Identification and characterization of α 1 ‐antitrypsin in fibrin clots

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.