Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis.
Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in inflammatory environments. Mast cells and neutrophils accumulate at sites of inflammation and vascular disease where they release proteinases as well as chemical mediators of these conditions. In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined. TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin. In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1:500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities. N-terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction. In contrast, alpha-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data indicate that TFPI is a novel and highly susceptible substrate of chymase. We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites.
Hamuro
Tsutomu
T
Therapeutic Protein Products Research Department, The Chemo-Sero-Therapeutic Research Institute, Kaketsuken, Japan. hamuro@kaketsuken.or.jp
Kido
Hiroshi
H
Asada
Yujiro
Y
Hatakeyama
Kinta
K
Okumura
Yuushi
Y
Kunori
Youichi
Y
Kamimura
Takashi
T
Iwanaga
Sadaaki
S
Kamei
Shintaro
S
D000818:Animals; D000991:Antithrombins / Q000737:chemistry; D016466:CHO Cells; D053818:Chymases / Q000737:chemistry*; D002918:Chymotrypsin / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D015951:Factor Xa / Q000737:chemistry; D065427:Factor Xa Inhibitors; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry*; D008407:Mast Cells / Q000201:enzymology*; D009504:Neutrophils / Q000201:enzymology*; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D013379:Substrate Specificity; D053802:Tryptases / Q000037:antagonists & inhibitors / Q000737:chemistry
D000991:Antithrombins; D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D053818:Chymases; D053802:Tryptases; D015951:Factor Xa