Tsutomu Fukuroi

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.

Platelet activation induced by an antiplatelet autoantibody against CD9 antigen and its inhibition by another autoantibody in immune thrombocytopenic purpura

Summary. In a patient with immune thrombocytopenic purpura (ITP), we found a novel platelet‐activating IgG (act‐IgG) and an inhibitory IgG (inhi‐IgG) that prevented activation induced by both CD9 monoclonal antibody (mAb) and the act‐IgG. Purified IgG from the patient plasma caused a rise in [Ca2+]i and the aggregation of normal platelets, and bound to a 24 kD membrane protein. This aggregation was inhibited by aspirin, staurosporine, an inhibitor of protein kinase C, and F(ab′)2 fragments of MALL13, a CD9 mAb. When the platelet count of this patient rose to normal range, the act‐IgG disappeared. About 2 weeks later, the relapse of thrombocytopenia was observed. The purified IgG obtained in this period did not activate platelets but inhibited both the rise in [Ca2+]i and platelet aggregation stimulated by NNKY 1–19, a CD9 mAb, as well as the act‐IgG, and bound to a 40 kD membrane protein. The inhi‐IgG prevented the binding of IV‐3, a mAb against Fcγ receptor II (FcγRII), but did not prevent the binding of NNKY 1–19 to its antigen. We suggest that the activating autoantibody recognized CD9 antigen and activated both the thromboxane‐ and phospholipase C‐dependent pathways, while the inhibitory autoantibody recognized the FcγRII and inhibited CD9 antibody‐induced platelet activation mediated via this receptor.

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.

Analysis of Idiopathic Thrombocytopenic Purpura Patients with Antiglycoprotein Ilb/IIIa or lb Autoantibodies

We analyzed the immunological characteristics of patients with idiopathic thrombocytopenic purpura (ITP) and antiglycoprotein (GP) IIb/IIIa or GPIb autoantibodies. Among 101 ITP patients, 32 had anti-GPIIb/IIIa and 19 had anti-GPIb autoantibodies. Thrombocytopenia was more severe in patients with anti-GPIb autoantibodies than in patients without these autoantibodies, whereas ITP patients with anti-GPIIb/IIIa autoantibodies did not develop severe thrombocytopenia. Patients with anti-GPIb autoantibodies showed significant increases of platelet-associated IgM and platelet-associated C3 in comparison with patients without the autoantibodies, despite there being no significant difference in the platelet-associated IgG levels. The lymphocyte subsets and the blastogenic response in patients with anti-GPIb autoantibodies were also significantly different from those in the patients without these autoantibodies. Furthermore, severe purpura and a poor response to prednisolone were far more common in the patients with anti-GPIb autoantibodies. Activation of the complement system and/or functional abnormalities of lymphocytes thus appear to be involved in the development of thrombocytopenia in ITP patients with anti-GPIb autoantibodies, and such antibodies may be associated with a particularly severe form of ITP.

Anti-phospholipid antibodies bind to platelet microparticles in idiopathic (autoimmune) thrombocytopenic purpura.

SummaryWe recently reported that IgM antibody-related microparticles exist in some patients with idiopathic thrombocytopenic purpura (ITP) [14]. In this study, we investigated the relationship between antiphospholipid (cardiolipin and phosphatidylinositol) antibodies and microparticles in 56 ITP patients. We used an ELISA to detect anti-phospholipid antibodies. IgG antibodies against cardiolipin and phosphatidylinositol were detected in 13 and 12 patients, respectively. The titers of IgG antibodies against these phospholipids did not correlate with the platelet-associated IgG level or the platelet count. Next, we investigated the binding of anti-phospholipid antibodies to platelets and microparticles. Microparticles were obtained by incubating washed platelets with collagen plus thrombin. ITP plasma containing IgG-class anti-phospholipid antibodies showed significantly increase binding to microparticles compared with plasma without such antibodies (p<0.001). Our results suggest that anti-phospholipid antibodies could affect the function of platelet microparticles in ITP.

Periodic Production of Antiplatelet Autoantibody Directed against GPIIIa in Cyclic Thrombocytopenia

We report here a female patient with cyclic thrombocytopenia associated with antiplatelet autoantibodies. There was an inverse relationship between the level of platelet-associated IgG and platelet count. Bone marrow megakaryocytes were normal in number even during the thrombocytopenia. The binding of monoclonal antibodies (mAbs) against glycoprotein (GP) IIb/IIIa to patient platelets was significantly inhibited in the thrombocytopenic phase, while these mAbs normally bound to patient platelets obtained during the normal platelet count. Western blotting and mAb-specific immobilization of platelet antigens showed that both plasma autoantibody and the eluted IgG from the patient platelets bound to GPIIIa. These results suggest that the periodic production of antiplatelet autoantibody against GPIIIa caused cyclic destruction of platelets in this patient.

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.

Effects of Kami-kihi-to (Jia-Wei-Gui-Pi-Tang) on autoantibodies in patients with chronic immune thrombocytopenic purpura

We studied the effect of Kami-kihi-to (Jia-Wei-Gui-Pi-Tang) on the production of autoantibodies in ten patients with chronic immune thrombocytopenic purpura. After administration of Kami-kihi-to, platelet count was increased in seven of the ten patients (p < 0.05). Using Western blotting, we demonstrated the disappearance of autoantibody reaction with antigen in one patient. However, platelet-associated IgG was decreased in eight of ten patients (p < 0.05). Kami-kihi-to appears to promote the suppression of autoantibodies in patients with chronic immune thrombocytopenic purpura. No side effects were observed in any patient. Thus, Kami-kihi-to may be a useful and safe drug in the management of chronic immune thrombocytopenia 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.