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We investigated the increase of platelet-associated IgG and complement component, ³CO caused by the in vitro action of anti-platelet MoAbs, and the effect of mouse and human IgG on these events, Anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib MoAbs caused a slight increase of C3, but not of platelet-associated IgG. In contrast, anti-CD9 and anti-Fey II receptor MoAbs caused an increase of both platelet-associated C3 and IgG. In particular, three MoAbs which activated the complement system caused a marked increase of C3. When platelet-rich plasma was treated with aspirin and prostaglandin, before incubation with antibodies, the increase of platelet-associated IgG was inhibited in all cases. In contrast, the increase of platelet-associated C3 was scarcely influenced. These results suggest that the binding to platelets of platelet-activating antibodies caused the increased expression of IgG molecules on the platelet surface and a possible increase of platelet-associated IgG, However, the increase of platelet-associated C3 appeared to depend on specific characteristics of the antibodies tested, such as a complement-activating effect. In addition, intact mouse or human IgG inhibited the increase of platelet-associated C3 caused by complement-activating antibodies, white F(ab') 2 mouse or human IgG had no such effect. This suggested that the Fc portion of IgG may block the increase of C3 mediated by anti-platelet antibodies. [ABSTRACT FROM AUTHOR]

We 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 Tyrode's 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. [ABSTRACT FROM AUTHOR]

Uremia causes a bleeding tendency associated with platelet dysfunction, and previous studies have shown abnormalities of platelet glycoprotein (GP) Ib or GPIIb/IIIa and a tendency for platelet activation in uremia. The present study compared the abnormalities of platelet function in uremia with (n = 1) or without (n = 18) associated Glanzmann's thrombasthenia. There was a significant difference between ristocetin-induced agglutination of platelets from the uremic patients without Glanzmann's thrombasthenia and platelets from healthy controls (n = 15). In addition, a reduction of GPIb expression by uremic platelets along with normal GPIIb/IIIa expression was confirmed using flow cytometry. Many coagulation markers were increased in the uremic patient with Glanzmann's thrombasthenia, suggesting that the coagulation was enhanced and the platelets were prone to activation. However, the thrombasthenic platelets actually showed little increase in the binding of a monoclonal anti-CD63 antibody directed against lysosomal integral membrane protein (which is expressed after platelet activation), while uremic platelets showed a marked increase. In addition, the expression of GPIb by thrombasthenic platelets was normal, while that of GPIIb/IIIa was markedly decreased. Our results suggest that thrombasthenic platelets are resistant to activation and to the degradation of GPIb under uremic condition and that this difference from 'ordinary' uremic platelets be related to the difference in GPIIb/IIIa.

The effects of cepharanthin and cytochalasin D on the internalization of anti-glycoprotein IIb/IIIa antibodies by platelets were investigated in 13 patients with chronic immune thrombocytopenic purpura who had circulating anti-glycoprotein IIb/IIIa autoantibodies. Unfixed platelets were incubated with a monoclonal anti-glycoprotein IIb/IIIa antibody (NNKY1-32) or with platelet-binding IgG from the patients (which contained anti-glycoprotein IIb/IIIa antibodies). Flow cytometry showed that the binding of NNKY1-32 to platelets was markedly decreased after incubation for 120 min compared with incubation for 10 min. This decrease was inhibited by cepharanthin but not by cytochalasin D. Platelet-binding IgG also showed markedly reduced binding after incubation for 120 min compared with 10 min, and this decrease was inhibited by both cepharanthin and cytochalasin D. Cytochalasin D inhibits platelet cytoskeletal activity while cepharanthin does not. Therefore, our results suggest that the internalization of anti-glycoprotein IIb/IIIa antibodies from the plasma of patients with immune thrombocytopenic purpura is related to platelet cytoskeletal reorganization, while the cytoskeleton did not participate in internalization of the monoclonal anti-glycoprotein IIb/IIIa antibody (NNKY1-32). Cepharanthin may be useful for studying the internalization and cycling of glycoprotein IIb/IIIa in human platelets, and it may also be potentially useful for the treatment of immune thrombocytopenic purpura.

We studied the effect of three Japanese kampo medicines on platelet activation by an anti-CD9 monoclonal antibody (NNKY1-19) and an anti-human Fc gamma receptor II monoclonal antibody (NNKY3-2). Sho-saiko-to (TJ-9) and Sairei-to (TJ-114) partially suppressed platelet aggregation induced by NNKY1-19, while Juzen-taiho-to (TJ-48) suppressed aggregation induced by NNKY3-2. TJ-9 and TJ-114 also suppressed collagen-induced aggregation, but TJ-48 did not. Flow cytometry showed that the three medicines did not affect antibody binding to the platelets. Thus, all three kampo medicines suppressed platelet activation by anti-platelet glycoprotein antibodies without inhibiting antibody binding.

We developed a new monoclonal antibody directed against platelet myosin (NNKY6-19). Using this antibody, we analyzed platelet cytoskeletal changes related to stimulation with thrombin and to long-term storage. Immunoelectron microscopy showed increased binding of NNKY6-19 to pseudopods and the open canalicular system during treatment with thrombin (0.1 U/ml) and during storage for 7 days. Flow cytometry also showed increased binding to platelets by NNKY6-19 and an antiactin monoclonal antibody during storage. The binding of NNKY6-19 showed an increase greater than that with the antiactin antibody after storage of platelets for 7 days and after thrombin treatment. These findings indicated that the increased binding of NNKY6-19 had some relationship to changes in intracellular myosin and platelet morphology. Thus use of NNKY6-19 allowed analysis of subtle changes related to platelet activation, which differed from those detected by antibodies against platelet glycoproteins or by the antiactin antibody. This antibody appears to provide a simple method for studying changes in platelet cytoskeletal and surface proteins.

We investigated the increase of platelet-associated IgG and complement component 3 (C3) caused by the in vitro action of anti-platelet MoAbs, and the effect of mouse and human IgG on these events. Anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib MoAbs caused a slight increase of C3, but not of platelet-associated IgG. In contrast, anti-CD9 and anti-Fc gamma II receptor MoAbs caused an increase of both platelet-associated C3 and IgG. In particular, three MoAbs which activated the complement system caused a marked increase of C3. When platelet-rich plasma was treated with aspirin and prostaglandin E1 before incubation with antibodies, the increase of platelet-associated IgG was inhibited in all cases. In contrast, the increase of platelet-associated C3 was scarcely influenced. These results suggest that the binding to platelets of platelet-activating antibodies caused the increased expression of IgG molecules on the platelet surface and a possible increase of platelet-associated IgG. However, the increase of platelet-associated C3 appeared to depend on specific characteristics of the antibodies tested, such as a complement-activating effect. In addition, intact mouse or human IgG inhibited the increase of platelet-associated C3 caused by complement-activating antibodies, while F(ab')2 mouse or human IgG had no such effect. This suggested that the Fc portion of IgG may block the increase of C3 mediated by anti-platelet antibodies.

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 Fc gamma receptor II (Fc gamma RII), 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 Fc gamma RII and inhibited CD9 antibody-induced platelet activation mediated via this receptor.

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.

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.

Flow cytometry was used to detect platelet-associated fibrinogen (PA-Fbg), platelet-associated fibronectin (PA-FN) and platelet-associated thrombospondin (PA-TSP) on the surface membrane of platelets and plasma (P)-TSP in 30 patients with lung cancer (16 case of adenocarcinoma and 14 of squamous cell carcinoma). ELISA was used to analyze beta-TG and PF4. In the lung cancer group, beta-TG and PF4 were higher than those of a normal control group. PA-Fbg values were correlated with beta-TG and PF4 values. Each adhesive protein had a higher value in the patient than in the normal control group, and the degree of the increase was related to the progression of clinical disease stage. In the squamous cell carcinoma group, the P-TSP value was significantly elevated. Platelet size increased as the clinical stage of the disease progressed. These results suggest the following: 1. An increase in PA-Fbg can indicate the presence of activated platelets. 2. In patients with lung cancer, activated platelets appear in the blood, and their numbers increase as the clinical stage of the disease progresses. 3. Differences in histologic type led to differences in binding adhesive protein.

We used flow cytometry to investigate the binding of platelet-binding IgG (PBIgG) to unfixed platelets in idiopathic thrombocytopenic purpura (ITP), including that of anti-glycoprotein (GP) IIb/IIIa antibodies. Anti-GPIIb/IIIa antibodies were detected in 13/64 ITP patients using antigen-capture ELISA and immunoblotting. When unfixed platelets were incubated with ITP plasma, the PBIgG level was significantly higher than after incubation with normal plasma. When 1 microM ADP was added to unfixed platelets, which were incubated with ITP plasma and washed, the PBIgG level increased additively. GMP-140 is a constituent of platelet alpha-granules, and a monoclonal antibody directed against this protein showed weak binding to platelets after 1 microM ADP stimulation. The increase of PBIgG produced by ADP was significantly greater when ITP plasma positive for anti-GPIIb/IIIa antibody was used compared with that obtained using antibody-negative ITP plasma. This increase of PBIgG was markedly inhibited by the removal of extracellular calcium with EDTA or the dissociation of the GPIIb/IIIa complex by EDTA treatment at 37 degrees C. These results suggest that anti-GPIIb/IIIa autoantibodies are internalized by unfixed ITP platelets and stored somewhere other than the alpha-granules. This stored antibody pool can be reversibly redistributed on the platelet surface by weak stimulants such as ADP and a functional GPIIb/IIIa complex appears to be necessary for this to occur.

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 Glanzmann's 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.

Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene-diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC-conjugated anti-GPIb antibody (NNKY5-5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EDTA at 37 degrees C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles).

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 patient's 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 patient's IgG was extremely slight. Phosphorylation of a 40 kDa protein was induced by the patient's 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 patient's IgG failed to induce aggregation itself, but enhanced ADP- or STA2-induced aggregation. Western blotting and immunoprecipitation experiments showed that the patient's 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.

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.

This report deals with a case of 23-year-old woman with chronic idiopathic thrombocytopenic purpura (ITP) showing a favorable response to vitamin C (VC) administration. She was treated with glucocorticoids and immunosuppressants, whereas inadequately controlled with its tolerable dosage. Next, she underwent splenectomy. However, there were only transient effects on platelet counts, so the therapy with ascorbate was carried out according to the report by Brox et al. The platelet counts increased to 20 x 10(4)/microliters after 4 months. Side effects of VC therapy did not occur.

Flow cytometry was used to detect fibrinogen (platelet associated fibrinogen: PAFbg) and fibronectin (PAFn) on the surface membrane of platelets in leukemia (9 cases), lung cancer (15 cases) and hepatoma (8 cases) patients (by one color analysis method), and simultaneously to investigate the binding of monoclonal anti-glycoprotein (GP) IIb/IIIa and anti-GP Ib antibodies (by two color analysis). All patient groups showed higher Fbg values than the normal control group, but no differences were found between patient groups. The values of Fbg and Fn showed a correlation, but the pattern of binding did not show a regular tendency in any patient group. There also was no significant correlation between Fbg values and the positive percentage of monoclonal anti-GPIIb/IIIa and anti-GPIb antibodies, and the binding of Fbg did not inhibit that of monoclonal antibody. The results suggest the following. 1. There are no differences in platelet activation in leukemia, lung cancer and hepatoma patients, and the degree of platelet activation is decided by the degree and the kind of stimulation. 2. The increase of both PAFbg and PAFn prove to the existence of activated platelet.