Abstract
Thrombin is an allosteric enzyme existing in two forms, slow and fast, that differ widely in their specificities toward synthetic and natural amide substrates. The two forms are significantly populated in vivo, and the allosteric equilibrium can be affected by the binding of effectors and natural substrates. The fast form is procoagulant because it cleaves fibrinogen with higher specificity; the slow form is anticoagulant because it cleaves protein C with higher specificity. Binding of thrombomodulin inhibits cleavage of fibrinogen by the fast form and promotes cleavage of protein C by the slow form. The allosteric properties of thrombin, which has targeted two distinct conformational states toward its two fundamental and competing roles in hemostasis, are paradigmatic of a molecular strategy that is likely to be exploited by other proteases in the blood coagulation cascade.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Amphlett G. W., Kisiel W., Castellino F. J. Interaction of calcium with bovine plasma protein C. Biochemistry. 1981 Apr 14;20(8):2156–2161. doi: 10.1021/bi00511a013. [DOI] [PubMed] [Google Scholar]
- Ayala Y., Di Cera E. Molecular recognition by thrombin. Role of the slow-->fast transition, site-specific ion binding energetics and thermodynamic mapping of structural components. J Mol Biol. 1994 Jan 14;235(2):733–746. doi: 10.1006/jmbi.1994.1024. [DOI] [PubMed] [Google Scholar]
- Bajaj S. P., Butkowski R. J., Mann K. G. Prothrombin fragments. Ca2+ binding and activation kinetics. J Biol Chem. 1975 Mar 25;250(6):2150–2156. [PubMed] [Google Scholar]
- Banfield D. K., MacGillivray R. T. Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2779–2783. doi: 10.1073/pnas.89.7.2779. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barshop B. A., Wrenn R. F., Frieden C. Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system. Anal Biochem. 1983 Apr 1;130(1):134–145. doi: 10.1016/0003-2697(83)90660-7. [DOI] [PubMed] [Google Scholar]
- DAVIE E. W., RATNOFF O. D. WATERFALL SEQUENCE FOR INTRINSIC BLOOD CLOTTING. Science. 1964 Sep 18;145(3638):1310–1312. doi: 10.1126/science.145.3638.1310. [DOI] [PubMed] [Google Scholar]
- Dang Q. D., Di Cera E. A simple activity assay for thrombin and hirudin. J Protein Chem. 1994 May;13(4):367–373. doi: 10.1007/BF01901692. [DOI] [PubMed] [Google Scholar]
- Davie E. W., Fujikawa K., Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991 Oct 29;30(43):10363–10370. doi: 10.1021/bi00107a001. [DOI] [PubMed] [Google Scholar]
- Doyle M. F., Mann K. G. Multiple active forms of thrombin. IV. Relative activities of meizothrombins. J Biol Chem. 1990 Jun 25;265(18):10693–10701. [PubMed] [Google Scholar]
- Esmon C. T., Owen W. G. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2249–2252. doi: 10.1073/pnas.78.4.2249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Esmon C. T. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem. 1989 Mar 25;264(9):4743–4746. [PubMed] [Google Scholar]
- Esmon N. L., DeBault L. E., Esmon C. T. Proteolytic formation and properties of gamma-carboxyglutamic acid-domainless protein C. J Biol Chem. 1983 May 10;258(9):5548–5553. [PubMed] [Google Scholar]
- Esmon N. L., Owen W. G., Esmon C. T. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem. 1982 Jan 25;257(2):859–864. [PubMed] [Google Scholar]
- Galvin J. B., Kurosawa S., Moore K., Esmon C. T., Esmon N. L. Reconstitution of rabbit thrombomodulin into phospholipid vesicles. J Biol Chem. 1987 Feb 15;262(5):2199–2205. [PubMed] [Google Scholar]
- Johnson A. E., Esmon N. L., Laue T. M., Esmon C. T. Structural changes required for activation of protein C are induced by Ca2+ binding to a high affinity site that does not contain gamma-carboxyglutamic acid. J Biol Chem. 1983 May 10;258(9):5554–5560. [PubMed] [Google Scholar]
- Le Bonniec B. F., Esmon C. T. Glu-192----Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7371–7375. doi: 10.1073/pnas.88.16.7371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MACFARLANE R. G. AN ENZYME CASCADE IN THE BLOOD CLOTTING MECHANISM, AND ITS FUNCTION AS A BIOCHEMICAL AMPLIFIER. Nature. 1964 May 2;202:498–499. doi: 10.1038/202498a0. [DOI] [PubMed] [Google Scholar]
- Mann K. G., Nesheim M. E., Church W. R., Haley P., Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood. 1990 Jul 1;76(1):1–16. [PubMed] [Google Scholar]
- Mathews I. I., Padmanabhan K. P., Tulinksy A., Sadler J. E. Structure of a nonadecapeptide of the fifth EGF domain of thrombomodulin complexed with thrombin. Biochemistry. 1994 Nov 22;33(46):13547–13552. doi: 10.1021/bi00250a006. [DOI] [PubMed] [Google Scholar]
- Mathur A., Schlapkohl W. A., Di Cera E. Thrombin-fibrinogen interaction: pH dependence and effects of the slow-->fast transition. Biochemistry. 1993 Jul 27;32(29):7568–7573. doi: 10.1021/bi00080a031. [DOI] [PubMed] [Google Scholar]
- Nayal M., Di Cera E. Predicting Ca(2+)-binding sites in proteins. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):817–821. doi: 10.1073/pnas.91.2.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ng A. S., Lewis S. D., Shafer J. A. Quantifying thrombin-catalyzed release of fibrinopeptides from fibrinogen using high-performance liquid chromatography. Methods Enzymol. 1993;222:341–358. doi: 10.1016/0076-6879(93)22023-9. [DOI] [PubMed] [Google Scholar]
- Rezaie A. R., Mather T., Sussman F., Esmon C. T. Mutation of Glu-80-->Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombin-thrombomodulin complex. J Biol Chem. 1994 Feb 4;269(5):3151–3154. [PubMed] [Google Scholar]
- Steiner S. A., Castellino F. J. Kinetic studies of the role of monovalent cations in the amidolytic activity of activated bovine plasma protein C. Biochemistry. 1982 Sep 14;21(19):4609–4614. doi: 10.1021/bi00262a015. [DOI] [PubMed] [Google Scholar]
- Toney M. D., Hohenester E., Cowan S. W., Jansonius J. N. Dialkylglycine decarboxylase structure: bifunctional active site and alkali metal sites. Science. 1993 Aug 6;261(5122):756–759. doi: 10.1126/science.8342040. [DOI] [PubMed] [Google Scholar]
- Tulinsky A. The structures of domains of blood proteins. Thromb Haemost. 1991 Jul 12;66(1):16–31. [PubMed] [Google Scholar]
- Wells C. M., Di Cera E. Thrombin is a Na(+)-activated enzyme. Biochemistry. 1992 Dec 1;31(47):11721–11730. doi: 10.1021/bi00162a008. [DOI] [PubMed] [Google Scholar]
- Wu Q. Y., Sheehan J. P., Tsiang M., Lentz S. R., Birktoft J. J., Sadler J. E. Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6775–6779. doi: 10.1073/pnas.88.15.6775. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zimmerle C. T., Frieden C. Analysis of progress curves by simulations generated by numerical integration. Biochem J. 1989 Mar 1;258(2):381–387. doi: 10.1042/bj2580381. [DOI] [PMC free article] [PubMed] [Google Scholar]