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Cloning and expression of rat coagulation factor VII
Shobha Seetharam1, Kathleen Murphy, Charity Atkins
1Cardiovacular Department, The Bristol Myers Squibb Company, P.O. Box 400, Experimental Station, Wilmington, DE 19880, USA. shoram2@yahoo.com
Thrombosis Research
|May 22, 2003
Summary
Researchers developed recombinant rat factor VII (FVII) to overcome challenges in studying antithrombotic drugs. This new tool enables easier investigation of blood coagulation in rats, aiding drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Rats are common models for antithrombotic drug evaluation.
- Purifying rat factor VII (FVII) from plasma is difficult due to low levels and interfering factors.
- Studying the extrinsic coagulation pathway in rats is hindered by FVII purification challenges.
Purpose of the Study:
- To isolate and express rat factor VII (FVII) cDNA.
- To establish cell lines that produce recombinant rat FVII.
- To facilitate research on the extrinsic coagulation pathway and antithrombotic drug development.
Main Methods:
- Isolated full-length rat factor VII (FVII) cDNA using polymerase-mediated DNA amplification.
- Transfected rat FVII cDNA into human embryonic kidney 293 cells to create stable expression lines.
- Screened cell culture media for functional recombinant rat FVII activity.
Main Results:
- Established stable cell lines constitutively expressing recombinant rat factor VII (FVII).
- Recombinant rat FVII normalized clotting time in FVII-depleted human plasma.
- Functional assays confirmed FVIIa activation and FXa generation in the presence of tissue factor.
Conclusions:
- Recombinant rat factor VII (FVII) provides a valuable tool for antithrombotic drug research.
- This method overcomes previous limitations in obtaining functional rat FVII.
- Facilitates studies on the extrinsic coagulation pathway and novel antithrombotic therapies.
