Identification and characterization of α1-antitrypsin in fibrin clots
Journal of Thrombosis and Haemostasis , Volume 11 - Issue 7 p. 1319- 1328
Background and Objectives: Preliminary studies indicated that α1-antitrypsin (A1AT) is the most abundant protein that is non-covalently bound to fibrin clots prepared from plasma. The aim of this study was to identify and characterize fibrin(ogen)-bound A1AT. Methods and Results: Plasma clots were prepared and extensively washed with saline. Clot-bound A1AT could only be extracted using denaturing agents such as urea, thiourea or SDS, pointing to an apparently strong association. Purified fibrinogen, but still containing A1AT as a contaminant, was gel filtered, which showed that the A1AT was bound to fibrinogen. A specific ELISA detected the presence of A1AT-fibrinogen complexes in both purified fibrinogen and pooled normal plasma. Finally, fibrin(ogen)-Sepharose chromatography indicated that A1AT purified from plasma contained a small fraction of fibrin(ogen)-binding A1AT. To study the inhibitory activity of fibrin(ogen)-bound A1AT, both fibrinogen containing A1AT and washed plasma clots were incubated with increasing amounts of elastase. SDS-PAGE and Western blotting showed under both conditions the generation of the A1AT-elastase complex as well as cleaved A1AT. The inhibitory activity of fibrin(ogen)-bound A1AT was also demonstrated by measuring elastase-induced lysis of fibrin clots. Conclusion: Fibrin clots contain strongly bound A1AT, which is functionally active as a serine protease inhibitor (serpin). This A1AT might play a role in the local regulation of proteases involved in coagulation or fibrinolysis and represent a novel link between the inflammatory and hemostatic systems.
|Western blotting, article, chromatography, clinical article, controlled study, enzyme linked immunosorbent assay, fibrin clot, human, human tissue, lysis, normal human, plasma, polyacrylamide gel electrophoresis, priority journal, protein analysis, protein binding|
|Journal of Thrombosis and Haemostasis|
|Organisation||Erasmus MC: University Medical Center Rotterdam|
Talens, S, Malfliet, J.J.M.C, van Hal, P.Th.W, Leebeek, F.W.G, & Rijken, D.C. (2013). Identification and characterization of α1-antitrypsin in fibrin clots. Journal of Thrombosis and Haemostasis, 11(7), 1319–1328. doi:10.1111/jth.12288