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Thromb Haemost 1995; 74(04): 1152-1162
DOI: 10.1055/s-0038-1649896
Original Article
Platelets
Schattauer GmbH Stuttgart

Inhibition of Platelet Aggregation by an SLE-Derived Human Hybridoma Autoantibody Against an Activation-Dependent Antigen[*]

H Xu
1   The Division of Rheumatology, The Montreal General Hospital Research Institute, Quebec, Canada
,
M M Frojmovic
2   The Department of Physiology, McGill University, Montreal, Quebec, Canada
,
T Wong
2   The Department of Physiology, McGill University, Montreal, Quebec, Canada
,
J Rauch
1   The Division of Rheumatology, The Montreal General Hospital Research Institute, Quebec, Canada
› Author Affiliations
Further Information

Publication History

Received 23 January 1995

Accepted after revision 29 June 1995

Publication Date:
09 July 2018 (online)

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Summary

Anti-platelet autoantibodies may be responsible for hematological complications in patients with systemic lupus erythematosus (SLE), but the mechanisms by which these antibodies cause abnormal hemostasis remain unknown. In the present study, using fluorescence activated cell sorter (FACS) analysis, we demonstrate that an SLE-derived human hybridoma autoantibody, 9604, recognizes a surface antigen expressed on platelets activated by ADP, calcium ionophore A23187, or phorbol myristate acetate (PMA), showing saturation with approximately 2,000 antibody molecules bound per platelet and a Kd of 41 nM. The binding of 9604 to activated platelets was significantly inhibited by EDTA, indicating partial dependence on divalent cations. It did not appear to be dependent on platelet secretion, nor did it directly affect α-granule or dense granule secretion. The protein antigen responsible for the binding of 9604 to activated platelets was characterized by Western blot and immunoprccipitation and shown to have a native molecular weight (M. W.) of greater than 400,000, with a 32,000 M. W. subunit (p 32). Antibody 9604 had little or no effect on the shape change and the initial rate of primary aggregation of normal platelets. In contrast, 9604 inhibited secondary aggregation of stirred platelet suspensions (IC50 ≥1 nM) following activation by ADP, thromboxane A2 mimetic U46619, or calcium ionophore A23187, but not PMA or thrombin. The inhibition of large platelet aggregate formation (secondary aggregation), with a major shift to smaller microaggregates and singlets, was confirmed by direct particle count and sizing studies. The functional inhibition of platelet aggregation by an SLE-derived human hybridoma autoantibody in vitro suggests one potential mechanism that may play a role in the hemostatic disorders found in SLE.

* This work was supported by operating grants from The Arthritis Society of Canada and The Medical Research Council of Canada and a Group Facilitation Grant from The Arthritis Society of Canada


 

  • References

  • 1 Karpatkin S, Strick N, Karpatkin MB, Siskind GW. Cumulative experience in the detection of antiplatelet antibody in 234 patients with idiopathic thrombocytopenic purpura, systemic lupus erythematosus and other clinical disorders. Am J Med 1972; 52: 776-785
    CrossrefPubMedGoogle Scholar
  • 2 McMillan R. Immune thrombocytopenias. Clin Haematol 1983; 12: 69-88
    PubMedGoogle Scholar
  • 3 Regan MG, Lackner H, Karpatkin S. Platelet function and coagulation profile in lupus erythematosus. Studies in 50 patients. Ann Intern Med 1974; 81: 462-468
    CrossrefPubMedGoogle Scholar
  • 4 Dorscli CA, Mcycrholf J. Mechanisms of abnormal platelet aggregation in systemic lupus erythematosus. Arthritis Rheum 1982; 25: 966-973
    CrossrefPubMedGoogle Scholar
  • 5 Woods VL, Kurata Y, Montgomery RR, Tani P, Mason D, Oh HE, McMillan R. Autoantibodies against platelet glycoprotein lb in patients with chronic immune thrombocytopenic purpura. Blood 1984; 64: 156-160
    PubMedGoogle Scholar
  • 6 Beardsley DS, Spiegel JE, Jacobs MM, llandin Rl, Lux SL IV. Platelet membrane glycoprotein Ilia contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias. J Clin Invest 1984; 74: 1701-1707
    CrossrefPubMedGoogle Scholar
  • 7 Woods VL, Oh EH, Mason D, McMillan R. Auto-antibodies against the platelet glycoprotein llh/llla complex in patients with chronic ITP. Blood 1984; 63: 368-375
    PubMedGoogle Scholar
  • 8 Hornby HI, Brown S, Wilkinson JM, Mattock C, Authi KS. Activation of human platelets by exposure to a monoclonal antibody, PM6/248, to glycoprotein Ilb-llla. Br J Haematol 1991; 79: 277-285
    CrossrefPubMedGoogle Scholar
  • 9 Balduini CL, Bertolino G, Noris P, Piovclla F, Sinigaglia F, Bellotti V, Samaden A, Torti M, Mazzini G. Defect of platelet aggregation and adhesion induced by autoantibodies against platelet glycoprotein IIIa. Thromb Haemost 1992; 68: 208-213
    Article in Thieme ConnectPubMedGoogle Scholar
  • 10 Frojmovic MM, Milton JG. Physical, chemical and functional changes following platelet activation in normal and ”giant” platelets. Blood Cells 1983; 9: 359-382
    PubMedGoogle Scholar
  • 11 Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation. J Biol Chem 1985; 260: 11107-11114
    PubMedGoogle Scholar
  • 12 Abrams CS, Ellison N, Budzynski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 1990; 75: 128-138
    PubMedGoogle Scholar
  • 13 Zamarron C, Ginsberg MH, Plow EF. Monoclonal antibodies specific for a conformationally altered state of fibrinogen. Thromb Haemost 1990; 64: 41-46
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Frelinger III AL, Cohen I, Plow EF, Smith MA, Roberts J, Lam SC T, Ginsberg MH. Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers. J Biol Chem 1990; 265: 6346-6352
    PubMedGoogle Scholar
  • 15 McEver RP, Martin NM. A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 1984; 259: 9799-9804
    PubMedGoogle Scholar
  • 16 Hsu-Lin SC, Berman CL, Furie BC, August D, Furie B. A platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets J Biol Chem 1984; 259: 9121-9126
    PubMedGoogle Scholar
  • 17 Metzelaar MJ, Heijnen HF G, Sixma JJ, Nieuwenhuis HK. Identification of a 33-Kd protein associated with the a-granule membrane (GMP-33) that is expressed on the surface of activated platelets. Blood 1992; 79: 372-379
    PubMedGoogle Scholar
  • 18 Febbraio M, Silverstein RL. Identification and characterization of LAMP-1 as an activation-dependent platelet surface glycoprotein. J Biol Chem 1990; 265: 18531-18537
    PubMedGoogle Scholar
  • 19 Silverstein RL, Febbraio M. Identification of Lysosome-associated membrane protein-2 as an activation-dependent platelet surface glycoprotein. Blood 1992; 80: 1470-1475
    PubMedGoogle Scholar
  • 20 Nieuwenhuis HK, van Oosterhout JJ G, Rozenmuller E, van Iwaarden F, Sixma JJ. Studies with a monoclonal antibody against activated platelets. Evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation Blood 1987; 70: 838-845
    PubMedGoogle Scholar
  • 21 Boukerche H, McGregor JL. Characterization of an anti-thrombospondin monoclonal antibody (P8) that inhibits human platelet functions. Eur J Biochem 1988; 171: 383-392
    CrossrefPubMedGoogle Scholar
  • 22 Leung LL K. Role of thrombospondin in platelet aggregation. J Clin Invest 1984; 74: 1764-1772
    CrossrefPubMedGoogle Scholar
  • 23 Thurlow PJ, Kenneally DA, Connellan JM. The role of fibronectin in platelet aggregation. Br J Haematol 1990; 75: 549-556
    CrossrefPubMedGoogle Scholar
  • 24 Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation Factor Va and express prothrombinase activity. J Biol Chem 1989; 263: 18205-18212
    PubMedGoogle Scholar
  • 25 Nugent DJ, Kunicki TJ, Berglund C, Bernstein ID. A human monoclonal autoantibody recognizes a neoantigen on glycoprotein IIIa expressed on stored and activated platelets. Blood 1987; 70: 16-22
    PubMedGoogle Scholar
  • 26 Abrams C, Shattil SJ. Immunological detection of activated platelets in clinical disorders. Thromb Haemost 1991; 65: 467-473
    Article in Thieme ConnectPubMedGoogle Scholar
  • 27 Coller BS. A new murine monoclonal antibody reports an activation- dependent change in the conformation and/or microenvironment of the platelet glycoprotein Ilb/IIIa complex. J Clin Invest 1985; 76: 101-109
    CrossrefPubMedGoogle Scholar
  • 28 Parmentier S, Catimel B, McGregor L, Leung LL K, McGregor JL. Role of glycoprotein IIa(β1subunit of very late activation antigens) in platelet functions. Blood 1991; 78: 2021-2026
    PubMedGoogle Scholar
  • 29 Parmentier S, McGregor L, Catimel B, Leung LL K, McGregor JL. Inhibition of platelet functions by a monoclonal antibody (LYP20) directed against a granule membrane glycoprotein (GMP-140/PADGEM). Blood 1991; 77: 1734-1739
    PubMedGoogle Scholar
  • 30 Denomme GA, Smith JW, Kelton JG, Bell DA. A human monoclonal autoantibody to platelet glycoprotein lib derived from normal human lymphocytes. Blood 1992; 79: 447-451
    PubMedGoogle Scholar
  • 31 Kunicki TJ, Furihata K, Kekomaki R, Scott JP, Nugent DJ. A human monoclonal autoantibody specific for human platelet glycoprotein lib (integrin a lib) heavy chain. Hum Antibod Hybridomas 1990; 1: 83-95
    PubMedGoogle Scholar
  • 32 Deckmyn H, Jingwu Z, Van Houtte E, Vermylen J. Production and nucleotide sequence of an inhibitory human IgM autoantibody directed against platelet glycoprotein Ia. Thromb Haemost. 1993 69. 913. Abstr
    PubMedGoogle Scholar
  • 33 Massicotte H, Rauch J, Shoenfeld Y, Tannenbaum H. Influence of fusion cell ratio and cell plating density on production of human-human hybridomas secreting anti-DNA autoantibodies from patients with systemic lupus erythematosus. Hybridoma 1984; 3: 215-222
    CrossrefPubMedGoogle Scholar
  • 34 Meng Q-h, Rauch J. A systemic lupus erythematosus-derived human hybridoma autoantibody reactive with antigens expressed on ADP- activated platelets. J Autoimmunity 1991; 4: 447-461
    CrossrefPubMedGoogle Scholar
  • 35 Rauch J, Xu H, Meng Q-h. An SLE-derived human hybridoma autoantibody reactive with antigens expressed on activated platelets. Thromb Haemost 1991; 65 6 1061 Abstr
    PubMedGoogle Scholar
  • 36 Xu H, Frojmovic MM, Wong T, Rauch J. p 32, a platelet autoantigen recognized by an SLE-derived autoantibody that inhibits platelet aggregation. J Autoimmunity 1995; 8: 97-119
    CrossrefPubMedGoogle Scholar
  • 37 Hayward CP M, Smith JW, Horsewood P, Warkentin TE, Kelton JG. P-155, a multimeric platelet protein that is expressed on activated platelets. J Biol Chem 1991; 266: 7114-7120
    PubMedGoogle Scholar
  • 38 McGregor JL, Catimel B, Parmentier S, Clezardin P, Dechavanne M, Leung LL K. Rapid purification and partial characterization of human platelet glycoprotein IIIb. Interaction with thrombospondin and its role in platelet aggregation J Biol Chem 1989; 264: 501-506
    PubMedGoogle Scholar
  • 39 Pidard D, Montgomery RR, Bennett JS, Kunicki TJ. Interaction of AP-2, a monoclonal antibody specific for human platelet glycoprotein Ilb-IIIa complex, with intact platelets. J Biol Chem 1983; 258: 12582-12586
    PubMedGoogle Scholar
  • 40 Gerrard JM, Lint D, Sims PJ, Wiedmer T, Fugate RD, McMillan E, Robertson C, Israels SJ. Identification of a platelet dense granule membrane protein that is deficient in a patient with Hermansky-Pudlak syndrome. Blood 1991; 77: 101-112
    PubMedGoogle Scholar
  • 41 Abrams CS, Ruggeri ZM, Taub R, Hoxie JA, Nagaswami C, Weisel JW, Shattil SJ. Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (gp) IIb-IIIa complex mimic gp Ilb-IIIa by recognizing fibrinogen. J Biol Chem 1992; 267: 2775-2785
    PubMedGoogle Scholar
  • 42 Pedvis LG, Wong T, Frojmovic MM. Differential inhibition of the platelet activation sequence: shape change, micro- and macro-aggregation by a stable prostacyclin analogue (Iloprost). Thromb Haemost 1988; 59: 323-328
    Article in Thieme ConnectPubMedGoogle Scholar
  • 43 Frojmovic M, Wong T, van de Ven T. Dynamic measurements of the platelet membrane glycoprotein Ilb-IIIa receptor for fibrinogen by flow cytometry. I. Methodology, theory and results for two distinct activators Biophys J 1991; 59: 815-827
    CrossrefPubMedGoogle Scholar
  • 44 Fay SP, Posner RG, Swann WN, Sklar LA. Real time analysis of the assembly of ligand, receptor and G protein by quantitative fluorescence flow cytometry. Biochem 1991; 30: 5066-5075
    CrossrefPubMedGoogle Scholar
  • 45 Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-315
    PubMedGoogle Scholar
  • 46 Rochon YP, Frojmovic MM, Mills EL. Comparative studies of microscopically-determined aggregation, degranulation, and light transmission after chemotactic activation of adult and new-born neutrophils. Blood 1990; 75: 2053-2060
    PubMedGoogle Scholar
  • 47 Sklar LA, Finney DA, Oades ZG, Jesaitis AJ, Painter RG, Cochrane CG. The dynamics of ligand-receptor interactions. J Biol Chem 1984; 259: 5661-5669
    PubMedGoogle Scholar
  • 48 Wong T, Pedvis L, Frojmovic M. Platelet size affects both micro- and macroaggregation: contributions of platelet number, volume fraction and cell surface. Thromb Haemost 1989; 62: 733-741
    Article in Thieme ConnectPubMedGoogle Scholar
  • 49 Cole SR, Ashman LK, Ey PL. Biotinylation: An alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates. Molecular Immunology 1987; 24: 699-705
    CrossrefPubMedGoogle Scholar
  • 50 Lombardo VT, Hodson E, Roberts JR, Kunicki TJ, Zimmerman TS, Ruggeri ZM. Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody. J Clin Invest 1985; 76: 1950-1958
    CrossrefPubMedGoogle Scholar
  • 51 Bell DN, Spain S, Goldsmith HL. Adenosine disphosphate-induced aggregation of human platelets in flow through tubes. 1. Measurement of concentration and size of single platelets and aggregates Biophys J 1989; 56: 817-828
    CrossrefPubMedGoogle Scholar
  • 52 Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987; 238: 491-497
    CrossrefPubMedGoogle Scholar
  • 53 Kelm RJ Jr, Mann KG. Human platelet osteonectin: release, surface expression, and partial characterization. Blood 1990; 75: 1105-1113
    PubMedGoogle Scholar
  • 54 Ruan CG, Du XP, Xi XD, Castaldi PA, Berndt MC. A murine antiglycoprotein lb complex monoclonal antibody, SZ2, inhibits platelet aggregation induced by both ristocetin and collagen. Blood 1987; 69: 570-577
    PubMedGoogle Scholar
  • 55 Adelman B, Carlson P, Powers P. von Willebrand factor binds to stimulated platelets in plasma. Blood 1987; 70: 1362-1369
    PubMedGoogle Scholar
  • 56 Kroll MH, Schafer AI. Biochemical mechanisms of platelet activation. Blood 1989; 74: 1181-1195
    PubMedGoogle Scholar
  • 57 Isenberg WM, Bainton DF, Newman PJ. Monoclonal antibodies bound to subunits of the integrin gpIIb/IIIa are internalized and interfere with filapodia formation and platelet aggregation. Blood 1990; 76: 1564-1571
    PubMedGoogle Scholar
  • 58 Ugarova TP, Budzynski AZ, Shattil SJ, Ruggeri ZM, Ginsberg MH, Plow EF. Conformational changes in fibrinogen elicited by its interaction with platelet membrane gpIIb-IIIa. J Biol Chem 1993; 268: 21080-21087
    PubMedGoogle Scholar
  • 59 Frojmovic MM, Milton JG. Human platelet size, shape, and related functions in health and disease. Physiol Rev 1982; 62: 185-261
    CrossrefPubMedGoogle Scholar
  • 60 Tuszynski GP, Rothman VL, Murphy A, Siegler K, Knudsen KA. Thrombospondin promotes platelet aggregation. Blood 1988; 72: 109-115
    PubMedGoogle Scholar
  • 61 Wolff R, Plow EF, Ginsberg MH. Interaction of thrombospondin with resting and stimulated human platelets. J Biol Chem 1986; 261: 6840-6846
    PubMedGoogle Scholar