Naoaki Sone

Influences of antiplatelet autoantibodies on platelet function in immune thrombocytopenic purpura

We investigated the characteristics of the antiplatelet autoantibodies in 60 patients with ITP. Using flow cytometry, the binding of monoclonal antibodies to the platelet glycoprotein (GP) IIb/IIIa complex and to GPIb was examined in these patients. The extent of binding was decreased in 15 patients (anti‐GPIIb/IIIa in 12 patients and both anti‐GPIIb/IIIa and anti‐GPIb in 3 patients). Western blotting revealed that 10 of these 15 patients had either anti‐GPIIb or anti‐GPIIIa and 2 had anti‐GPIb autoantibodies. ADP‐induced aggregation of normal platelets was inhibited by autoantibodies in 12 of 60 patients, and 11 of these had anti‐GPIIb/IIIa antibodies. Ristocetin‐induced aggregation was inhibited in 4 of these patients, and 2 with prominent inhibition had anti‐GPIb antibodies. There was a significant relationship between platelet‐associated IgG value and ATP secretion. These results suggest that some antiplatelet autoantibodies can affect platelet function and thus have an influence on the pathophysiology of ITP.

Synergistic action in platelet activation induced by an antiplatelet autoantibody in ITP

Summary. We detected an autoantibody which activated normal platelets in a patient with immune thrombocytopenic purpura and investigated the mechanism by which this autoantibody mediated platelet activation. The patients IgG induced platelet aggregation and ATP secretion in normal platelet‐rich plasma (PRP). IgG‐induced aggregation was inhibited by aspirin (ASA), apyrase, a protein kinase C (PKC) inhibitor and two anti‐platelet glycoprotein (GP) IIb/IIIa monoclonal antibodies. The increase of aequorin‐detected intraplatelet Ca2+ induced by the patients IgG was extremely slight. Phosphorylation of a 40 kDa protein was induced by the patients IgG without any obvious phosphorylation of a 20 kDa protein, and was inhibited by a PKC inhibitor but not by ASA. With ASA‐treated normal PRP, the patients IgG failed to induce aggregation itself, but enhanced ADP‐ or STA2‐induced aggregation. Western blotting and immuno‐precipitation experiments showed that the patients IgG reacted to a protein of 36 kDa. These results suggest that the platelet activation induced by this autoantibody depended on both the selective activation of PKC and the slight Ca2+ mobilization induced by thromboxane A2 synthesis, while the aggregation depended on secretion induced by the synergistic action of the above two mechanisms and was mediated through GP IIb/IIIa.

Kinetics of Platelets in Dogs with Thrombocytopenia Induced by Antiglycoprotein IIb/IIIa Receptor Monoclonal Antibody

To experimentally assess the kinetics of platelets in thrombocytopenia, we constructed a canine model using 111In-oxine labeled autologous platelets and an intact antiplatelet monoclonal antibody (MAb) NNKY2-11 (IgG2a). With the infusion of radiolabeled autologous platelets into dogs, the peripheral platelet count and blood radioactivity level were examined, and the radioactivity in the liver, spleen and heart was determined with scintigraphic analysis. Thereafter, i.v. injection of 100 micrograms/kg of NNKY2-11 had no effect on platelet counts or the biodistribution of radiolabeled platelets. However, 200 and 300 micrograms/kg of MAb reduced the platelets, and the radioactivity of the liver and spleen augmented clearly after injection of MAb. Platelet radioactivity in serum, which had decreased after MAb infusion, did not recover, even when peripheral platelet counts returned to the normal levels, indicating that these new platelets might be derived from the platelet-storage pool or new thrombocytogenesis. This model of antiplatelet MAb induced thrombocytopenia seems to be useful for analyzing the kinetics of platelets in thrombocytopenia.