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Separation of Factor V Leiden Molecule, a Mutated Form of Factor V, from Plasma of Homozygous Patient

  • Samim Rezania
  • Kyung A. Kang
Part of the Advances In Experimental Medicine And Biology book series (AEMB, volume 614)

Abstract

Factor V (FV) is a coagulant in plasma. The FV molecule consists of a heavy chain and a light chain, and Factor V Leiden (FVL) is mutated FV at a single amino acid in the heavy chain. FVL patients are in a dangerous hyper-coagulation state in their body. Current FVL diagnosis is done by DNA analysis, which is expensive and time consuming. Our group has been developing a real-time, cost effective immuno-optical biosensor for FVL diagnosis. For the sensor development, pure FVL, which is not currently available, is needed. Here, we have attempted FVL purification from FVL patient’s plasma. Since plasma contains many proteins and some proteins are structurally homologous to FV, the purification must be done by a very specific method, such as immuno-affinity chromatography. However, an antibody that does not react with FV is not currently available. Because the mutation is in the heavy chain and the amino acid sequence of the light chain of FVL is identical to that of FV, antibodies generated against the light chain of FV were tested for purifying FVL. Plasma was obtained from a homozygous FVL patient. First, the plasma was pretreated by barium citrate and polyethylene glycol 6000, to remove the vitamin K-dependent proteins, alpha globulins, and other smaller than 6 kDa molecular weight proteins. The yield in the process was 54%. Immuno-affinity purification of FVL from patient plasma was then performed using an anti-FV light chain antibody immobilized CNBr-Sepharose, and the purification yield was 25%. In summary, the antibody against the light chain of FV was able to purify the single point mutated form of FV (FVL) from plasma with an overall yield of 14%. The same principle can probably be used for purification of the other single point mutated proteins.

Keywords

Light Chain Heavy Chain Factor Versus Factor Versus Leiden Immobilization Efficiency 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    C. T. Esmon, The subunit structure of thrombin-activated factor V: Isolation of activated factor V, separation of subunits, and reconstitution of biological activity. J. Biol. Chem. 254, 964–973 (1979).PubMedGoogle Scholar
  2. 2.
    W. H. Kane, P. W. Majerus, Purification and characterization of human coagulation factor V. J. Biol. Chem. 256, 1002–1007 (1981).PubMedGoogle Scholar
  3. 3.
    B. Dahlback, Human coagulation factor V purification and thrombin-catalyzed activation. J. Clin Invest. 66, 583–591 (1980).PubMedCrossRefGoogle Scholar
  4. 4.
    J. Rosing, and G. Tans, Factor V, Int J. Biochem.29, 1123–1126 (1997).CrossRefGoogle Scholar
  5. 5.
    E. Castoldi, J. M. Brugge, G. A. Nicolaes, D. Girelli, G. Tans, J. Rosing, Impaired APC cofactor activity of factor V plays a major role in the APC resistance associated with the factor V Leiden (R506Q) and R2 (H1299R) mutations, Blood 103, 4173–4179 (2004).PubMedCrossRefGoogle Scholar
  6. 6.
    M. Wilmer, C. Stocker, B. Buhler, B. Conell, A. Calatzis, Improved distinction of factor V wild-type and factor V Leiden using a novel prothrombin-based activated protein C resistance assay, Am J Clin Pathol. 122, 836–842 (2004).PubMedCrossRefGoogle Scholar
  7. 7.
    R. J. Alexander, T. C. Detwiler, Quantitative adsorption of platelet glycoprotein G (thrombin-sensitive protein, thrombospondin) to barium citrate. Biochem. J. 217, 67–71 (1984).PubMedGoogle Scholar
  8. 8.
    H. A. Donald, K. C. Ingham, Mechanism of precipitation of proteins by Polyethylene Glycol. J. Biol. Chem. 256, 12108–1211766 (1981).Google Scholar
  9. 9.
    J. A. Katzmann, M. E. Nesheim, L. S. Hibbard, K. G. Mann, Isolation of functional human coagulation factor V by using a hybridoma antibody. Proc. Natl. Acad. Sci. USA. 78, 162–166 (1981).PubMedCrossRefGoogle Scholar
  10. 10.
    K. A. Kang, D. Ryu, W. M. Drohan and C. L. Orthner, Effect of matrices on affinity purification of protein C. Biotechnol Bioeng. 39, 1086–1096 (1992).CrossRefPubMedGoogle Scholar
  11. 11.
    S. Rezania, D. G. Ahn, and K. A. Kang, Separation of protein C from Cohn fraction IV-1 by mini-antibody, Proceedings of the Annual 2005 ISOTT meeting. Adv. Exp. Med. Biol.: Oxygen Transport to Tissue XXVIII, 599, (Maguire, D.J., Bruley, D.F., Harrison, D.K., eds.), p. 125–132, 2007.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Samim Rezania
    • 1
  • Kyung A. Kang
    • 1
  1. 1.Department of Chemical EngineeringUniversity of LouisvilleLouisville

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