG) of intact platelets to that of lysed platelets has been studied using a competitive ELISA. In platelets from 20 normal individuals mean surface PA-IgG constituted 35% of mean total PA-IgG and showed a significant linear relationship with total PA-IgG. In platelets from 61 patients with various forms of thrombocytopenia including that with immune causes, 38 showed a similar proportion of PA-IgG on the surface after storage at 2 degrees C, as seen in normal platelets, while in 23, levels of surface and total PA-IgG were equal. Normal platelets activated with thrombin or calcium ionophore A23187, also had levels of surface PA-IgG close to total PA-IgG. Supernatants of platelet suspensions activated with aggregating agents contained increased amounts of IgG and when the platelets were washed, both total and surface PA-IgG were decreased. Liberation of IgG from platelets was slower than that of [14C]serotonin but was decreased by release reaction inhibitors. Platelets treated at 2 degrees C with soluble heat-aggregated IgG, which binds to the Fc gamma receptor, showed increased surface PA-IgG, but, after incubation at 37 degrees C, although [14C]serotonin was released, PA-IgG levels were no longer increased. Thus since platelet activation, including that mediated by IgG binding to the Fc gamma receptor, causes PA-IgG release, levels of both surface-located and total PA-IgG may be affected by platelet activation, either in vivo, or in vitro during sample preparation and assay.
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