Role of thrombin exosites in inhibition by heparin cofactor II

We determined the role of specific thrombin "exosites" in the mechanism of inhibition by the plasma serine proteinase inhibitors heparin cofactor II (HC) and antithrombin (AT) in the absence and presence of a glycosaminoglycan by comparing the inhibition of alpha-thrombin to epsilon- and g...

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Bibliographic Details
Published in:The Journal of biological chemistry 1992-02, Vol.267 (6), p.3613-3617
Main Authors: ROGERS, SJ, PRATT, CW, WHINNA, HC, CHURCH, FC
Format: Article
Language:English
Subjects:
Amino Acid Chloromethyl Ketones - metabolism
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Animals
Antithrombins - pharmacology
Binding Sites
Biochemistry & Molecular Biology
Biological and medical sciences
Cattle
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Glycosaminoglycans - pharmacology
heparin cofactor II
Heparin Cofactor II - pharmacology
Hirudins - pharmacology
Humans
Hydrolases
inhibition
Life Sciences & Biomedicine
man
mechanisms
Molecular Sequence Data
Peptide Fragments - pharmacology
Protein Conformation
requirements
Science & Technology
serum proteins
thrombin
Thrombin - antagonists & inhibitors
Thrombin - metabolism
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Summary:We determined the role of specific thrombin "exosites" in the mechanism of inhibition by the plasma serine proteinase inhibitors heparin cofactor II (HC) and antithrombin (AT) in the absence and presence of a glycosaminoglycan by comparing the inhibition of alpha-thrombin to epsilon- and gamma T-thrombin (produced by partial proteolysis of alpha-thrombin by elastase and trypsin, respectively). All of the thrombin derivatives were inhibited in a similar manner by AT, either in the absence or presence of heparin, which confirmed the integrity of both heparin binding abilities and serpin reactivities of epsilon- and gamma T-thrombin compared to alpha-thrombin. Antithrombin activities of HC in the absence of a glycosaminoglycan with alpha-, epsilon, and gamma T-thrombin were similar with rate constants of 3.5, 2.4, and 1.2 x 10(4) M-1 min-1, respectively. Interestingly, in the presence of glycosaminoglycans the maximal inhibition rate constants by HC with heparin and dermatan sulfate, respectively, were as follows: 30.0 x 10(7) and 60.5 x 10(7) for alpha-thrombin, 14.6 x 10(7) and 24.3 x 10(7) for epsilon-thrombin, and 0.017 x 10(7) and 0.034 x 10(7) M-1 min-1 for gamma T-thrombin. A hirudin carboxyl-terminal peptide, which binds to anion-binding exosite-I of alpha-thrombin, dramatically reduced alpha-thrombin inhibition by HC in the presence of heparin but not in its absence. We analyzed our results in relation to the recently determined x-ray structure of D-Phe-Pro-Arg-chloromethyl ketone-alpha-thrombin (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., and Hofsteenge, J. (1989) EMBO J. 8, 3467-3475). Our results suggest that the beta-loop region of anion-binding exosite-I in alpha-thrombin, which is not present in gamma T-thrombin, is essential for the rapid inhibition reaction by HC in the presence of a glycosaminoglycan. Therefore, alpha-thrombin and its derivatives would be recognized and inhibited differently by HC and AT in the presence of a glycosaminoglycan.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(19)50568-3