Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8

Anna C. Cunningham, Karen A. Hasty, Jan J. Enghild, Alan E. Mast

Research output: Contribution to journalArticlepeer-review

39 Scopus citations


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/Km ∼ 75 M-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.

Original languageEnglish (US)
Pages (from-to)451-458
Number of pages8
JournalBiochemical Journal
Issue number2
StatePublished - Oct 15 2002
Externally publishedYes


  • Blood coagulation
  • Neutrophil collagenase
  • Proteolysis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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