Factor VIII - Reducing Agents, Copper Ions, and Stability

Every couple of months or so I will take weekend off from writing and attack one of the 'Problems" in my files. This week I am again with factor VIII with two (or perhaps three) questions.

Factor VIII is a large protein which is (1) expressed as heterogeneous product either as the native protein or the recombinant protein (an exception is the B-domain-deleted protein) and (2) functions as a cofactor in a enzyme-catalyzed proteolytic cleavage. The precise function of factor VIII in the "tenase" reaction is poorly understood; in siimple terms, factor VIII after cleavage by thrombin increases the reaction velocity (kcat). The linear sequence of amino acids in factor VIII has been determined and there is some understanding of the structural biology [1,2]. The x-ray structure of the B-domain-deleted factor VIII [2] provides information on metal ion binding sites, the role of such sites on stabilization of factor VIII and factor VIIIa, and further identification of sites of interaction between factor VIIIa and factor IXa. The structure does not provide any further insight into the mechism(s) by which factor VIIIa enhances the catalytic activity of factor IXa (see previous posting on musing on factor VIII within this site).

The presence of at least one free sulfhydryl group (as with albumin) was suggested from the cDNA studies in the cloning of factor VIII. This observation combined with variable results on the chemistry of sulfhydryl modification in factor VIII as well as susceptiblity to oxidation suggested the following questions

1. Is factor VIII inactivated by oxidation? Implicit in this question is the question as to whether reducing agents stabilize factor VIII?

2. What is the function of copper ions? and is there a relationship between copper ions and sulfhydryl groups?

Background

Austen in 1970[3] showed that bovine or human factor VIII was inactivated (two-stage assay; see footnote 1) by treatment with iodine, hydrogen peroxide, iodoacetamide, and p-chloromercuribenzoate; the inactivation by p-chloromercuribenzoate was reversed by treatment with cysteine. This would suggest that modification of factor VIII with reagents which are reasonably specific for cysteine[4] results in the loss of activity. Subsequently Kaelin [5]reported that the treatment of factor VIII with sodium periodate increased activity (two-stage assay). Sodium periodate is used most frequently for the cleavage of cis-diol functions in glycoproteins; reaction with cysteine can result in the formation of disulfide bonds from suitably placed residues [6-9]. Savidge and coworkers [10] reported a slight reduction of factor VIII activity in plasma with iodoacetic acid. Manning and coworkers [11] reported that reaction with bifunctional maleimides did not result in loss of factor VIII activity (there was a loss of thiols measured with Ellman's reagent)(see footnote 2); activity was lost with hydrogen peroxide. Peroxide can modify methionine [4]. Related observations include the unpublished work of Lundblad [11] where reaction with sodium cyanoborohyride increased factor VIII activity. Sodium cyanoborohydride is used most often to reduce Schiff bases in proteins formed between aldhydes and amino groups. Thus, while it is a reducing agent, it is not clear that there is any effect of cystine, cysteine, or methionine. Osterberg and Fatouros [12] demonstrated factor VIII stability is increased in solution by storage under an inert gas (nitrogen preferred) in the presence of glutathione. Nordfang and Ezben [13] reported that the presence of mercaptoethanol enhanced association of factor VIII light chain and factor VIII heavy chain to form function factor VIII. Finally, human factor VIII contains three cysteine residues [14] which have been suggested to important for copper ion binding by factor VIII [2]. Factor VIII contains cupric ions [15] which are thought to be important for subunit association [16]. A related observation is provided by Nielson and coworkers [17] who showed that peroxynitrite decreased factor VIII activity in plasma. The observations of Kashita-Iwatsuki and coworkers [18] suggest that this might be to S-nitrosylation of cysteine which decrease copper binding in albumin. Peroxynitrite also oxidizes cysteine [19]; oxidation of albumin reduces copper binding [20]. It is noted that intrinsic copper ion can oxidize cysteine residues in protein in the presence of oxygen [21-23]. Copper ions also increase the rate of cysteine oxidation by hydrogen peroxide [24]. Unpublished work [25] showed that ascorbic acid rapidly inactivated human factor VIII. Ascorbate does inactivate enzymes by modification of sulfhydryl groups [26-28].

Conclusion: Factor VIII is sensitive to oxidation; the chemistry is consistent with the modifiction of cysteine and/or methionine. The loss of activity may reflect decreased binding of copper ion with consequent dissociation of the protein into component chains. The participation of singlet oxygen mediation through bound copper must be considered. Formulation of factor VIII in a liquid formulation should consider these observations . The presence of bound copper might increase susceptibility to oxidation.

Footnotes

1. The assay for factor VIII can be complicated and the reader is referred to Lundblad, RL,  Kingdon, HS, Mann, KG, and White, GW, Issues with the assay of Factor VIII Activity in Plasma and Factor VIII Concentrates.  Thrombosis and Haemostasis 84, 942-948, 2001.

2. While there are differences in reaction rates, most free thiol groups will react with either maleimides(i.e. N-ethylmaleimide) or alkylating agents (i.e. iodoacetate), there can be differences as illustrated below. Also, while iodoacetate/iodoacetamide will react with thiourea, there is no reaction with N-ethylmaleimide.

References

1. Gilbert, G.E. and Baleja, J.D., Membrane-binding peptide from the C2 domain of factor VIII forms an amphipathic structure as determined by NMR spectroscopy, Biochemistry 34, 3022-3031, 1995.

2. Ngo, J.C., Huang, M., Roth, D.A., et al., Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex, Structure 16, 597-606, 2008

3. Austen, D.E.G., Thiol groups in the blood clotting action of factor VIII, Brit.J.Haematol. 19, 477-484, 1970

4. Lundblad, R.L., Chemical Reagents for the Modification of Protens, CRC Press, Boca Raton, FL, USA, 2004

5. Kaelin, A.C., Sodium periodate modification of factor VIII procoagulant activity, Brit.J.Haematol. 31, 349-359, 1975

6. Husain, M. and Bienarz, C., Fc site-specific labeling of immunoglobulins with calf intestinal alkaline phosphatase, Bioconjug.Chem. 5, 482-490, 1994

7. Presentini, R. and Terrana, B., Influence of the antibody-peroxidase coupling methods on the conjugate stability and on the methodologies for the preservation of the activity in time, J.Immunoassay 16, 309-324, 1995

8. Beppu, M., Takahashi, T., Hayashi, T., et al., Mechanism of macrophage recognition of SH-oxidized erythrocytes: recognition of glycophorin A on erythrocytes by a macrophage receptor for sialosaccharides, Biochim.Biophys.Acta 1223, 47-56, 1994

9. Montazerozohori, M., Joohari, S., Karami, B., et al., Fast and higly efficient solid state oxidation of thiols, Molecules 12, 694-702, 2007

10. Savidge, G., Carlebjork, G., Thorell, L., et al., Reduction of factor VIII and other coagulation factors by the thioredoxin system, Thromb.Res. 16, 587-599, 1979

11. Lundblad, R.L., unpublished observation, 1989-1991

12. Osterberg, T. and Fatouros, A., Oxygen-reduced aqueous solution of factor VIII, U.S. Patent 5,962,680, October 5, 1999

13. Nordfang, O. and Ezban, M., Generation of active coagulation factor VIII from isolated subunits, J.Biol.Chem. 263, 1115-1118, 1988

14. McMullen, B.A., Fujikawa, K., Davie, E.W., et al., Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor), Protein Sci. 4, 740-746, 1995

15. Bihoreau, N., Pin, S., de Kersabiec, A.M., et al., Copper-atom identification in the active and inactive forms of plasma-derived FVIII and recombinant FVIII-delta II, Eur.J.Biochem. 222, 41-48, 1994

16. Wakabayashi, H., Zhou, Q., Nogami, K., et al., pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+, Biochim.Biophys.Acta 1764, 1094-1101, 2006

17. Nielsen, V.G., Crow, J.P., Mogal, A., et al., Peroxynitrite decreases hemostasis in human plasma In Vitro, Anesth.Analg. 99, 21-26, 2004

18. Kashiba-Iwatsuki, M., Miyamoto, M., and Inoue, M., Effect of nitric oxide on the ligand-binding activity of albumin, Arch.Biochem.Biophys. 345, 237-242, 1997

19. Landino, L.M.., Protein thiol modification by peroxynitrite anion and nitric oxide donors, Methods Enzymol. 440, 95-109, 2008

20. Bourdon, E., Loreau, N., Lagrost, L., and Blache, D., Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin, Free Radic. Res. 39, 15-20, 2005

21. Pamp, K., Bramey, T., Kirsch, M., et al., NAD(H) enhances the Cu (II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups, Free Radic.Res. 39, 31-40, 2005

22. Arnesano, F., Balatri, E., Banci, L., et al., Folding studies of Cox17 reveal an important interplay of cysteine oxidation and copper binding, Structure 13, 713-722, 2005

23. Houghton, E.A. and Nicholas, K.M., In vitro reactive oxygen species production by histatins and copper (I, II), J.Biol.Inorg.Chem. 14, 243-251, 2009

24. Luo, D., Smith, S.W., and Anderson, B.D., Kinetics and mechanism of the reaction of cysteine and hydrogen peroxide in aqueous solution, J.Pharm.Sci. 94, 304-316, 2005

25. Lundblad, R.L., unpublished observations, University of North Carolina, 1990.

26. Orr, C.W.M., Studies on ascorbic acid. I. Factors influencing the ascorbate-mediated of catalase, Bichemistry 6, 2995-3000, 1967

27. Sok, D.E., Ascorbate-induced oxidative inactivation of Zn2+-glycerophosphocholine choline phosphodiesterase, J.Neurochem. 70, 67-74, 1998

28. Hussain, S., Noor, R., and Igbal, J., Studies on the inactivation of soluble and immobilized papain by the ascorbic acid-Cu2+ system: a model to propose the effect of free radicals on membrane-bound enzymes in vivo, Biotechnol.Appl.Biochem. 34, 205-209, 2001