In vitro interaction of C1-inhibitor with thrombin.
Previous observations of increased generation of thrombin during acute attacks of angioedema in plasma of patients with C1-inhibitor (C1-INH) deficiency prompted us to evaluate the interaction of C1-INH with thrombin in both purified systems and human plasma. For this purpose, we used several methods: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis; (2) enzyme-linked immunosorbent assays to measure complexes between C1-INH and thrombin and inactivated C1-INH; and (3) kinetic studies using a chromogenic assay. We found that the interaction of purified C1-INH with thrombin is associated with the formation of bimolecular complexes of molecular weight (Mr) 130 000 and 120 000 as well as with the appearance of a cleaved form of C1-INH of Mr 97 000. The kinetic studies of inhibition of thrombin by C1-INH showed an average second-order rate constant of 19/s per mol/l, which was significantly increased in the presence of heparin. The addition of thrombin to human plasma was not associated with detectable C1-INH-thrombin complex formation or with cleavage of C1-INH. In conclusion, our data demonstrate that C1-INH upon interaction with thrombin, in part, forms enzyme-inhibitor complexes and, in part, is cleaved. The low second-order rate constant and the lack of a significant interaction in plasma suggest that the inhibition of thrombin by C1-INH has a minor role in circulating blood; however, its role might be important at the endothelial surface, where high concentrations of glycosaminoglycans occur.
Cugno
M
M
Department of Internal Medicine, IRCCS Maggiore Hospital, University of Milan, Milan, Italy. massimo.cugno@unimi.it
Bos
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I
Lubbers
Y
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Hack
C E
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Agostoni
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A
D003174:Complement C1 Inactivator Proteins / Q000737:chemistry / Q000378:metabolism*; D050718:Complement C1 Inhibitor Protein; D006801:Humans; D007118:Immunoassay; D007700:Kinetics; D011485:Protein Binding; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D013917:Thrombin