Hirokazu Nagata

Effects of EDTA on the membrane glycoproteins IIb−IIIa complex. Analysis using flow cytometry

We developed new monoclonal antibodies (NNKY1-32, NNKY2-6, NNKY2-18) that react with a determinant specific to the GPIIb-IIIa complex, and studied the distribution of GPIIb/IIIa on the platelet surface in vivo and some factors that influence the structure of GPIIb/IIIa using flow cytometry. 1. The surface of large platelets is richer in GPIIb/IIIa than that of small platelets. 2. Incubation of intact platelets with EDTA at 37 degrees C causes progressive dissociation of GPIIb-IIIa complexes, and this influence is marked in small platelets. 3. Addition of Ca2+ or Mg2+ to dissociated GPIIb-IIIa complexes causes reassociation, and additionary ADP after the cation treatment further increases the binding of monoclonal antibodies. 4. Prolonged incubation of intact platelets with EDTA at 37 degrees C induces a peculiar change of structure in the GPIIb/IIIa.

Antiplatelet autoantibody-related microparticles in patients with idiopathic (autoimmune) thrombocytopenic purpura

SummaryWe used flow cytometry to detect antiplatelet antibody-related microparticles (MP) in 56 patients with idiopathic thrombocytopenic purpura (ITP). We measured MP in platelets following various types of stimulation in two experimental systems. In one system washed platelets were incubated with normal serum which included the complement system, and in the other, washed platelets were incubated with Tyrodes buffer. There were no differences between the two measurement systems in the degree of increase in MP using various agonists. An increase in MP using ITP plasma was found in 12 out of 56 patients. In particular, four patients showed a significant increase in MP in washed platelets (WP) plus serum. Furthermore, the increase in platelet-associated IgM (PAIgM) was significant in these patients. There was also a definite positive correlation between PAIgM and the percentage of MP of WP plus serum. On the other hand, no specificity for MP formation with anti-GPIIb/IIIa or anti-GPIb autoantibodies was observed. IgM antibody-related MP appear to exist in some patients with ITP.

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.

Microparticle generation during in vitro platelet activation by anti-CD9 murine monoclonal antibodies

We used flow cytometry and two anti-CD9 murine monoclonal antibodies (NNKY1-19, MALL13) to investigate the glycoprotein composition and the potential functions of microparticles (MP) released by platelets exposed to these antibodies in vitro. NNKY1-19 produced aggregation with characteristics similar to those noted in previous reports. The action of MALL13 on platelets in platelet-rich plasma (PRP), however, differs from that of other anti-CD9 antibodies. The normal fluctuation in the MALL13-induced change in optical density disappeared when complement was present. MALL13-induced effect for platelet in PRP was not inhibited by preincubation with monoclonal anti-GPIIb/IIIa antibody, but was inhibited in washed platelets (WP). Furthermore, following MALL13 stimulation in PRP platelets, the amount of buffer LDH markedly increased and electron microscopy findings showed vacuoles appearing inside the platelets. These results suggest that MALL13 has at least two effects on platelets that differ for PRP platelets and WP. The number of MP released was increased by the addition of anti-CD9 antibodies. MP surfaces were found to be rich in CD9 protein. MALL13 stimulation lead to a significant increase in the binding of C1q and C3 to platelets and caused the production of MP to occur more rapidly than it did the exposure of fibrinogen binding sites in the presence of complement. The analysis of the relationship of MP to anti-CD9 monoclonal antibody may be useful in the investigation of the relationship between platelet function and coagulation regulation.

New monoclonal anti‐human Fc gamma receptor II antibodies induce platelet aggregation

We developed two new monoclonal antibodies, designaled NNKY3‐2 and NNKY4‐7, that recognized a 40‐kD platelet protein. They appeared to be monoclonal anti‐Fc gamma receptor II (FC‐/RII) antibodies from the results of flow cytometric binding inhibition studies using another monoclonal anti‐FcγRII antibody (2E1). The addition of NNKY3‐2 or NNKY4‐7 to platetet‐rich plasma (PRP) led to a typical aggregation pattern preceded by a lag phase, but their addition to washed platelets did not induce aggregation. The aggregation of PRP by these antibodies was inhibited by prostaglandin E1 (PGE1) or staurosporine (protein kinase C inhibitor), whereas it was only slightly affected by a monoclonal anti‐GPIIb/IIIa antibody or Arg‐Gly‐Asp‐Ser, Furthermore, these antibodies induced the aggregation of washed platelets plus normal serum, but not that of washed platelets plus heat‐treatcd serum (destruction of complement activity). These results suggest that NNKY3‐2 or NNKY4‐7‐induced aggregation involves an unusual pathway independent of fibrinogen, and that the important factor is the participation of complement. NNKY3‐2 and NNKY4‐7 may be useful to study the relationship between autoantibodies, the Fc receptor, and complement in idiopathic thrombocytopenic purpura.

Antithrombotic effect of an anti-glycoprotein IIb/IIIa antibody in primate lethal thrombosis

We investigated the antithrombotic effect of anti-glycoprotein (GP) IIb/IIIa antibody in a primate model of lethal thrombosis. Eight monkeys were injected intravenously with an anti-CD9 antibody (MALL13). They died within 5 min and displayed severe thrombocytopenia. Histological examination showed multiple platelet thrombi in the pulmonary microvasculature, but no thrombi in the liver, kidneys, or spleen. In contrast, monkeys pretreated with an anti-GPIIb/IIIa antibody (NNKY1-32) at 30 min before MALL13 administration did not die, and the thrombocytopenia in these animals did not develop as rapidly or become as severe. These results suggest that the antiCD9 antibody caused lethal pulmonary thrombosis in vivo, and that pretreatment with the anti-GPIIb/IIIa antibody was able to prevent this thrombosis.

Effects of ticlopidine on monoclonal anti-CD9 antibody-induced platelet aggregation and microparticle generation.

We analyzed the effects of ticlopidine on platelet aggregation and on microparticle (MP) formation when platelets were exposed to a monoclonal anti-CD9 antibody (NNKY1-19) in vitro. Even when NNKY1-19-induced platelet aggregation was completely inhibited by preincubation with anti-GPIIb/IIIa antibody or Arg-Gly-Asp-Ser, or by using washed platelets from a Glanzmanns thrombasthenia patient, the formation of MP was still observed. Prostaglandin E1 and protein kinase C antagonists (H-7 and staurosporine) inhibited both NNKY1-19-induced aggregation and MP formation. Ticlopidine or aspirin plus apyrase scarcely affected NNKY1-19-induced platelet aggregation, except to prolong the lag time. However, ticlopidine significantly inhibited MP formation (p less than 0.01). These results suggest that ticlopidine inhibits NNKY1-19-induced MP formation by a different mechanism to that of the other antagonists, and that this mechanism is unrelated to the inhibition of platelet aggregation.