Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Oct 1;101(4):1434–1441. doi: 10.1083/jcb.101.4.1434

Mapping of epitopes for monoclonal antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing

PMCID: PMC2113943  PMID: 2413043

Abstract

A panel of monoclonal antibodies (Mab's) has been raised against human platelet thrombospondin (TSP). One Mab, designated A2.5, inhibits the hemagglutinating activity of TSP and immunoprecipitates the NH2 terminal 25 kD heparin binding domain of TSP (Dixit, V.M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Biochemistry, in press). Another Mab, C6.7, blocks the thrombin-stimulated aggregation of live platelets and immunoprecipitates an 18-kD fragment distinct from the heparin binding domain (Dixit, V. M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Proc. Natl. Acad. Sci. 82: 3472-3476). To determine the relative locations of the epitopes for these Mabs in the three-dimensional structure of TSP, we have examined TSP-Mab complexes by electron microscopy of rotary- shadowed proteins. The TSP molecule is composed of three 180-kD subunits, each of which consists of a small globular domain (approximately 8 nm diam) and a larger globular domain (approximately 16 nm diam) connected by a thin, flexible strand. The subunit interaction site is on the thin connecting strands, nearer the small globular domains. Mab A2.5 binds to the cluster of three small domains, indicating that this region contains the heparin binding domain and thus represents the NH2 termini of the TSP peptide chains. Mab C6.7 binds to the large globular domains on the side opposite the point at which the connecting strand enters the domain, essentially the maximum possible distance from the A2.5 epitope. Using high sensitivity automated NH2 terminal sequencing of TSP chymotryptic peptides we have ordered these fragments within the TSP peptide chain and have confirmed that the epitope for C6.7 in fact lies near the extreme COOH terminus of the peptide chain. In combination with other data, we have been able to construct a map of the linear order of the identified domains of TSP that indicates that to a large extent, the domains are arranged co- linearly with the peptide chain.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Baenziger N. L., Brodie G. N., Majerus P. W. Isolation and properties of a thrombin-sensitive protein of human platelets. J Biol Chem. 1972 May 10;247(9):2723–2731. [PubMed] [Google Scholar]
  2. Coligan J. E., Slayter H. S. Structure of thrombospondin. J Biol Chem. 1984 Mar 25;259(6):3944–3948. [PubMed] [Google Scholar]
  3. Dixit V. M., Grant G. A., Frazier W. A., Santoro S. A. Isolation of the fibrinogen-binding region of platelet thrombospondin. Biochem Biophys Res Commun. 1984 Mar 30;119(3):1075–1081. doi: 10.1016/0006-291x(84)90884-2. [DOI] [PubMed] [Google Scholar]
  4. Dixit V. M., Grant G. A., Santoro S. A., Frazier W. A. Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin. J Biol Chem. 1984 Aug 25;259(16):10100–10105. [PubMed] [Google Scholar]
  5. Dixit V. M., Haverstick D. M., O'Rourke K. M., Hennessy S. W., Grant G. A., Santoro S. A., Frazier W. A. A monoclonal antibody against human thrombospondin inhibits platelet aggregation. Proc Natl Acad Sci U S A. 1985 May;82(10):3472–3476. doi: 10.1073/pnas.82.10.3472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dixit V. M., Haverstick D. M., O'Rourke K., Hennessy S. W., Broekelmann T. J., McDonald J. A., Grant G. A., Santoro S. A., Frazier W. A. Inhibition of platelet aggregation by a monoclonal antibody against human fibronectin. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3844–3848. doi: 10.1073/pnas.82.11.3844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gartner T. K., Gerrard J. M., White J. G., Williams D. C. Fibrinogen is the receptor for the endogenous lectin of human platelets. Nature. 1981 Feb 19;289(5799):688–690. doi: 10.1038/289688a0. [DOI] [PubMed] [Google Scholar]
  8. Gartner T. K., Gerrard J. M., White J. G., Williams D. C. The endogenous lectin of human platelets is an alpha-granule component. Blood. 1981 Jul;58(1):153–157. [PubMed] [Google Scholar]
  9. Gartner T. K., Phillips D. R., Williams D. C. Expression of thrombin-enhanced platelet lectin activity is controlled by secretion. FEBS Lett. 1980 May 5;113(2):196–199. doi: 10.1016/0014-5793(80)80590-4. [DOI] [PubMed] [Google Scholar]
  10. Gartner T. K., Walz D. A., Aiken M., Starr-Spires L., Ogilvie M. L. Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation. Biochem Biophys Res Commun. 1984 Oct 15;124(1):290–295. doi: 10.1016/0006-291x(84)90950-1. [DOI] [PubMed] [Google Scholar]
  11. Gartner T. K., Walz D. A., Aiken M., Starr-Spires L., Ogilvie M. L. Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation. Biochem Biophys Res Commun. 1984 Oct 15;124(1):290–295. doi: 10.1016/0006-291x(84)90950-1. [DOI] [PubMed] [Google Scholar]
  12. Haverstick D. M., Dixit V. M., Grant G. A., Frazier W. A., Santoro S. A. Characterization of the platelet agglutinating activity of thrombospondin. Biochemistry. 1985 Jun 18;24(13):3128–3134. doi: 10.1021/bi00334a009. [DOI] [PubMed] [Google Scholar]
  13. Haverstick D. M., Dixit V. M., Grant G. A., Frazier W. A., Santoro S. A. Localization of the hemagglutinating activity of platelet thrombospondin to a 140 000-dalton thermolytic fragment. Biochemistry. 1984 Nov 6;23(23):5597–5603. doi: 10.1021/bi00318a033. [DOI] [PubMed] [Google Scholar]
  14. Jaffe E. A., Leung L. L., Nachman R. L., Levin R. I., Mosher D. F. Thrombospondin is the endogenous lectin of human platelets. Nature. 1982 Jan 21;295(5846):246–248. doi: 10.1038/295246a0. [DOI] [PubMed] [Google Scholar]
  15. Lawler J. W., Slayter H. S., Coligan J. E. Isolation and characterization of a high molecular weight glycoprotein from human blood platelets. J Biol Chem. 1978 Dec 10;253(23):8609–8616. [PubMed] [Google Scholar]
  16. Lawler J. W., Slayter H. S. The release of heparin binding peptides from platelet thrombospondin by proteolytic action of thrombin, plasmin and trypsin. Thromb Res. 1981 May 1;22(3):267–279. doi: 10.1016/0049-3848(81)90119-5. [DOI] [PubMed] [Google Scholar]
  17. Lawler J., Chao F. C., Cohen C. M. Evidence for calcium-sensitive structure in platelet thrombospondin. Isolation and partial characterization of thrombospondin in the presence of calcium. J Biol Chem. 1982 Oct 25;257(20):12257–12265. [PubMed] [Google Scholar]
  18. Lawler J., Derick L. H., Connolly J. E., Chen J. H., Chao F. C. The structure of human platelet thrombospondin. J Biol Chem. 1985 Mar 25;260(6):3762–3772. [PubMed] [Google Scholar]
  19. Leung L. L., Nachman R. L. Complex formation of platelet thrombospondin with fibrinogen. J Clin Invest. 1982 Sep;70(3):542–549. doi: 10.1172/JCI110646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leung L. L. Role of thrombospondin in platelet aggregation. J Clin Invest. 1984 Nov;74(5):1764–1772. doi: 10.1172/JCI111595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mumby S. M., Raugi G. J., Bornstein P. Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen. J Cell Biol. 1984 Feb;98(2):646–652. doi: 10.1083/jcb.98.2.646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Phillips D. R., Jennings L. K., Prasanna H. R. Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets. J Biol Chem. 1980 Dec 25;255(24):11629–11632. [PubMed] [Google Scholar]
  23. Raugi G. J., Mumby S. M., Ready C. A., Bornstein P. Location and partial characterization of the heparin-binding fragment of platelet thrombospondin. Thromb Res. 1984 Oct 15;36(2):165–175. doi: 10.1016/0049-3848(84)90338-4. [DOI] [PubMed] [Google Scholar]
  24. Roberts D. D., Haverstick D. M., Dixit V. M., Frazier W. A., Santoro S. A., Ginsburg V. The platelet glycoprotein thrombospondin binds specifically to sulfated glycolipids. J Biol Chem. 1985 Aug 5;260(16):9405–9411. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES