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Identification and characterization of α 1 ‐antitrypsin in fibrin clots
Author(s) -
Talens S.,
Malfliet J. J. M. C.,
Hal P. Th. W.,
Leebeek F. W. G.,
Rijken D. C.
Publication year - 2013
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.12288
Subject(s) - fibrin , fibrinogen , chemistry , fibrinolysis , biochemistry , elastase , microbiology and biotechnology , sepharose , affinity chromatography , enzyme , immunology , biology , medicine , psychiatry
Summary Background and Objectives Preliminary studies indicated that α 1 ‐antitrypsin ( A 1 AT ) 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 A 1 AT . Methods and Results Plasma clots were prepared and extensively washed with saline. Clot‐bound A 1 AT could only be extracted using denaturing agents such as urea, thiourea or SDS , pointing to an apparently strong association. Purified fibrinogen, but still containing A 1 AT as a contaminant, was gel filtered, which showed that the A 1 AT was bound to fibrinogen. A specific ELISA detected the presence of A 1 AT ‐fibrinogen complexes in both purified fibrinogen and pooled normal plasma. Finally, fibrin(ogen)‐ S epharose chromatography indicated that A 1 AT purified from plasma contained a small fraction of fibrin(ogen)‐binding A 1 AT . To study the inhibitory activity of fibrin(ogen)‐bound A 1 AT , both fibrinogen containing A 1 AT and washed plasma clots were incubated with increasing amounts of elastase. SDS ‐ PAGE and W estern blotting showed under both conditions the generation of the A 1 AT ‐elastase complex as well as cleaved A 1 AT . The inhibitory activity of fibrin(ogen)‐bound A 1 AT was also demonstrated by measuring elastase‐induced lysis of fibrin clots. Conclusion Fibrin clots contain strongly bound A 1 AT , which is functionally active as a serine protease inhibitor (serpin). This A 1 AT 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.