Abstract
α-Thrombin is a trypsin-like serine proteinase involved in blood coagulation and wound repair processes. Thrombin interacts with many macromolecular substrates, cofactors, cell-surface receptors, and blood plasma inhibitors. The three-dimensional structure of human α-thrombin shows multiple surface “exosites” for interactions with these macromolecules. We used these coordinates to probe the interaction of thrombin's active site and two exosites, anion-binding exosite-I and -II, with the blood plasma serine proteinase inhibitors (serpins) antithrombin (AT), heparin cofactor II (HC), and protein C inhibitor (PCI). Heparin, a widely used anticoagulant drug, accelerates the rate of thrombin inhibition by AT, PCI, and HC. Thrombin Quick II is a dysfunctional thrombin mutant with a Gly 226 → Val substitution in the substrate specificity pocket. We found that thrombin Quick II was inhibited by HC, but not by AT or PCI. Molecular modeling studies suggest that the larger Val side chain protrudes into the specificity pocket, allowing room for the smaller P1 side chain of HC (Leu) but not the larger P1 side chain of AT and PCI (both with Arg). λT −-Thrombin and thrombin Quick I (Arg 67 → Cys) are both altered in anion-binding exosite-I, yet bind to heparin-Sepharose and can be inhibited by AT, HC, and PCI in an essentially normal manner in the absence of heparin. In the presence of heparin, inhibition of these altered thrombins by HC is greatly reduced compared to both AT and PCI. α-Thrombin with chemically modified lysines in both anion-binding exosite-I and -II has no heparin accelerated thrombin inhibition by either AT or HC. Thrombin lysine-modified in the presence of heparin has protected residues in anion-binding exosite-II and the loss of heparin-accelerated inhibition by HC is greater than that by AT. Collectively, these results suggest differences in serpin reactive site recognition by thrombin and a more complicated mechanism for heparin-accelerated inhibition by HC compared to either AT or PCI.
Similar content being viewed by others
References
Banner, D. W., and Hadväry, P. (1991).J. Biol. Chem. 266 20,085–20,093.
Berliner, L. J. (1984).Mol. Cell. Biochem. 61 159–172.
Bezeaud, A., Denninger, M.-H., and Guillen, M.-C. (1985).Eur. J. Biochem. 153 491–496.
Blinder, M. A., Marasa, J. C., Reynolds, C. H., Deaven, L. L., and Tollefsen, D. M. (1988).Biochemistry 27 752–759.
Blinder, M. A., and Tollefsen, D. M. (1990).J. Biol. Chem. 265 286–291.
Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., and Hofsteenge, J. (1989).EMBO J. 8 3467–3475.
Bode, W., Turk, D., and Karshikov, A. (1992).Protein Sci. 1 426–471.
Brower, M. S., Walz, D. A., Garry, K. E., and Fenton, J. W., II (1987).Blood 69 813–819.
Casu, B. (1985).Adv. Carbohydr. Chem. Biochem. 43 51–134.
Casu, B. (1989).Ann. N.Y. Acad. Sci. 556 1–17.
Church, F. C., and Griffith, M. J. (1984).Biochem. Biophys. Res. Commun. 124 745–751.
Church, F. C., Noyes, C. M., and Griffith, M. J. (1985).Proc. Natl. Acad. Sci. USA 82 6431–6434.
Church, F. C., Pratt, C. W., and Hoffman, M. (1991).J. Biol. Chem. 266 704–709.
Church, F. C., Pratt, C. W., Noyes, C. M., Kalyanamit, T., Sherrill, G. B., Tobin, R. B., and Meade, J. M. (1989).J. Biol. Chem. 264 18,419–18,425.
Church, F. C., and Whinna, H. C. (1986).Anal. Biochem. 157 77–83.
de Agostini, A. I., Watkins, S. C., Slayter, H. S., Youssoufian, H., and Rosenberg, R. D. (1990).J. Cell Biol. 111 1293–1304.
Derechin, V., Blinder, M. A., and Tollefsen, D. M. (1990).J. Biol. Chem. 265 5623–5628.
Fenton, J. W., II (1988).Sem. Thromb. Hemost 14 234–240.
Fenton, J. W., II, Ofosu, F. A., Moon, D. G. and Maraganore, J. M. (1991).Blood Coag. Fibrinol. 2 69–75.
Fenton, J. W., II, Olson, T. A., Zabinski, M. P., and Wilner, G. D. (1988).Biochemistry 27 7106–7112.
Furie, B., and Furie, B. C. (1992).N. Engl. J. Med. 326 800–806.
Griffith, M. J. (1983).Proc. Natl. Acad. Sci. USA 80 5460–5465.
Griffith, M. J., Noyes, C. M., and Church, F. C. (1985a).J. Biol. Chem. 260 2218–2225.
Griffith, M. J., Noyes, C. M., Tyndall, J. A., and Church, F. C. (1985b).Biochemistry 24 6777–6782.
Grutter, M. G., Priestle, J. P., Rahuel, J., Grossenbacher, H., Bode, W., Hofsteenge, J., and Stone, S.R. (1990).EMBO J. 9 2361–2365.
Henriksen, R. A., and Mann, K. G. (1988).Biochemistry 27 9160–9165.
Henriksen, R. A., and Mann, K. G. (1989).Biochemistry 28 2078–2082.
Henriksen, R. A., and Owen, W. G. (1987).J. Biol. Chem. 262 4664–4669.
Hofsteenge, J., Braun, P. J., and Stone, S. R. (1988).Biochemistry 27 2144–2151.
Hortin, G., Tollefsen, D. M., and Strauss, A. W. (1986).J. Biol. Chem. 261 15,827–15,830.
Hortin, G. L., Tollefsen, D. M., and Benutto, B. M. (1989).J. Biol. Chem. 264 13,979–13,982.
Huber, R., and Carrell, R. W. (1989).Biochemistry 28 8951–8966.
Kawabata, S., Morita, T., Iwanaga, S., and Igarashi, H. (1985).J. Biochem. 97 325–331.
Kuhn, L. A., Griffin, J. H., Fisher, C. L., Greengard, J. S., Bouma, B. N., Espana, F., and Tainer, J. A. (1990).Proc. Natl. Acad. Sci. USA,87 8506–8510.
Laemmli, U. K. (1970).Nature 227 680–685.
Lundblad, R. L., Noyes, C. M., Featherstone, G. L., Harrison, J. H., and Jenzano, J. W. (1988).263, 3729–3734.
Lundblad, R. L. (1971).Biochemistry 10 2501–2506.
Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P., and Krishnaswamy, S. (1990).Blood 76 1–16.
Nesheim, M. E. (1983).J. Biol. Chem. 258 14,708–14,717.
Nienaber, V. L., and Berliner, L. J. (1991).Thromb. Haemost. 65 40–45.
Noe, G., Hofsteenge, J., Rovelli, G., and Stone, S. R. (1988).J. Biol. Chem. 263 11,729–11,735.
Olson, S. T. (1988).J. Biol. Chem. 263 1698–1708.
Olson, S. T., and Shore, J. D. (1986).J. Biol. Chem. 261 13,151–13,159.
Parker, K. A., and Tollefsen, D. M. (1985).J. Biol. Chem. 260 3501–3505.
Phillips, J. E., Shirk, R. A., Whinna, H. C., Henriksen, R. A., and Church, F. C. (1993).J. Biol. Chem. 268 3321–3327.
Pratt, C. W., and Church, F. C. (1991).Sem. Hematol. 28 3–9.
Pratt, C. W., and Church, F. C. (1992).J. Biol. Chem. 267 8789–8794.
Pratt, C. W., and Church, F. C. (1993).Blood Coag. Fibrinol. 4 (in press).
Pratt, C. W., Macik, B. G., and Church, F. C. (1989a).Thromb. Res. 53 595–602.
Pratt, C. W., Whinna, H. C., and Church, F. C. (1992).J. Biol. Chem. 267 8795–8801.
Pratt, C. W., Whinna, H. C., Meade, J. M., Treanor, R. E., and Church, F. C. (1989b).Ann. N.Y. Acad. Sci. 556 104–114.
Ragg, H., Ulshofer, T., and Gerewitz, J. (1990a).J. Biol. Chem. 265 22,386–22,391.
Ragg, H., Ulshofer, T., and Gerewitz, J. (1990b).J. Biol. Chem. 265 5211–5218.
Rogers, S. J., Pratt, C. W., Whinna, H. C., and Church, F. C. (1992).J. Biol. Chem. 267 3613–3617.
Rydel, T. J., Ravichandran, K. G., Tulinsky, A., Bode, W., Huber, R., Roitsch, C., and Fenton, J. W., II (1990).Science 249 277–280.
Rydel, T. J., and Tulinsky, A. (1991).J. Mol. Biol. 221 583–601.
Schechter, I., and Berger, A. (1967).Biochem. Biophys. Res. Commun. 27 157–162.
Skrzypczak-Jankun, E., Carperos, V. E., Ravichandran, K. G., Tulinsky, A., Westbrook, M., and Maraganore, J. M. (1991).J. Mol. Biol. 221 1379–1393.
Stone, S. R., Braun, P. J., and Hofsteenge, J. (1987).Biochemistry 26 4617–4624.
Suzuki, K., Deyashiki, Y., Nishioka, J., Kurachi, K., Akira, M., Yamamoto, S., and Hashimoto, S. (1987).J. Biol. Chem. 262 611–616.
Suzuki, K., Deyashiki, Y., Nishioka, J., and Toma, K. (1989).Thromb. Haemostas. 61 337–342.
Suzuki, K., Nishioka, J., and Hashimoto, S. (1983).J. Biol. Chem. 258 163–168.
Teien, A. N., Abildgaard, U., and Höök, M. (1976).Thromb. Res.,8 859–867.
Tollefsen, D. M., and Blank, M. K. (1981).J. Clin. Invest. 68 589–596.
Tollefsen, D. M., Majerus, D. W., and Blank, M. K. (1982).J. Biol. Chem. 257 2162–2169.
van Deerlin, V. M. D., and Tollefsen, D. M. (1991).J. Biol. Chem. 266 20,223–20,231.
Whinna, H. C., Blinder, M. A., Szewczyk, M., Tollefsen, D. M., and Church, F. C. (1991).J. Biol. Chem. 266 8129–8135.
Author information
Authors and Affiliations
Additional information
Abbreviations used: AT, antithrombin; HC, heparin cofactor II; PCI, protein C inhibitor; serpin(s), serine proteinase inhibitor(s); FPRck, D-Phe-Pro-Arg-chloromethyl ketone; FPLck, D-Phe-Pro-Leu-chloromethyl ketone; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HNP, 20mM HEPES, 150mM NaCl, 0.1% (w/v) poly(ethyleneglycol) (Mr = 8000) buffer atpH 7.4; Unp-PLPT, unprotected pyridoxal 5′phosphate modified-thrombin; HPPLPT, heparin-protected pyridoxal 5′phosphate modifiedthrombin.
Rights and permissions
About this article
Cite this article
Whinna, H.C., Church, F.C. Interaction of thrombin with antithrombin, heparin cofactor II, and protein C inhibitor. J Protein Chem 12, 677–688 (1993). https://doi.org/10.1007/BF01024926
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01024926