Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1980 Dec;66(6):1311–1318. doi: 10.1172/JCI109983

Isolation and Quantitation of the Platelet Membrane Glycoprotein Deficient in Thrombasthenia Using a Monoclonal Hybridoma Antibody

Rodger P McEver 1,2, Nancy Lewis Baenziger 1,2, Philip W Majerus 1,2
PMCID: PMC371616  PMID: 6449521

Abstract

We used the hybridoma technique to characterize further the platelet glycoprotein abnormality in Glanzmann's thrombasthenia. Spleen cells from Balb/c mice immunized with human platelets were fused to mouse myeloma cell line Sp2/0-Ag14. Hybridoma lines producing a variety of antiplatelet antibodies were isolated by hypoxanthine-aminopterin-thymidine selection and cloned, and purified monoclonal IgG from six lines was prepared. One of these lines, 8aB5-9, produced an antibody, Tab, that binds to a protein on normal but not thrombasthenic platelets. We isolated this protein from Triton X-100 solubilized normal platelet membranes by affinity chromatography on Tab-Sepharose. As determined by SDS polyacrylamide gel electrophoresis, the isolated protein is a complex of glycoproteins IIb and IIIa, because the two subunits comigrate with glycoproteins IIb and IIIa of whole platelets and show identical changes in mobility after disulfide bond reduction. We prepared 125I-Tab to determine the number of glycoprotein IIb-IIIa complexes on normal and thrombasthenic platelets by a direct binding assay. Platelets from 17 normal donors bound 39,000±4,600 (SD) Tab molecules/platelet. Platelets from four patients with thrombasthenia lacked Tab binding sites (<5%). Five obligate and four presumed heterozygotes for thrombasthenia bound 24,500±5,800 Tab molecules/platelet. The platelet alloantigen, PlAl, is not that recognized by Tab, because platelets from three PlAl-negative subjects bound Tab normally. Studies with the Tab antibody have (a) enabled quantitation of the number of glycoprotein IIb-IIIa complexes on normal platelet membranes, (b) demonstrated that thrombasthenic homozygotes lack and heterozygotes have a partial deficiency of this complex, and (c) made possible the isolation of this membrane protein which may be required for normal platelet aggregation and clot retraction.

Full text

PDF
1312

Images in this article

1315Image
on p.1315

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BRINKHOUS K. M., READ M. S., MASON R. G. PLASMA THROMBOCYTE-AGGLUTINATING ACTIVITY AND FIBRINOGEN. SYNERGISM WITH ADENOSINE DIPHOSPHATE. Lab Invest. 1965 Apr;14:335–342. [PubMed] [Google Scholar]
  2. Bennett J. S., Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest. 1979 Nov;64(5):1393–1401. doi: 10.1172/JCI109597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett V., Branton D. Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin. J Biol Chem. 1977 Apr 25;252(8):2753–2763. [PubMed] [Google Scholar]
  4. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  5. CROSS M. J. EFFECT OF FIBRINOGEN ON THE AGGREGATION OF PLATELETS BY ADENOSINE DIPHOSPHATE. Thromb Diath Haemorrh. 1964 Dec 31;12:524–527. [PubMed] [Google Scholar]
  6. Coller B. S. Interaction of normal, thrombasthenic, and Bernard-Soulier platelets with immobilized fibrinogen: defective platelet-fibrinogen interaction in thrombasthenia. Blood. 1980 Feb;55(2):169–178. [PubMed] [Google Scholar]
  7. Davidson R. L., Gerald P. S. Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol. Somatic Cell Genet. 1976 Mar;2(2):165–176. doi: 10.1007/BF01542629. [DOI] [PubMed] [Google Scholar]
  8. Degos L., Dautigny A., Brouet J. C., Colombani M., Ardaillou N., Caen J. P., Colombani J. A molecular defect in thrombasthenic platelets. J Clin Invest. 1975 Jul;56(1):236–240. doi: 10.1172/JCI108074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Galfre G., Howe S. C., Milstein C., Butcher G. W., Howard J. C. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature. 1977 Apr 7;266(5602):550–552. doi: 10.1038/266550a0. [DOI] [PubMed] [Google Scholar]
  10. Gerrard J. M., Schollmeyer J. V., Phillips D. R., White J. G. alpha-Actinin deficiency in thrombasthenia: possible identity of alpha-actinin and glycoprotein III. Am J Pathol. 1979 Mar;94(3):509–528. [PMC free article] [PubMed] [Google Scholar]
  11. Goodfellow P. N., Levinson J. R., Williams V. E., 2nd, McDevitt H. O. Monoclonal antibodies reacting with murine teratocarcinoma cells. Proc Natl Acad Sci U S A. 1979 Jan;76(1):377–380. doi: 10.1073/pnas.76.1.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Green D., Rossi E. C. Disorders of platelet function. Med Clin North Am. 1972 Jan;56(1):35–46. doi: 10.1016/s0025-7125(16)32421-x. [DOI] [PubMed] [Google Scholar]
  13. Hagen I., Nurden A., Bjerrum O. J., Solum N. O., Caen J. Immunochemical evidence for protein abnormalities in platelets from patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome. J Clin Invest. 1980 Mar;65(3):722–731. doi: 10.1172/JCI109719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hawiger J., Parkinson S., Timmons S. Prostacyclin inhibits mobilisation of fibrinogen-binding sites on human ADP- and thrombin-treated platelets. Nature. 1980 Jan 10;283(5743):195–197. doi: 10.1038/283195a0. [DOI] [PubMed] [Google Scholar]
  15. Jamieson G. A., Okumura T., Fishback B., Johnson M. M., Egan J. J., Weiss H. J. Platelet membrane glycoproteins in thrombasthenia, Bernard-Soulier syndrome, and storage pool disease. J Lab Clin Med. 1979 Apr;93(4):652–660. [PubMed] [Google Scholar]
  16. Kunicki T. J., Aster R. H. Deletion of the platelet-specific alloantigen PlA1 from platelets in Glanzmann's thrombasthenia. J Clin Invest. 1978 May;61(5):1225–1231. doi: 10.1172/JCI109038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kunicki T. J., Aster R. H. Isolation and immunologic characterization of the human platelet alloantigen, P1A1. Mol Immunol. 1979 Jun;16(6):353–360. doi: 10.1016/0161-5890(79)90100-7. [DOI] [PubMed] [Google Scholar]
  18. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  19. LITTLEFIELD J. W. SELECTION OF HYBRIDS FROM MATINGS OF FIBROBLASTS IN VITRO AND THEIR PRESUMED RECOMBINANTS. Science. 1964 Aug 14;145(3633):709–710. doi: 10.1126/science.145.3633.709. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lemke H., Hammerling G. J., Hohmann C., Rajewsky K. Hybrid cell lines secreting monoclonal antibody specific for major histocompatibility antigens of the mouse. Nature. 1978 Jan 19;271(5642):249–251. doi: 10.1038/271249a0. [DOI] [PubMed] [Google Scholar]
  22. Lusher J. M., Barnhart M. I. Congenital disorders affecting platelets. Semin Thromb Hemost. 1977 Fall;4(2):123–186. doi: 10.1055/s-0028-1087131. [DOI] [PubMed] [Google Scholar]
  23. March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
  24. Marguerie G. A., Edgington T. S., Plow E. F. Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem. 1980 Jan 10;255(1):154–161. [PubMed] [Google Scholar]
  25. Marguerie G. A., Plow E. F., Edgington T. S. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem. 1979 Jun 25;254(12):5357–5363. [PubMed] [Google Scholar]
  26. Miletich J. P., Jackson C. M., Majerus P. W. Interaction of coagulation factor Xa with human platelets. Proc Natl Acad Sci U S A. 1977 Sep;74(9):4033–4036. doi: 10.1073/pnas.74.9.4033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miletich J. P., Jackson C. M., Majerus P. W. Properties of the factor Xa binding site on human platelets. J Biol Chem. 1978 Oct 10;253(19):6908–6916. [PubMed] [Google Scholar]
  28. Mustard J. F., Kinlough-Rathbone R. L., Packham M. A., Perry D. W., Harfenist E. J., Pai K. R. Comparison of fibrinogen association with normal and thrombasthenic platelets on exposure to ADP or chymotrypsin. Blood. 1979 Nov;54(5):987–993. [PubMed] [Google Scholar]
  29. Mustard J. F., Packham M. A., Kinlough-Rathbone R. L., Perry D. W., Regoeczi E. Fibrinogen and ADP-induced platelet aggregation. Blood. 1978 Aug;52(2):453–466. [PubMed] [Google Scholar]
  30. Nurden A. T., Caen J. P. An abnormal platelet glycoprotein pattern in three cases of Glanzmann's thrombasthenia. Br J Haematol. 1974 Oct;28(2):253–260. doi: 10.1111/j.1365-2141.1974.tb06660.x. [DOI] [PubMed] [Google Scholar]
  31. Nurden A. T., Caen J. P. The different glycoprotein abnormalities in thrombasthenic and Bernard-Soulier platelets. Semin Hematol. 1979 Jul;16(3):234–250. [PubMed] [Google Scholar]
  32. Parham P. Purification of immunologically active HLA-A and -B antigens by a series of monoclonal antibody columns. J Biol Chem. 1979 Sep 25;254(18):8709–8712. [PubMed] [Google Scholar]
  33. Peerschke E. I., Zucker M. B., Grant R. A., Egan J. J., Johnson M. M. Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood. 1980 May;55(5):841–847. [PubMed] [Google Scholar]
  34. Phillips D. R., Agin P. P. Platelet membrane defects in Glanzmann's thrombasthenia. Evidence for decreased amounts of two major glycoproteins. J Clin Invest. 1977 Sep;60(3):535–545. doi: 10.1172/JCI108805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Phillips D. R., Agin P. P. Platelet plasma membrane glycoproteins. Evidence for the presence of nonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis. J Biol Chem. 1977 Mar 25;252(6):2121–2126. [PubMed] [Google Scholar]
  36. Phillips D. R., Jennings L. K., Edwards H. H. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol. 1980 Jul;86(1):77–86. doi: 10.1083/jcb.86.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shulman M., Wilde C. D., Köhler G. A better cell line for making hybridomas secreting specific antibodies. Nature. 1978 Nov 16;276(5685):269–270. doi: 10.1038/276269a0. [DOI] [PubMed] [Google Scholar]
  38. Solter D., Knowles B. B. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U S A. 1978 Nov;75(11):5565–5569. doi: 10.1073/pnas.75.11.5565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Solum N. O., Stormorken H. Influence of fibrinogen on the aggregation of washed human blood platelets induced by adenosine diphosphate, thrombin, collagen, and adrenaline. Scand J Clin Lab Invest. 1965;17(Suppl):170+–170+. [PubMed] [Google Scholar]
  40. Stern P. L., Willison K. R., Lennox E., Galfrè G., Milstein C., Secher D., Ziegler A. Monoclonal antibodies as probes for differentiation and tumor-associated antigens: a Forssman specificity on teratocarcinoma stem cells. Cell. 1978 Aug;14(4):775–783. doi: 10.1016/0092-8674(78)90333-1. [DOI] [PubMed] [Google Scholar]
  41. Tollefsen D. M., Feagler J. R., Majerus P. W. Induction of the platelet release reaction by phytohemagglutinin. J Clin Invest. 1974 Jan;53(1):211–218. doi: 10.1172/JCI107540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Williams A. F., Galfrè G., Milstein C. Analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes. Cell. 1977 Nov;12(3):663–673. doi: 10.1016/0092-8674(77)90266-5. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES