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A NEW CONVENIENT METHOD FOR FIBRINOGEN PURIFICATION BASED ON THE AFFINITY OF STAPHYLOCOCCUS AUREUS CLUMPING FACTOR A TO FIBRINOGEN
Abstract number: P-S-399
Liu1 C.Z., Cheng2 H.J., Chang1 L.Y.
11Department of Pharmacology, Tzu Chi University, Hualien City 22Department of Pharmacy, Mackay Memorial Hospital (Main Branch), Taipei, Taiwan
How-to-cite Liu CZ, Cheng HJ, Chang LY. A NEW CONVENIENT METHOD FOR FIBRINOGEN PURIFICATION BASED ON THE AFFINITY OF STAPHYLOCOCCUS AUREUS CLUMPING FACTOR A TO FIBRINOGEN. J Thromb Haemost 2007; 5 Supplement 2: P-S-399
Abstract
Introduction: Fibrinogen participates in several physiological and pathological events, including haemostasis, thrombosis, inflammation, wound healing and angiogenesis. As a result, fibrinogen is one of the important materials in the study of these fields. Herein, we report a new convenient method for the rapid purification of fibrinogen based on the affinity of Staphylococcus aureus clumping factor A (ClfA) to fibrinogen.
Methods: The fibrinogen-binding activity of ClfA resides in the residues from 221 to 550. It was produced in fusion to the C-terminus of glutathione S-transferase (GST) with recombinant technology. GST-ClfA221-550 fusion protein was first incubated with citrated and heparinized human plasma, followed by adding glutathione Sepharose 4B beads to pull down GST-ClfA221-550 to which the plasma fibrinogen was bound. After washing beads with Tris-buffered saline, fibrinogen was eluted down from beads by a citrate solution (50 mM, pH 5.0). The purity of fibrinogen was examined by SDS-PAGE and the biological function of fibrinogen was demonstrated with its capacity to support platelet aggregation and form fibrin clot in response to thrombin.
Results: Highly purified fibrinogen was obtained with this method as indicated by SDS-PAGE. It was shown to support aggregation of washed human platelets by ADP and convert into fibrin clot following thrombin treatment, in which the formation of gamma-gamma dimer did not occur. These results indicate that ClfA221-550-purified fibrinogen is functionally active and free from factor XIII activity. This method can also apply to experimental animals, such as mouse and rat, for fibrinogen purification.
Conclusions: By virtue of its simplicity and wide application, ClfA221-550-based method will make fibrinogen purification more easily to acess in laboratory. It is useful to the scientists who are interested in the researches regarding fibrinogen and fibrin.
To cite this abstract use the following format:
Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
Session Details
| Date: |
01/08/2007
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| Time: |
00:00-00:00
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| Session name: |
XXIst ISTH Congress |
| Subject: |
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| Location: |
Oxford, UK |
| Presentation type: |
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