Identification of mimotopes of platelet autoantigens associated with autoimmune thrombocytopenic purpura

GPIIb/IIIa, the human platelet glycoprotein complex, is the autoantigen most commonly recognized by autoantibodies in autoimmune thrombocytopenic purpura (AITP). Two murine monoclonal antibodies (mAbs), namely Y2/51 and 5B12, directed against gpIIIa and gpIIb/IIIa, respectively, and rabbit anti-human platelet polyclonal antibodies have been used to select AITP-related epitopes from a phage display peptide library expressing random dodecapeptides in the pIII coat protein of M13 phage. The selected phage clones were tested by ELISA for binding to rabbit anti-human platelet antibodies as well as to sera from AITP patients. Seven clones reacted strongly with rabbit anti-human platelet antibodies, and four clones reacted with sera from AITP patients. Some homology between peptide inserts sequences of selected clones and human platelet gpIIIa and gpIb were found.     

A GPIIb/IIIa bioreactor for specific treatment of immune thrombocytopenic purpura, an autoimmune disease. Preparation, in vitro characterization, and preliminary proof-of-concept animal studies

Immune thrombocytopenic purpura (ITP) is an autoimmune disease that affects thousands of Americans each year. The resulting thrombocytopenia, which develops from destruction of platelets (PLT) by anti-PLT autoantibodies (APAb), is often associated with hemorrhagic complications. Existing therapies are not effective and are associated with significant morbidity. Recently, a new treatment modality using plasmapheresis with a Protein-A column has shown some clinical promise. Yet, although this method would remove the pathogenic APAb, it would also deplete protective antibodies, thereby weakening the body's self-defense system. Because about 80% of patients with ITP develop APAb against the GPIIb/IIIa antigens on PLT, a novel approach of attaching a GPIIb/IIIa-linked bioreactor with an extracorporeal circuit is suggested herein to achieve highly effective/specific APAb removal and overcome shortcomings of plasmapheresis in treating ITP. A hollow fiber-based bioreactor device was fabricated, and GPIIb/IIIa antigens were immobilized onto the inner lumens of the hollow fibers by using the epichlorohydrin activation method. An optimized bioreactor containing a loading of 1.63 mg GPIIb/IIIa/g fibers and adsorption capacity of 1.9 mg 7E3/g fibers was developed. Preliminary proof-of-concept investigation using a 7E3-induced thrombocytopenic rat model (which mimicked clinical ITP) was carried out. A complete (100%) return of PLT counts to their initial levels was observed in rats within 6 h after the GPIIb/IIIa bioreactor treatment. In addition, a rapid restoration of WBC counts in the treated rats was also found. These preliminary findings shed light of promise of using the GPIIb/IIIa bioreactor approach in achieving highly improved ITP therapy.     

MHC class I-associated peptides identified from normal platelets and from individuals with idiopathic thrombocytopenic purpura

Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8(+) T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I-associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4-12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I.     

Anti-glycoprotein IIb/IIIa autoantibodies are reversibly internalized into platelets in idiopathic (autoimmune) thrombocytopenic purpura.

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.     

Recombinant anti-idiotypic antibodies inhibit human natural anti-glycoprotein (GP)IIb/IIIa autoantibodies

Anti-idiotypic antibodies (anti-Id) have been described against idiotypes expressed on various autoantibodies. Since an immunoregulatory effect has been postulated for anti-Id, modulation of the anti-Id response in autoimmune disease may be of interest. In chronic immune thrombocytopenic purpura (AITP), autoantibodies directed mainly against platelet membrane glycoprotein (GP) IIb/IIIa cause platelet destruction by Fc-mediated phagocytosis or by complement lysis. We have previously reported on the generation of two recombinant anti-GPIIb/IIIa autoantibody fragments (PDG-X, PDG-B), that are specific for conformationally intact GPIIb/IIIa and inhibit binding of autoantibodies from patients with AITP. In the present study, we show that anti-GPIIb/IIIa specificities are not limited to a single individual by isolating five additional anti-GPIIb/IIIa autoantibody fragments from a second phagemid Fab library of an unrelated healthy donor. Using soluble Fab of PDG-X and PDG-B as antigens for panning Fab phagemid libraries from healthy human individuals, we isolated anti-Id phage clones specific for PDG-X or PDG-B. In addition they inhibited the binding of PDG-X or PDG-B to GPIIb/IIIa. Amino acid sequence comparison between these specific antiId and GPIIb/IIIa was performed. Generation of these anti-Id directed against pathologically relevant anti-GPIIb/IIIa autoantibodies may represent a new suitable and specific therapeutic option for the treatment of antibody-mediated AITP.     

用单克隆抗体对ITP自身抗血小板抗体及其相关抗原的初步研究

本文应用三种抗血小板膜糖蛋白单克隆抗体,按ELISA 间接法、ELISA双抗体夹心法、ELISA竞争法,对ITP患者自身抗血小板抗体及其相关抗原进行了初步研究。实验结果表明,抗血小板抗体的相关抗原呈多态性,包括:GPⅠb、GPⅡb及/或 GPⅢa,以 GPⅡb及/或 GPⅢa占多数,还可能包括血小板膜蛋白以外的其它抗原成分。这提示,ITP可能是一组复杂的、不均一的自身免疫性疾病。     

人类白细胞抗原DRB1 等位基因与儿童特发性血小板减少性紫癜的相关性研究

目的研究人类白细胞抗原(human leukocyte antigen, HLA)DRB1等位基因与儿童特发性血小板减少性紫癜(idiopathic thrombocytopenic purpura, ITP)的关系.方法用聚合酶链反应-序列特异的寡核苷酸探针杂交技术对42例ITP患儿进行了HLA-DRB1等位基因分型,同时用改良的血小板抗原单抗特异性固相化法检测了其中36例ITP患儿血清中的抗GPⅡb/Ⅲa和抗GPIb/Ⅰx自身抗体.结果与健康对照相比,ITP患儿HLA-DRB1*17基因频率显著升高(P<0.05,RR＝2.76,EF=0.1064),而HLA-DRB1*1202基因频率显著降低(P<0.025,RR＝0.20,PF=0.7616);慢性难治性ITP患儿与非难治性患儿相比,HLA-DRB1*11基因频率显著升高(χ2＝6.091,P<0.025),且具有DRB1*11的患儿主要(5/6)为女性年长患儿;抗GPⅡb/Ⅲa及抗GPIb/Ⅰx自身抗体的阳性率都与HLA-DRB1*02(15/16)基因显著相关(P分别为0.02和0.01),但难治性和非难治性ITP患儿间抗体阳性率差异无显著性(P>0.1).结论 DRB1*17可能与儿童ITP的易感性有关,而DRB1*1202则可能对儿童ITP的发病具有保护作用;具有DRB1*11基因的患儿易发展为慢性难治性ITP;血小板自身抗体与抗原表位的反应可能受DRB1*02限制,但自身抗体阳性与否并不能提示ITP患儿的预后.     

Tryptic peptides of human thyroglobulin: II. Immunoreactivity with sera from patients with thyroid diseases.

Tryptic peptides of human thyroglobulin (Tg) were analysed by Western immunoblot for their reactivity to circulating autoantibodies from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and thyroid carcinoma, and from normal human controls. Low molecular weight peptides were released after 4 h incubation of Tg with trypsin. The sera of thyroid disease patients reacted with several peptides, but predominantly bound three peptides with apparent molecular weights (MWap) of 25 kD, 20 kD, and 15 kD; the sera of normal individuals did not bind these fragments of Tg. The pattern of tryptic peptides recognized by the majority of sera from GD patients differed from that recognized by sera from most patients with HT. Autoantibodies from both groups of patients recognized a 15-kD peptide with a high frequency, but the sera from 26/43 (60%) GD patients also recognized a peptide with MWap of 25 kD, whereas the sera from 22/35 (63%) of HT patients recognized a 20-kD peptide. A few sera from patients with thyroid carcinoma reacted with peptides with MWap of 15 and 20-kD, and none bound the 25-kD peptide. The immunoreactivity of autoantibodies in HT sera to the 20-kD peptide paralleled the competitive inhibition of the MoAb 137C1 by these sera. In addition, MoAb 137C1 and Hashimoto's sera showed the same Western immunoblot-binding pattern to Tg tryptic peptides, suggesting that a Hashimoto-associated epitope and the 137C1-binding site are found on the same peptide. These findings suggest that distinct peptides are recognized by Tg autoantibodies from patients with different thyroid diseases.     

The binding of human blood platelets to fibrinogen-, fibronectin-, and Arg-Gly-Asp-derivatized Sephadex G-10

The adhesive proteins fibrinogen (FG) and fibronectin (FN) were immobilized to glycine-Sephadex G-10. The derivatized Sephadex G-10 gels were used to bind human blood platelets. For comparison, Gly-Arg-Gly-Asp-Ser-Pro(GRGDSP)-derivatized Gly-Sephadex G-10 was used. FG-, FN-, and GRGDSP-Gly-Sephadex G-10 each bound a substantial number of activated blood platelets (5 X 10⁸ ml^-1 gel) while non-activated platelets were not bound. Binding of ADP-treated blood platelets to the affinity adsorbents was dependent on the ADP-concentration which was used, reaching a near-maximal value at about 10 pM ADP. Platelet binding to the three types of affinity gels could be completely inhibited by dissolved GRGDSP as well as monoclonal anti-platelet glycoprotein IIb/IIIa (GPIIb/IIIa) antibody CLB-C17, which demonstrates that platelet binding specifically involves the fibrinogen binding site on GPIIb/IIIa. Platelet binding to all three affinity gels required free Ca²⁺ and Mg²⁺ ions: platelet binding in the absence of these divalent cations was considerably lower than platelet binding in buffer containing 2 mM Ca²⁺ and 1 mM Mg²⁺. Moreover, activated ethylenediamine-tetraacetate (EDTA)-treated platelets did not bind at all to the affinity gels. The finding that non-activated platelets did not bind to the affinity gels is thought to be related to both the high hydrophilicity of the Sephadex basic material and to the native state of the gel-bound fibrinogen and fibronectin.     

Anti-platelet antibodies associated with the Canale-Smith syndrome bind to the same platelet glycoprotein complexes as those of idiopathic thrombocytopenic purpura patients

The Canale-Smith syndrome (CSS) is an inherited disease characterized by massive lymphadenopathy, hepatosplenomegaly and systemic autoimmunity to erythrocytes and platelets. Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease in which approximately 60-80% of patients have anti-platelet antibodies directed against specific platelet glycoprotein complexes (GPCs) located on their membrane: GP IIb/IIIa, GPIb/IX, and GPIa/IIa. Almost all (95-100%) of the antibody-positive patients have antibodies directed against GPIIb/IIIa alone, or in combination with other glycoprotein targets. Our objective was to determine the specificities of the anti-platelet antibodies in CSS patients. The detection of anti-platelet antibodies was performed using a commercially available ELISA, the Pak-AUTO (GTI, Brookfield, WI), in which highly purified GPIIb/IIIa, GPIb/IX, and GPIa/IIa are immobilized on microtitre plates, incubated with serum or plasma, and subsequently developed with an antihuman polyclonal immunoglobulin. Of 14 CSS patients tested, 11 (79%) had anti-platelet antibodies in their serum directed toward at least one of the three major GPC, nine (82%) of which were against GPIIb/IIIa alone or in combination. Antibodies detected in the sera of ITP patients had similar specificities. No such antibodies were detected in samples from 25 consecutive normal controls. These results demonstrate that a genetically defined defect in lymphocyte apoptosis results in a humoral autoimmune response with anti-platelet specificities very similar to the common idiopathic form of autoimmune thrombocytopenia.     

Quantitative comparisons of antibody-binding sites of platelet glycoprotein IIb/IIIa in aplastic anemia and idiopathic thrombocytopenic purpura

The numbers of antibody-binding sites of platelet glycoprotein (GP) IIb/IIIa on circulating platelets were analyzed using 4 kinds of antibodies in 34 aplastic anemia (AA) patients, 20 idiopathic thrombocytopenic purpura (ITP) patients, and 14 normal controls. The numbers of antibody-binding sites of CD41, CD41a, CD41b, and CD61 on platelets of the AA patients were less than in the normal controls (p <0.001). In the ITP patients, the numbers of sites for CD41 and CD41a were less than in normal controls (p <0.05). There were significant positive correlations between CD41 and CD41a, CD41b, and CD61 in the 3 groups. There were significant negative correlations between CD41 and CD41b and between CD41a and CD41b in the normal controls, but not in the AA or ITP patients. In summary, the numbers of the 4 antibody-binding sites of GPIIb/IIIa on platelets of AA and ITP patients are different from those in normal controls. Measurements of the antibody-binding sites of GPIIb/IIIa are not necessary for the differential diagnosis of AA and ITP. However, the differences in correlations between the numbers of epitopes in AA and ITP patients suggest that the epitopes of GPIIb/IIIa are altered in these diseases.     

Identification and characterization of antibodies that bind GPIIb/IIIa: antagonist complexes

A potential limitation of anti-thrombotic therapies directed at platelet GPIIb/IIIa is immune mediated thrombocytopenia. Reagents that mimic the behavior of patient antibodies would provide a valuable tool for studies directed at understanding the basis of the immune mechanism involved in GPIIb/IIIa antagonist induced thrombocytopenia. Such reagents would bind epitopes that are exposed when the conformation of the receptor is modified in response to inhibitor binding. We describe the production and characterization of monoclonal antibodies that were raised against platelet GPIIb/IIIa bound to a potent antagonist, XP280. These antibodies have high affinity and specificity for XP280 bound GPIIb/IIIa using either purified protein or human platelets. We have demonstrated that the antibodies recognize a conformationally altered form of the receptor, that both subunits are required for binding, and that the antagonist itself does not form part of the binding epitope. Competition experiments indicate that multiple drug-dependent epitopes are exposed on the receptor in response to antagonist binding. The antibodies bind with high specificity to some but not all GP IIb/IIIa/antagonist complexes indicating that different conformational epitopes are exposed when GP IIb/IIIa is bound to different antagonists.     

Identification of the platelet glycoprotein IIb/IIIa complex as a target antigen in primary biliary cirrhosis-associated autoimmune thrombocytopenia. Evidence that platelet-reactive autoantibodies can also bind to the mitochondrial antigen M2

A 67-year-old woman with a 4-year history of primary biliary cirrhosis (PBC) unexpectedly developed autoimmune thrombocytopenia. The platelet-bound IgG antibody was eluted from the patient's platelets to determine the platelet target antigen. The autoantibodies were found to precipitate the platelet glycoprotein complex IIb/IIIa of autologous and allogeneic platelets. A further precipitate of 70 kDa was detectable under reducing conditions. In addition, platelet-reactive antibodies bound to the 70 kDa mitochondrial antigen M2. No cross-absorption studies were performed to confirm that a single antibody reacted with both antigens. Computer analysis of published peptide sequences of the mitochondrial protein and the platelet GPIIb/IIIa complex showed partial amino acid sequence homology suggesting the possibility of a common antibody binding site. These findings suggest a relationship between the development of autoimmune thrombocytopenia in PBC and the underlying liver disease.     

Recognition of synthetic peptides of Sm-D autoantigen by lupus sera.

The reactivity of autoantibodies present in the serum of patients with systemic lupus erythematosus (SLE) was investigated by ELISA using seven overlapping synthetic peptides representing the entire sequence of the polypeptide D component of 'Sm antigen'. Of the 165 SLE sera tested, 59% were found to contain IgG antibodies able to bind to peptide 1-20, while 37% of the sera reacted with peptide 44-67. All sera reacting with peptide 44-67 also reacted with peptide 1-20. These two peptides were only seldom recognized by the sera of 187 patients with other rheumatic autoimmune diseases or by 53 sera of normal individuals. In a parallel study using sera that reacted with the D band in immunoblotting, most of the sera recognized peptides 44-67 (89%) and 1-20 (67%), while 33% of them reacted with peptide 97-119. The use of these synthetic peptides in ELISA may be of considerable help for detecting anti Sm autoantibodies.     

Autoantibodies from patients with systemic lupus erythematosus bind a shared sequence of SmD and Epstein-Barr virus-encoded nuclear antigen EBNA I

SmD is one of the small nuclear ribonucleoproteins frequently targeted by autoantibodies in systemic lupus erythematosus. We isolated and characterized the antibodies present in lupus sera that are specific for the C-terminal region of SmD (sequence 95–119). This region is highly homologous to sequence 35-58 of the EBNA I antigen, one of the nuclear antigens induced by infection with Epstein-Barr virus. Antibodies affinity purified over a peptide 95–119 column were able to recognize this sequence in the context of the whole SmD molecule, as they reacted with blotted recombinant SmD. Anti-SmD 95-119 antibodies bound also the EBNA I 35–58 peptide and detected the EBNA I molecule in a total cell extract from Epstein-Barr virus-infected lines. A population of anti-SmD antibodies is, therefore, able to bind an epitope shared by the autoantigen and the viral antigen EBNA I. To investigate the involvement of this shared epitope in the generation of anti-SmD antibodies, we immunized mice with the EBNA I 35-58 peptide. Sera from immunized animals displayed the same pattern of reactivity of spontaneously produced anti-SmD antibodies. They reacted in fact with the EBNA peptide as well as with SmD 95-119 and recombinant SmD. These data suggest that molecular mimicry may play a role in the induction of anti-SmD autoantibodies.     

抗血小板糖蛋白Ⅱb/Ⅲa Fab抗体的原核表达及其生物活性

目的：研制抗血小板膜糖蛋白Ⅱb／Ⅲa Fab抗体。方法：通过问接免疫荧光试验和血小板聚集抑制试验，选取鼠源抗血小板糖蛋白Ⅱb／Ⅲa单克隆抗体(mAb P140)。从分泌该mAb的杂交瘤细胞株(P140)中，克隆到抗体轻链基因和重链Fd段基因，构建原核表达重组质粒p3MH／P140x-Fd，并在XLI-Blu菌株中进行表达。采用钴亲和层析法对P140 Fab抗体进行纯化，用SDS-PAGE、ELISA和Western blot等方法，对P140 Fab抗体进行检测，并通过血小板聚集抑制试验，观察P140 Fab抗体的抗栓活性。结果：SDS-PAGE和Western blot表明，纯化的P140Fab抗体的相对分子质量(Mr)约为47000。ELISA的结果显示，P140 Fab抗体可与人血小板特异性结合。在体外ADP诱导的血小板聚集试验中，P140 Fab抗体对血小板聚集的抑制作用成剂量依赖性，IC50的平均值为16．85mg/L。结论：成功地研制出具有抗栓活性的抗血小板膜糖蛋白Ⅱb／Ⅲa的Fab抗体。     

Human Anti-Ro Autoantibodies Bind Peptides Accessible to the Surface of the Native Ro Autoantigen

The relationship between fine specificity of linear epitopes and conformational determinants has been explored in a naturally arising human autoimmune response. In particular, the hypothesis tested is that the linear epitopes of the human Ro autoantigen are components of its conformational epitopes. Twenty groups among the 531 overlapping octapeptides 60kDa Ro are variably bound by anti-Ro precipitin positive lupus sera whose reactivity was easily distinguished from sera of normal controls and of anti-Ro precipitin negative lupus patients. The specific activities of anti-peptide antibodies and of anti-native Ro autoantibodies are similarly increased after affinity enrichment using native human Ro as ligand. Moreover, affinity-enriched anti-native Ro autoantibodies bind virtually the same 20 groups of epitopes recognized by whole anti-Ro positive sera. Two peptides (residues 274–290 and 480–494) from the defined 60 kDa Ro octapeptide epitopes have been prepared and used as ligands for affinity purification of peptide specific autoantibodies. The binding of both whole IgG and affinity-enriched peptide specific autoantibodies is inhibited by native Ro autoantigen. Thus, none of the available data can be construed to support the existence of cryptic linear epitopes in this system. Indeed, the data are only consistent with the conclusion that all of the anti-Ro octapeptide autoantibodies are part of the population of anti-native Ro autoantibodies in this naturally arising autoimmune response.     

Relationship between autoepitope and DNA-binding site on a histone H1 molecule.

Autoepitope and DNA-binding domain on a histone H1 molecule were compared using truncated histone H1 peptides as antigens. At least two epitopes (epitope A, N-terminal side; epitope B, C-terminal side) were found both of which were composed of approximately 20 amino acids. IgM from all 17 anti-histone H1-positive SLE sera reacted with epitope A. IgG from 12 sera reacted with epitope A and IgG from 4 sera reacted with epitope B. In one case, no IgG anti-histone H1 reactivities were found while IgM from the same patient reacted with epitope A. Epitope A had the ability to bind DNA. The reactivities against histone H1 of affinity-purified antiepitope A autoantibodies were inhibited by DNA. These data suggest that some anti-histone H1 antibodies are directed against a histone H1 DNA-binding site, raising the possibility that an idiotype/anti-idiotype network, at least in part, is involved in the generation of anti-histone H1 autoantibodies.     

Isolation and characterization of human monoclonal autoantibodies to glutamic acid decarboxylase

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M(r) 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2S from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M(r) chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 x 10(9), 5.8 x 10(9), 1.3 x 10(10) mol/l(-1); affinities of the mouse monoclonals (n = 5) ranged from 1.1 x 10(8) to 5.4 x 10(10) mol/l(-1). The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2S suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.     

Ca2+ influx and platelet membrane glycoproteins

When aequorin-loaded platelets were stimulated with thrombin, the luminescence of aequorin showed two peaks. From experiments with 1 mM external Ca2+ or 1 mM EGTA, both one-half of the first peak and the entire second peak reflected the influx of Ca2+ from the external medium, and the remaining half of the first peak reflected the mobilization of Ca2+ from its storage site. A monoclonal antibody (TM83), that recognizes the GPIIb/IIIa complex which has binding sites for fibrinogen, and synthetic peptide GRGDSP are known to inhibit fibrinogen binding and platelet aggregation. Both of them eliminated the second peak of intracellular free calcium. Similar effects were observed during activation by collagen, but not by TPA. Also dihydrocytochalasin B inhibited the second peak of Ca2+ influx by thrombin, suggesting that the signal, which was caused by fibrinogen-binding to GPIIb/IIIa (aggregation) in thrombin-activated platelets, is transferred to the inner sites of GPIIb/IIIa complex and induces the cytoskeletal reorganization such as actin polymerization. This in turn, induces the secondary increase in [Ca2+] i of platelets. It is interesting that ticlopidine inhibited the Ca2+ influx through the GPIIb/IIIa complex. This result suggests the importance of such kinds of antiplatelet drugs to prevent thrombus formation.