MMP-8 cleaves TFPI and decreases its anticoagulant activity

Research output: Contribution to journalArticle

Abstract

Vascular injury results in activation of coagulation and 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. We investigated the ability of neutrophil collagenase (MMP-8), a predominant metalloproteinase in healing wounds, to degrade tissue factor pathway inhibitor (TFPI), a trivalent Kunitz-type inhibitor and major regulator of tissue factor initiated coagulation. MMP-8 cleaves following Serl74 between the second and third Kunitz domains of TFPI. Western analysis of MMP-8 cleaved TFPI with an antibody against the C-terminus indicates that the C-terminal fragment remains intact from Thrl75 to the C-terminus. The ktil/Km for cleavage at Serl74 by MMP-8 is -164 M 1s1, a rate 15-fold slower than MMP-8 cleaves human type I collagen (kcar/Km-2538 M-'s'1), but similar to that for human type III collagen cleavage (kcat/Km-130 M 's '). TFPI is a slow, tight-binding inhibitor of factor Xa. As such, it forms an immediate encounter complex which slowly isomerizes into a final tight complex. The affinity of the initial encounter complex between factor Xa and MMP-8 cleaved TFPI is reduced with the K.(initial) increasing from 8 nM to 91 nM. The affinity of the final complex is also decreased with the K.(fmal) increasing from 68 pM to 950 pM following cleavage. In factor Xa initiated plasma clotting assays, the apparent inhibition of factor Xa by MMP8 cleaved TFPI is reduced 14-fold when compared to native TFPI. These data indicate that the region between Thrl75 and the C-terminus is necessary for rapid, tight inhibition of factor Xa by the second Kunitz domain, either by maintaining the second Kunitz domain in an optimal inhibitory conformation or by providing secondary binding sites for factor Xa. We further characterized the interaction of the C-terminal fragment of TFPI with factor Xa using SDS-PAGE followed by blots with I25l-bovine factor Xa. Under non-reducing conditions, factor Xa binds avidly to the N-terminal fragment that contains the second Kunitz domain. Under reducing conditions, binding to the N-terminal fragment does not occur, but binding to the C-terminal fragment can now be observed. Our data indicate TFPI degradation by MMP-8 may limit its effectiveness as an anticoagulant in a healing wound and also suggest that factor Xa directly interacts with a portion of TFPI between Thrl75 and the C-terminus through molecular contacts that do not require an intact Kunitz domain.

Original languageEnglish (US)
JournalBlood
Volume96
Issue number11 PART I
StatePublished - Dec 1 2000
Externally publishedYes

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Matrix Metalloproteinases
Anticoagulants
Factor Xa
Coagulation
Wound Healing
Peptide Hydrolases
lipoprotein-associated coagulation inhibitor
Matrix Metalloproteinase 8
Connective Tissue Cells
Collagen Type III
Vascular System Injuries
Thromboplastin
Metalloproteases
Blood Coagulation
Collagen Type I
Conformations
Blood Proteins
Polyacrylamide Gel Electrophoresis
Assays
Neutrophils

ASJC Scopus subject areas

  • Hematology

Cite this

MMP-8 cleaves TFPI and decreases its anticoagulant activity. / Edmondson, Anna Cunningham.

In: Blood, Vol. 96, No. 11 PART I, 01.12.2000.

Research output: Contribution to journalArticle

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N2 - Vascular injury results in activation of coagulation and 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. We investigated the ability of neutrophil collagenase (MMP-8), a predominant metalloproteinase in healing wounds, to degrade tissue factor pathway inhibitor (TFPI), a trivalent Kunitz-type inhibitor and major regulator of tissue factor initiated coagulation. MMP-8 cleaves following Serl74 between the second and third Kunitz domains of TFPI. Western analysis of MMP-8 cleaved TFPI with an antibody against the C-terminus indicates that the C-terminal fragment remains intact from Thrl75 to the C-terminus. The ktil/Km for cleavage at Serl74 by MMP-8 is -164 M 1s1, a rate 15-fold slower than MMP-8 cleaves human type I collagen (kcar/Km-2538 M-'s'1), but similar to that for human type III collagen cleavage (kcat/Km-130 M 's '). TFPI is a slow, tight-binding inhibitor of factor Xa. As such, it forms an immediate encounter complex which slowly isomerizes into a final tight complex. The affinity of the initial encounter complex between factor Xa and MMP-8 cleaved TFPI is reduced with the K.(initial) increasing from 8 nM to 91 nM. The affinity of the final complex is also decreased with the K.(fmal) increasing from 68 pM to 950 pM following cleavage. In factor Xa initiated plasma clotting assays, the apparent inhibition of factor Xa by MMP8 cleaved TFPI is reduced 14-fold when compared to native TFPI. These data indicate that the region between Thrl75 and the C-terminus is necessary for rapid, tight inhibition of factor Xa by the second Kunitz domain, either by maintaining the second Kunitz domain in an optimal inhibitory conformation or by providing secondary binding sites for factor Xa. We further characterized the interaction of the C-terminal fragment of TFPI with factor Xa using SDS-PAGE followed by blots with I25l-bovine factor Xa. Under non-reducing conditions, factor Xa binds avidly to the N-terminal fragment that contains the second Kunitz domain. Under reducing conditions, binding to the N-terminal fragment does not occur, but binding to the C-terminal fragment can now be observed. Our data indicate TFPI degradation by MMP-8 may limit its effectiveness as an anticoagulant in a healing wound and also suggest that factor Xa directly interacts with a portion of TFPI between Thrl75 and the C-terminus through molecular contacts that do not require an intact Kunitz domain.

AB - Vascular injury results in activation of coagulation and 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. We investigated the ability of neutrophil collagenase (MMP-8), a predominant metalloproteinase in healing wounds, to degrade tissue factor pathway inhibitor (TFPI), a trivalent Kunitz-type inhibitor and major regulator of tissue factor initiated coagulation. MMP-8 cleaves following Serl74 between the second and third Kunitz domains of TFPI. Western analysis of MMP-8 cleaved TFPI with an antibody against the C-terminus indicates that the C-terminal fragment remains intact from Thrl75 to the C-terminus. The ktil/Km for cleavage at Serl74 by MMP-8 is -164 M 1s1, a rate 15-fold slower than MMP-8 cleaves human type I collagen (kcar/Km-2538 M-'s'1), but similar to that for human type III collagen cleavage (kcat/Km-130 M 's '). TFPI is a slow, tight-binding inhibitor of factor Xa. As such, it forms an immediate encounter complex which slowly isomerizes into a final tight complex. The affinity of the initial encounter complex between factor Xa and MMP-8 cleaved TFPI is reduced with the K.(initial) increasing from 8 nM to 91 nM. The affinity of the final complex is also decreased with the K.(fmal) increasing from 68 pM to 950 pM following cleavage. In factor Xa initiated plasma clotting assays, the apparent inhibition of factor Xa by MMP8 cleaved TFPI is reduced 14-fold when compared to native TFPI. These data indicate that the region between Thrl75 and the C-terminus is necessary for rapid, tight inhibition of factor Xa by the second Kunitz domain, either by maintaining the second Kunitz domain in an optimal inhibitory conformation or by providing secondary binding sites for factor Xa. We further characterized the interaction of the C-terminal fragment of TFPI with factor Xa using SDS-PAGE followed by blots with I25l-bovine factor Xa. Under non-reducing conditions, factor Xa binds avidly to the N-terminal fragment that contains the second Kunitz domain. Under reducing conditions, binding to the N-terminal fragment does not occur, but binding to the C-terminal fragment can now be observed. Our data indicate TFPI degradation by MMP-8 may limit its effectiveness as an anticoagulant in a healing wound and also suggest that factor Xa directly interacts with a portion of TFPI between Thrl75 and the C-terminus through molecular contacts that do not require an intact Kunitz domain.

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