Factor VIII Function

            I published my first scientific paper 45 years ago in Biochemistry[1].  The title was “The activation of antihemophilic factor (Factor VIII) by activated Christmas factor (activated factor IX).”  While the conclusion was incorrect, the paper has a respectable citation record (101 citations).   Factor VIII is not converted to an enzyme but rather forms a complex[2,3] with factor IXa, calcium ions, and a “phospholipid surface.”   In vivo the ‘phospholipid surface” is provided by platelets.   Factor VIII must be converted to factor VIIIa by thrombin and platelets needed to be activated, presumably by thrombin, to participate in this process.  The complex formed with factor IXa, factor VIIIa, calcium ions, and phospholipid/platelets is called tenase in analogy with the complexed formed with factor Xa, factor Va, calcium ions, and phospholipid/platelets which is called prothrombinase[4].  Platelets also have a problem in that Peter Walsh observed in 1978[5] that there are different requirements for intrinsic factor-Xa forming activity (tenase) and platelet factor 3 (prothrombinase function).   Now, some 45 years later, we don’t much more about factor VIII function except Hemker and colleagues[3] have suggest that the effect is on Vmax (catalytic efficiency) as opposed to a Km effect which is ascribed to platelets.   The process by which Factor VIIIa increases the activity of factor IXa is, to be generous, poorly understood.   Several investigators have suggested that enzymes are large to prevent wobble in highly precise molecular interactions.  Despite the above considerations, the work of Ken Mann and associates gives confidence in the application of the data obtained with isolated systems to the process in whole blood[6]. 
There are at least three other considerations which should fit somewhere in this discussion. 

• First, factor VIII circulates as a complex with von Willebrand Factor and there is reason to think that the complex occurs with a specific subset of von Willebrand Factor.  During the preparation of plasma-derived factor VIII, the bulk of the factor VIII remains in solution during separation of the cyro-insoluble fraction and Bill Fricke demonstrated the immunopurified human factor VIII contained a unique subset of von Willebrand factor[7].   The nature of the association may well be important for the work on the gene therapy of hemophilia as the point at which the physiological association of factor VIII and vWF may be critical to secretion of the complex[8].
• Von Willebrand Factor appears to have an effect on the assay (and perhaps in vivo) function of factor VIII[9].
•  Third, factor VIII occurs as a heterogeneous mixture of polypeptides derived from a single polypeptide chain; it is not clear when proteolysis occurs during the processing of factor VIII.  A active, single chain form of factor VIII can be isolated from plasma[10]; preparations with a greater degree of proteolysis appear to have greater activity[11]. 

Notwithstanding a lack of understanding of molecular function, there is no question about the value of Factor VIII as a therapeutic agent in the treatment of hemophilia A.   The issues important for consumers (patients, treaters) are (1) product safety, (2) product effectiveness, and (3) east of administration.  Great progress in safety and effectiveness has been made in the past thirty years[12].  That leaves ease of administration as a challenge.   There are two ways of addressing the ease of administration problem: extending circulatory half-life or non-parenteral administration.  The methods for extension of circulatory half-life have been reviewed recently by Lillicrap[13].   Some of these approaches were previously critiqued by Lundblad and Bradshaw[14].  One major problem is that it has not been possible to chemically modify factor VIII without the loss of activity; modification of lysine residues results in the loss of activity while the modification of the single sulfhydryl group yield equivocal data.  While a fusion protein approach has been useful for factor VIIa[15]. such appears to be ineffective for factor IX[16].   The process of making a peptide/protein larger by this technique or by modification with poly(ethylene) glycol likely affects renal clearance more than other catabolic processes and is thus without strong rationale in the case of Factor VIII.
I am not ignoring gene therapy for hemophilia A.  Gene therapy for hemophilia A has been promising for the past two decades[17,18].  It is my sense that gene therapy will be achieved for hemophilia A when there is concerted effort to coordinate the expression of vWF and factor VIII; success may be achieved when more consideration in given to the work of Montgomery and colleagues[8,19,20]

References .   

1. Lundblad RL, Davie EW:  The Activation of Antihemophilic Factor (Factor VIII) by Activated Christmas Factor (Activated Factor IX).  Biochemistry 3:1720-1725, 1964.

2. van Dieijen,  G., van Rijn, J.L., Govers-Riemslag, J.W., et al., Assembly of the intrinsic factor X activating complex - - interactions between factor IXa, factor VIIIa and phospholipid, Thromb.Haemost. 53, 396-400, 1985

3. van Dieijen, G., Tans, G., Rosing, J., and Hemker, H.C., The role of phospholipid and factor VIIIa in the activation of bovine factor X, J.Biol.Chem. 256, 3433-3442, 1981

4.  Mann, K.G., Membrane-bound enzyme complexes in blood coagulation,  Prog.Hemost.Thromb. 7, 1-23, 1984      

5. Walsh, P.N., Camp, E. and Dende, D., Different requirements for intrinsic factor – Xa forming activity and platelet factor 3 activity and their relationship to platelet aggregation and secretion, Brit.J.Haematol. 40, 311-331, 1978'

6. Mann, K.G., Brummel-Ziedins, K., Orfeo, T., and Butenas, S., Models of blood coagulation, Blood Cells Mol.Dis. 36, 108-117, 2006

7. Fricke, W.A. and Yu, M.Y., Characterization of von Willebrand factor in factor VIII concentrates, Am.J.Hematol. 31, 41-45, 1989

8. Over, J., Bouma, B.N., Sixma, J.J. et al., Heterogeneity of human factor VIII. III. Transitions between forms of factor VIII present in cryoprecipitate and in cyrosupernatant plasma, J.Lab.Clin.Med. 95, 323-334, 1980

9. Haberichter, S.L., Shit, Q., and Montgomery, R.R., Regulated release of VWF and FVIII and the biologic implications, Pediatr.Blodd Cancer 46, 547-553, 2006

10. Lundblad, R.L., Kingdon, H.S., Mann, K.G., and Mann, G.C., Issues with the assay of factor VIII activity in plasma and concentrates, Thomb.Haemostas. 84, 942-948, 2000

11. Bihoreau, N., Sauger, A., Yon, J.M. et al., Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor, Eur.J.Biochem. 185, 111-118, 1989

12. Kingdon, H.S. and Lundblad, R.L., An adventure in biotechnology; the development of therapeutics from whole blood transfusion to recombinant DNA to gene therapy, Biotechnol.Appl.Biochem. 35, 141-148, 2003

13. Lillicrap, D., Extending half-life to coagulation factors: where do we stand?, Thromb.Res. 122(suppl 4), 52-58, 2008

14. .Lundblad, R.L. and Bradshaw, R.A., Addressing product improvement using chemical modification in biopharmaceutical manufacture.  A case study in blood coagulation factor VIII, Bioprocess International, September 2006

15. Weimer, T., Wormsbächer, W., Kronthaler, U., et al., Prolonger in-vivo half-life of factor VIIa by fusion to albumin, Thromb.Haemost. 99, 659-667, 2008

16. Sheffield, W.P., Mamdani, A., Hortelano, G., et al., Effects of genetic fusion of factor IX to albumin on in vivo clearance in mice and rabbits, Br.J.Haematol. 126, 565-573, 2004

17.  Lozier, J.N. and Brinkhous, K.M., Gene therapy and the hemophiliac, JAMA 271, 47-51, 1994

18. Youjin, S. and Jun, Y., The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy, Biotechnol.Lett. 31, 321-328, 2009

19. Wilcox, D.A., Shi, Q., Nurden, P., et al., Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII, J.Thromb.Haemost. 1, 2477-2489, 2003

20. Shi, Q., Fahs, S.A., Wilcox, D.A., et al., Syngeneic transplantation of hematopoietic stem cells that are genetically modified to express factor VIII in platelets restores hemostasis to hemophilila A mice with preexisting FVIII immunity, Blood 112, 2713-2721, 2008