Vascular injury results in the activation of coagulation and the release of proteolytic enzymes from neutrophils and connective- tissue cells. High concentrations of these inflammatory proteinases may destroy blood coagulation proteins, contributing to coagulation and bleeding disorders associated with severe inflammation. Matrix metalloproteinase-8 (MMP-8) is released from neutrophils at sites of inflammation and vascular disease. We have investigated the effect of MMP-8 degradation on the anticoagulant function of tissue factor pathway inhibitor (TFPI) as a potential pathological mechanism contributing to coagulation disorders. MMP-8 cleaves TFPI following Ser174 within the connecting region between the second and third Kunitz domains (kcat/Km75M-1·s-1) as well as following Lys20 within the NH2-terminal region. MMP-8 cleavage of TFPI decreases the anticoagulant activity of TFPI in factor Xa initiated clotting assays as well as the ability of TFPI to inhibit factor Xa in amidolytic assays. Yet, MMP-8 cleavage does not alter the ability of TFPI to inhibit trypsin. Since the inhibition of both factor Xa and trypsin is mediated by binding to the second Kunitz domain, these results suggest that regions of TFPI other than the second Kunitz domain may directly interact with factor Xa. 125I-factor Xa ligand blots of TFPI fragments generated following MMP-8 degradation were used for probing binding interactions between factor Xa and regions of TFPI, other than the second Kunitz domain. In experiments performed under reducing conditions that disrupt the Kunitz domain structure, 125I-factor Xa binds to the C-terminal fragment of MMP-8-degraded TFPI. This fragment contains portions of TFPI distal to Ser174, which include the third Kunitz domain and the basic C-terminal region. An altered form of TFPI lacking the third Kunitz domain, but containing the C-terminal region, was used to demonstrate that the C-terminal region directly interacts with factor Xa.
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October 2002
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Research Article|
October 15 2002
Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8
Anna C. CUNNINGHAM;
Anna C. CUNNINGHAM
∗Department of Pathology, The University of Tennessee, Memphis, TN 38163, U.S.A.
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Karen A. HASTY;
Karen A. HASTY
†Research and Pathology Services, Department of Veterans Affairs, Memphis, TN 38104, U.S.A.
‡Department of Anatomy and Neurobiology, The University of Tennessee, Memphis, TN 38163, U.S.A.,
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Jan J. ENGHILD;
Jan J. ENGHILD
§Department of Molecular and Structural Biology, University of Arhus, Arhus, Denmark
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Alan E. MAST
Alan E. MAST
1
∗Department of Pathology, The University of Tennessee, Memphis, TN 38163, U.S.A.
†Research and Pathology Services, Department of Veterans Affairs, Memphis, TN 38104, U.S.A.
1To whom correspondence should be addressed, at Research Service-151, VA Hospital, 1030 Jefferson Ave., Memphis, TN 38104, U.S.A. (e-mail alan.mast3@med.va.gov).
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Publisher: Portland Press Ltd
Received:
May 02 2002
Revision Received:
June 18 2002
Accepted:
July 12 2002
Accepted Manuscript online:
July 12 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 367 (2): 451–458.
Article history
Received:
May 02 2002
Revision Received:
June 18 2002
Accepted:
July 12 2002
Accepted Manuscript online:
July 12 2002
Citation
Anna C. CUNNINGHAM, Karen A. HASTY, Jan J. ENGHILD, Alan E. MAST; Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8. Biochem J 15 October 2002; 367 (2): 451–458. doi: https://doi.org/10.1042/bj20020696
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