In vitro interaction of C1-inhibitor with thrombin

Previous observations of increased generation of thrombin during acute atta cks of angioedema in plasma of patients with C1-inhibitor (C1-INH) deficien cy prompted us to evaluate the interaction of CI-INH with thrombin in both purified systems and human plasma. For this purpose, we used several method s: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immuno blotting analysis; (2) enzyme-linked immunosorbent assays to measure comple xes between CI-INH and thrombin and inactivated C1-INH; and (3) kinetic stu dies using a chromogenic assay. We found that the interaction of purified C I-INH with thrombin is associated with the formation of bimolecular complex es of molecular weight (M-r) 130000 and 120000 as well as with the appearan ce of a cleaved form of C1-INH of M-r 97000. The kinetic studies of inhibit ion of thrombin by C1-INH showed an average second-order rate constant of 1 9/s per mol/l, which was significantly increased in the presence of heparin . The addition of thrombin to human plasma was not associated with detectab le C1-INH-thrombin complex formation or with cleavage of C1-INH. In conclus ion, our data demonstrate that C1-INH upon interaction with thrombin, in pa rt, forms enzyme-inhibitor complexes and, in part, is cleaved. The low seco nd-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 circu lating blood; however, its role might be important at the endothelial surfa ce, where high concentrations of glycosaminoglycans occur. (C) 2001 Lippinc ott Williams & Wilkins.