Thrombin binding polypeptides

Details Thrombin binding polypeptides Thrombin binding polypeptides

1991-01-31

2001-05-29

Furman, Keith C. (Department: 1652)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S380000, C530S350000, C530S322000, C530S324000, C514S012200, C435S069100, C435S069200, C435S069600

Reexamination Certificate

active

06239101

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to synthetic polypeptides, and in particular to peptides having the ability to inhibit the clotting activity of thrombin and to accelerate the activation of protein C, and to peptides having the ability to inhibit the clotting activity of thrombin without accelerating the activation of protein C, all derived from thrombomodulin.
Thrombomodulin is an endothelial cell surface glycoprotein that forms a high affinity complex with thrombin. When thrombin binds to thrombomodulin there is at least a one thousand-fold increase in the activation rate of protein C which forms the anticoagulant enzyme activated protein C. In addition, when thrombin is bound to thrombomodulin, thrombin no longer works as a procoagulant enzyme. Specifically, thrombin-catalyzed fibrin formation, Factor V activation and platelet activation, are all inhibited in the presence of thrombomodulin. Thus, thrombomodulin converts thrombin into a physiological anticoagulant.
Thrombomodulin was first identified in 1980 by C. T. Esmon et al., in a heart perfusion system [Proc. Natl. Acad. Sci. U.S.A., 78:2249-2252 (1981)]. Thrombomodulin has been isolated and purified from rabbit lung [Esmon et al., J. Biol. Chem. 261:859-864 (1982)], from bovine lung [Jakubowski et al., J. Biol. Chem. 261:3876-3882 (1986); Suzuki et al., Biochim. Biophys. Acta, 882:343-352 (1986)], and from human placenta [Salem et al., J. Biol. Chem. 259:12246-12251 (1984); Kurosawa et al., Thromb. Res. 37:353-364 (1985)]. The human thrombomodulin cDNA has been reported providing a structural picture of thrombomodulin. [Suzuki et al., EMBO J. 6:1891-1897 (1987); Wen et al., Biochem. 26:43504357 (1987); Jackman et al., Proc. Natl. Acad. Sci. U.S.A. 84:6425-6429 (1987)]. A partial CDNA sequence of bovine thrombomodulin also has been reported, [Jackman et al., Proc. Natl. Acad. Sci. U.S.A. 82:8834-8838 (1986)], as well as mouse, by Dittman, et al., Nucleic Acids Res. 17:802 (1989).
As the previous reports have shown, the thrombomodulin molecule is organized into five regions: an amino terminal, hydrophobic region, residues 1-244; a cysteine-rich region, residues 245-480; a serine/threonine/proline-rich region with O-glycosylation sites, residues 481-514; a hydrophobic transmembrane region, residues 515-537; and a cytosolic tail containing the remaining 38 residues.
The cysteine-rich region, consisting of the 246 amino acid residues from 234 to 480, has been shown to include six repeated structures homologous to the epidermal growth factor precursor, called EGF-like or EGF-homology domains.
Although human, mouse and bovine thrombomodulin have been studied and characterized with respect to structure and function, the specific functional regions of the thrombomodulin molecule have heretofore remained unknown.
It is therefore an object of the present invention to provide polypeptides derived from regions of thrombomodulin having anticoagulant activity that are suitable for administration to a patient.
It is a further object of the present invention to provide polypeptides which can be used to form non-thrombogenic coatings on the surfaces of medical devices contacting blood.
SUMMARY OF THE INVENTION
Selected polypeptide fragments of the thrombomodulin molecule including residues 234 to 486, 310 to 486 and 407 to 486, respectively, polypeptides comprising amino acid sequences corresponding thereto, and functionally equivalent analogs thereof inhibiting the clotting activity of thrombin and increasing protein C activation, thereby serving as anticoagulants when administered in combination with a pharmaceutically acceptable carrier to a patient, are disclosed. Also disclosed are the use of these polypeptides to coat the surfaces of articles or devices exposed to blood which could otherwise induce clotting.
Methods for using these compositions to inhibit the clotting activity of thrombin without increasing the activation of protein C, and methods to inhibit the clotting activity of thrombin and increase the activation of protein C are also described.


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