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. 1993 Feb 1;289(Pt 3):903–909. doi: 10.1042/bj2890903

Characterization of the antiplasmin activity of human thrombospondin-1 in solution.

P K Anonick 1, J K Yoo 1, D J Webb 1, S L Gonias 1
PMCID: PMC1132261  PMID: 7679575

Abstract

These studies demonstrate relatively rapid association of plasmin with thrombospondin and the effects of this interaction on plasmin activity towards D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) and the proteinase inhibitors alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M). Binding of plasmin to thrombospondin reached an apparent reversible equilibrium within 3 min at 22 degrees C. The amidase activity of bound plasmin was inhibited. An analysis of S-2251 hydrolysis indicated that thrombospondin is a linear mixed-type plasmin inhibitor. The dissociation constant (KD) for the binding of plasmin to thrombospondin was 0.5 microM, assuming one plasmin binding site per thrombospondin homotrimer. Plasmin and miniplasmin slowly cleaved thrombospondin, yielding products which were comparable with those generated by other proteinases. Tranexamic acid inhibited the digestion of thrombospondin by plasmin and miniplasmin, suggesting an important role for the kringle-5 domain in this process. When plasmin was incubated first with thrombospondin and then with alpha 2AP, plasmin that was apparently bound to thrombospondin reacted with alpha 2AP at a decreased rate; however, within 20 min, all of the plasmin was recovered in complex with alpha 2AP. Similar results were obtained with alpha 2M. Transfer of plasmin from thrombospondin to alpha 2AP or alpha 2M probably required plasmin-thrombospondin-complex dissociation. A low level of reaction of alpha 2AP with thrombospondin-associated plasmin could not be ruled out. These results demonstrate that the activity of plasmin, when bound to thrombospondin, is greatly diminished or eliminated. The plasmin-thrombospondin complex, which is formed within 3 min, is fully reversible and the associated plasmin is in a latent form protected from proteinase inhibitors. Therefore, thrombospondin may regulate plasmin activity in a manner which is distinct from conventional proteinase inhibitors and other extracellular-matrix proteins.

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Selected References

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  1. Anonick P. K., Gonias S. L. Soluble fibrin preparations inhibit the reaction of plasmin with alpha 2-macroglobulin. Comparison with alpha 2-antiplasmin and leupeptin. Biochem J. 1991 Apr 1;275(Pt 1):53–59. doi: 10.1042/bj2750053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anonick P. K., Vasudevan J., Gonias S. L. Antifibrinolytic activities of alpha-N-acetyl-L-lysine methyl ester, epsilon-aminocaproic acid, and tranexamic acid. Importance of kringle interactions and active site inhibition. Arterioscler Thromb. 1992 Jun;12(6):708–716. doi: 10.1161/01.atv.12.6.708. [DOI] [PubMed] [Google Scholar]
  3. Asch A. S., Nachman R. L. Thrombospondin: phenomenology to function. Prog Hemost Thromb. 1989;9:157–176. [PubMed] [Google Scholar]
  4. 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]
  5. Castellino F. J. Biochemistry of human plasminogen. Semin Thromb Hemost. 1984 Jan;10(1):18–23. doi: 10.1055/s-2007-1004404. [DOI] [PubMed] [Google Scholar]
  6. Chase T., Jr, Shaw E. p-Nitrophenyl-p'-guanidinobenzoate HCl: a new active site titrant for trypsin. Biochem Biophys Res Commun. 1967 Nov 30;29(4):508–514. doi: 10.1016/0006-291x(67)90513-x. [DOI] [PubMed] [Google Scholar]
  7. DePoli P., Bacon-Baguley T., Kendra-Franczak S., Cederholm M. T., Walz D. A. Thrombospondin interaction with plasminogen. Evidence for binding to a specific region of the kringle structure of plasminogen. Blood. 1989 Mar;73(4):976–982. [PubMed] [Google Scholar]
  8. Deutsch D. G., Mertz E. T. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970 Dec 4;170(3962):1095–1096. doi: 10.1126/science.170.3962.1095. [DOI] [PubMed] [Google Scholar]
  9. Dixit V. M., Hennessy S. W., Grant G. A., Rotwein P., Frazier W. A. Characterization of a cDNA encoding the heparin and collagen binding domains of human thrombospondin. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5449–5453. doi: 10.1073/pnas.83.15.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Forsgren M., Råden B., Israelsson M., Larsson K., Hedén L. O. Molecular cloning and characterization of a full-length cDNA clone for human plasminogen. FEBS Lett. 1987 Mar 23;213(2):254–260. doi: 10.1016/0014-5793(87)81501-6. [DOI] [PubMed] [Google Scholar]
  11. Gonias S. L. Alpha 2-macroglobulin: a protein at the interface of fibrinolysis and cellular growth regulation. Exp Hematol. 1992 Mar;20(3):302–311. [PubMed] [Google Scholar]
  12. Hall S. W., Humphries J. E., Gonias S. L. Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin. J Biol Chem. 1991 Jul 5;266(19):12329–12336. [PubMed] [Google Scholar]
  13. Hogg P. J., Stenflo J., Mosher D. F. Thrombospondin is a slow tight-binding inhibitor of plasmin. Biochemistry. 1992 Jan 14;31(1):265–269. doi: 10.1021/bi00116a036. [DOI] [PubMed] [Google Scholar]
  14. Imber M. J., Pizzo S. V. Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin. J Biol Chem. 1981 Aug 10;256(15):8134–8139. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Lawler J., Hynes R. O. The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J Cell Biol. 1986 Nov;103(5):1635–1648. doi: 10.1083/jcb.103.5.1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lawler J. The structural and functional properties of thrombospondin. Blood. 1986 May;67(5):1197–1209. [PubMed] [Google Scholar]
  19. 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]
  20. Margossian S. S., Lawler J. W., Slayter H. S. Physical characterization of platelet thrombospondin. J Biol Chem. 1981 Jul 25;256(14):7495–7500. [PubMed] [Google Scholar]
  21. Mosher D. F. Physiology of thrombospondin. Annu Rev Med. 1990;41:85–97. doi: 10.1146/annurev.me.41.020190.000505. [DOI] [PubMed] [Google Scholar]
  22. Santoro S. A., Frazier W. A. Isolation and characterization of thrombospondin. Methods Enzymol. 1987;144:438–446. doi: 10.1016/0076-6879(87)44193-1. [DOI] [PubMed] [Google Scholar]
  23. Silverstein R. L., Leung L. L., Harpel P. C., Nachman R. L. Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator. J Clin Invest. 1984 Nov;74(5):1625–1633. doi: 10.1172/JCI111578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Silverstein R. L., Nachman R. L., Leung L. L., Harpel P. C. Activation of immobilized plasminogen by tissue activator. Multimolecular complex formation. J Biol Chem. 1985 Aug 25;260(18):10346–10352. [PubMed] [Google Scholar]
  25. Steiner J. P., Migliorini M., Strickland D. K. Characterization of the reaction of plasmin with alpha 2-macroglobulin: effect of antifibrinolytic agents. Biochemistry. 1987 Dec 15;26(25):8487–8495. doi: 10.1021/bi00399a068. [DOI] [PubMed] [Google Scholar]
  26. Wiman B. Affinity-chromatographic purification of human alpha 2-antiplasmin. Biochem J. 1980 Oct 1;191(1):229–232. doi: 10.1042/bj1910229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wiman B., Collen D. On the kinetics of the reaction between human antiplasmin and plasmin. Eur J Biochem. 1978 Mar 15;84(2):573–578. doi: 10.1111/j.1432-1033.1978.tb12200.x. [DOI] [PubMed] [Google Scholar]
  28. Wiman B., Lijnen H. R., Collen D. On the specific interaction between the lysine-binding sites in plasmin and complementary sites in alpha2-antiplasmin and in fibrinogen. Biochim Biophys Acta. 1979 Jul 25;579(1):142–154. doi: 10.1016/0005-2795(79)90094-1. [DOI] [PubMed] [Google Scholar]

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