Plasmin degradation of the alpha chain of fibrinogen/fibrin: improved activation constant and activity determination in assays for tissue plasminogen activator
Objectives. The aim of this investigation was to increase the efficiency of ternary complex formation (fibrin-plasminogen-tissue-plasminogen activator) in the degradation process of the three-dimensional soluble fibrin monomer. Materials and methods. Fibrinogen was purified from human plasma by repe...
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Published in | Iatreia (Medellín, Colombia) Vol. 26; no. 3; pp. 291 - 301 |
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Main Authors | , , |
Format | Journal Article |
Language | English Portuguese |
Published |
Medellín
Universidad de Antioquía
01.07.2013
Universidad de Antioquia |
Subjects | |
Online Access | Get full text |
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Summary: | Objectives. The aim of this investigation was to increase the efficiency of ternary complex formation (fibrin-plasminogen-tissue-plasminogen activator) in the degradation process of the three-dimensional soluble fibrin monomer. Materials and methods. Fibrinogen was purified from human plasma by repeating precipitation six times, using different concentrations of cold ethanol. Fibrinogen was converted to DesAAfibrinogen by degradation with bathroxobin. Human plasminogen was purified by affinity and ion-exchange chromatography, and activated to plasmin by incubation with urokinase. Digested DesAAfibrinogen was prepared by controlled digestion with plasmin. Results. This study demonstrates that the α-chains of DesAAfibrinogen sterically hinder the formation of the ternary complex and are first degraded by plasmin. The degradation of fibrin(ogen) facilitates the in vitro determination of tissue plasminogen activator activity. Finally, release of fibrinopeptide A from bathroxobin-cleaved fibrinogen was confirmed, optimized and evaluated by various methods. Conclusions. Use of digested desAAfibrinogen with plasmin yielded a more stable activation constant of the ternary complex than that of undigested DesAAfibrinogen. |
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ISSN: | 0121-0793 2011-7965 |