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 EFTA at 37°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). © 1992 Wiley‐Liss, Inc.
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.
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 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.
We 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 less than 0.001). Our results suggest that anti-phospholipid antibodies could affect the function of platelet microparticles in ITP.
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