Factor VIII: Past, Present, and Future

Factor VIII and hemophilia are bit strange in the world of commercial biotechnology.   First, the market is relatively  small; perhaps 400,000 to 500,000 worldwide of which about a quarter receive "state-of-the-art" treatment.1,2  Second, the cost of drug is the dominant factor in treatment3 representing the participation of only several companies in provision of product.a    Third, there is division of opinion on the value of prophylactic care versus reactive care.4,5 Fourth, only factor VIII concentrates are effective for the treatment of hemophilia A and there are no other uses for factor VIII concentrates.  Despite these constraints, the global market for factor VIII is between 1 and 2 billion dollars (US)/year; this figure does not include supporting products associated with inhibitor therapy.
The escalating costs of treatment3 present significant challenges to the hemophilia treatment community.6  Advances in the factor VIII product over the last 50 years has resulted in a major increase in quality of life such that management of hemophilia is a challenge in the older population.7-9  This progress has, however, occurred with considerable increases in cost.3 such that advanced therapeutics are available only in the "developed" world and a lack of adequate treatment modalities in the developing world.1,2 The presence of strong patient advocate groups in the "developed" world has driven accelerated approval of factor VIII therapeutics.
Excluding the desire for corporate income as a driver, there are two major issues which drove the development of factor VIII products produced with recombinant DNA technology.  The first is product safety in that recombinant products are assumed to be free of pathogens such as HIV and the second is ease of product administration.  Future advances are driven by the second of these issues,  product administration;  a rather broad view is taken of product administration including gene therapy, in-dwelling pumps, non-parenteral administration, and extension of therapeutic half-life of the factor VIII product.
The development of effective gene therapy for hemophilia A would obviate the need for any product development but progress in this area has been painfully slow.10   Recent work from Robert Montgomery's laboratory discussed below  would suggest that the expression of factor VIII is linked to that of von Willebrand factor.  Gene therapy is also complicated by product definition; will gene therapy be a service, a product, or a product combined with a service such as that seen with intravenous immunoglobulin?11,12
The author posits that future research on factor VIII is complicated by a profound lack of understanding of the molecular action of factor VIII.   Despite early considerations of factor VIII as a proenzyme,13 it is clear that factor is a cofactor for the action of factor IXa on factor Xa  in what been described as the "tenase" complex14 following on the earlier characterization of the "prothrombinase" complex.15  The classical enzymologist looks at cofactors as small, organic compounds16-19 which assist an enzyme in its action; factor VIII does not fit into this category.  It should be noted that the manufacture of factor VIII as a recombinant protein means that much information about factor VIII which would have been gathered during classical protein purification and characterization is not available.   In addition, the site and mechanism of factor VIII synthesis is still poorly understood. It is the purpose of this paper to discuss some of the questions about factor VIII which are important for development of improved therapy for hemophilia A. 
Factor VIII is a large (ca. 240-300 kDa) glycoprotein found in blood at relatively low concentration (0.2 μg/mL in plasma, approximately 1 nM).20,21  Factor VIII is associated with von Willebrand factor (vWF) in the circulation22 such that there is very little free proteins (less that 6%).21  Von Willebrand factor  is also a large protein formed from monomers of 250 kDa.23,24  There is approximately 50  times as much vWF in plasma as factor VIII.22   When factor is isolated from human plasma using immunoaffinity chromatography (antibody directed against factor VIII), there is vWF associated with the factor VIII.25  However, this vWF is of relatively low molecular weight as compared to the high molecular weight multimers associated with the hemostatic effect of vWF.24   Recombinant factor VIII binds rapidly to immobilized factor VIII (50% of factor VIII bound in 2 seconds)26 although another report suggests that 20% of recombinant factor VIII is unable to bind to vWF.27  Finally, recombinant factor VIII appears to bind randomly to vWF in plasma.28  Work from Montgomery's laboratory29  and Merten's laboratory30 suggests coexpression of vWF and factor VIII  This brief summary suggests two questions concerning the interaction of factor VIII and vWF of importance for hemophilia therapy.

It is now useful to briefly explore the history of hemophilia and the development of therapeutics.   Reports of bleeding consistent with hemophilia has been reported as early as 400-500 A.D.31 The treatment of hemophilia first required the development of transfusion medicine working from the discovery of the concept of circulation by William Harvey in 1628 to the first successful blood transfusion by James Blundell in 1818.32-34  The first blood transfusion for the treatment of hemophilia was reported by Lane in 1940.35  These early studies were performed either by direct donor to subject transfusion or collection of blood into a container for short period prior to infusion.  On occasion, a clot was removed from blood by physical defibrillation36 as well as the use of sodium phosphate.36-38 Transfusion medicine did not become practical until the introduction of citrate which allowed the separation of donor and recipient in space and glucose which allowed the same separation in time.39  However, the effective use of blood and/or blood products would have to wait until the development of blood banking in the 1930's40  and the development of plasma production and fractionation in WWII.41  There were some interesting observations on the treatment of hemophilia prior to the development of plasma fractionation as discussed below. 
Eley, Green and McKhann described the use of a blood coagulant prepared from human placenta for the treatment of hemophilia in 1936.42   The blood coagulant was prepared by the sequential extraction of human placenta (chopped in a food blender) with cold isotonic (0.145 M) NaCl and alkaline (pH 8.5) deionized water. After filtration through cheesecloth followed by centrifugation to remove red blood cells, extract was taken to pH 5 with HCl and the resulting precipitate which contained the blood coagulant activity obtained on standing.  This material was washed twice and stored in 0.2 % sodium bicarbonate at pH 7.5.  The activity of the material was estimated by the effect on the clotting time of recalcified citrated bovine or human plasma in glass tubes.  Activity was lost in alkaline or acid solution, on oxidation, or by Seitz filtration (Seitz filtration is used to remove large particles and viruses43).   Activity was also lost more rapidly in the presence of human serum.   The placental extract was more effective in the clotting of human plasma than bovine plasma.  The intravenous injection of this material  into experimental animals resulted in intravascular coagulation while intraperitoneal, intramuscular, or subcutaneous injections were not lethal but did result in the shortening of the capillary clotting time.44 The oral administration of this blood coagulant to a normal subject resulted in a decrease in the capillary blood clotting time from 6 minutes to 1.5 minutes; the coagulation time returned to normal after five hours.   Oral administration of this material to  one hemophilia patient resulted in a decrease in the clotting time from 3.5 hours to 10 minutes (considered to be the upper limit of normal); results in other patients was not as striking.  Intramuscular administration was also successful.  The duration of the effect lasted from several hours to 7 days.  It is likely that the blood coagulant material was crude human tissue thromboplastin.45
Bendien and van Crevald46 reported that the oral administration of bovine serum globulin had some hemostatic effect in one hemophilia subject.   The coagulation time was decreased and there was an improvement in his clinical condition.   A similar result was obtained on the administration of human serum.   A similar response was observed in another subject but not in a third subject.   The intramuscular injection of serum globulin was also effective.  The persistence of the effect was variable but not inconsistent with our understanding of the pharmacokinetics of factor VIII; the effect was somewhat longer than that observed for factor VIIa47 but this is speculation.  Patek and Stetson48 also showed that infusion of serum decreased the clotting time of hemophiliac blood; fresh serum was required as the infusion of aged serum had no effect on of the clotting time of hemophiliac blood.  The infusion of plasma also resulted in the correction of the clotting time of hemophiliac blood; the plasma factor was more stable than the serum factor. 
What can be gathered from the above which might be of use of in the Factor VIII story?

The development of blood banks in the 1930's and need for plasma and blood fractions in WWII resulted in an extraordinary collection of researchers who worked of blood and blood proteins in the period from 1938 through 1945.   Some of this work was collected by James Tullis52 and other materials appeared in the literature53-61 as the work was cleared for publication.b  It is noted that many of the scientists involved these studies went on distinguished scientific careers; notable in this group were John Edsall, George Scatchard, Philip Boyer, James Luck, and Beatrice Kassell.  Elsewhere, Hans Neurath was working with fibrinogen at Duke University62 as were Erwin Chargaff and Aaron Bendich at Columbia University.63 
The work of these individuals provided the basis for modern protein chemistry64 which was essential for the development of therapeutics for hemophilia. In addition to the increase in the knowledge of the protein molecule, the development of large-scale fractionation methods65 for plasma by E.J. Cohn and coworkers was of value in providing a concentrated source of factor VIII for therapeutic use.66-70 While the availability of a concentrated source of factor VIII was a major advancement, the serendipitous discovery of cryoprecipitate  by Judith Pool and colleagues71 was a more significant advance as it provided a facile method for local preparation of a therapeutic product.72  The cryoprecipitate concept also found use in the manufacturing of advanced factor VIII therapeutic products73-74 as it proved to be a "cleaner" starting material than Cohn Fraction I. The factor VIII product obtained by immunoaffinity chromatography73,74 is essential homogeneous and represented the ultimate product which could be derived from plasma.  There were several advances in the development of factor VIII products between Cohn I and the immunoaffinity-purified materials which have been reviewed elsewhere.75 I would also be remiss if I did mention another influence on the study of blood coagulation in the 1960-1980 time period.  As with the WWII period investigators above, the 1960-1980 period saw the entrance of investigators such as Earl Davie, Frank Castellino, Ken Mann, and Michael Griffith from basic science disciplines such as physical biochemistry, protein chemistry, and enzymology; these individuals were not hampered by the prejudices of previous investigators and brought major advancement to the field.   More recent work has seen the use of recombinant DNA technology for the production sophisticated factor VIII products.76,77 It is of some interest that this progress was accomplished with minimal information about the structure and function of factor VIII.
The last fifty years have seen great progress in the development of products for the treatment of hemophilia A in moving from relatively crude plasma protein preparations such as Cohn Fraction I to essentially homogeneous recombinant factor VIII preparations available in protein-free formulations. 78-80.  Progress in therapeutics will come from advances in the mechanism of delivery of the therapeutic including consideration of vehicles such as liposomes.81 For the purpose of the current discussion, gene therapy is considered a form of drug delivery.    Considering the state of the art then, what are the challenges in advancing hemophilia A therapy.

Some aspects of factor VIII were briefly discussed above.  While we have understanding of the molecular events (peptide bonds cleaved and conformational change(s) as judged by spectroscopy) involved in the thrombin activation of factor VIII and the physical interaction of factor VIIIa with factor IXa, we still do not understand how the combination of the various factors in the "tenase" complex results in the marked enhanced catalytic efficiency of factor IXa.  This is not intended to be critical of the considerable work of Fay, Mertens, and others but rather an indication of lack of understanding of the chemistry of factor VIII and factor IXa.  Factor VIII presents a considerable challenge to the protein chemist.

It is not possible to cover all of these issues within the scope of the current document.  I am choosing to focus on two topics, copper ions and the free sulfhydryl groups,  which I feel are related and important to improvements in delivery of therapeutic effect in hemophilia A. 
Elucidation of the structure of human factor VIII via cDNA technology by Vehar and coworkers in 1984 suggested homology with ceruloplasmin89 and  identification of a single bound copper ion in human factor VIII by Bihoreau and coworkers occurred in 1994.90  Additional support for a single copper binding site was obtained by modeling91 and a role is suggested for copper ion in the association of the heterodimer.92  While most of the work focused on cupric ions Cu (II), Tagliavacca and coworkers93 have suggested a role for cuprous Cu (I) ions.88  Finally, while there is structural homology with blue copper proteins, there is no evidence for the characteristic UV spectra of type 1 Cu (II)  proteins (lmax 597 nm)94 with factor VIII; the copper might be present in a type 2 Cu (II) binding site91 or present, as suggested by Taglivacca and coworkers,93 as Cu (I) ions.   However, definitive for a role for copper ions or, for that matter, calcium ions, has not been rigorously established.   The metals ions might be physically involved in the association of the heterodimer (or heterotrimer) or the metal ions might maintain conformation of the component polypeptide chains which are amenable for association into heterodimer or heterotrimer.  Gross and coworkers reported on the importance of the copper center in the stability of plastocyanin.95  This latter study is of importance as it provides support for copper ions in the stabilization of a protein.  Fay and coworkers92,96 have argued that copper ions are important for bridging the component chains of the heterodimer (heterotrimer) complex consistent with the modeling studies from UCSF.86  This would be consistent with a  type 1 copper binding site (blue copper binding site) which is composed of two histidine residues and a cysteine; a methionine might also be involved.  The intense blue color (lmax ≈ 600 nm) is a product of the interaction of the copper with the cysteine residue.94  A type 2 copper binding sites consist mostly of histidine residues with the coordination sphere completed by methionine, glutamate, or tyrosine97 and is not characterized by the 600 nm absorbance but rather by weaker absorbance at 330 nm.  Recent crystallographic studies on factor VIII suggest two type 1 binding sites in factor VIII.98,99

The copper-Factor VIII interaction is a bit of a puzzle.  While a bit of an oversimplification, a metal ion can serve to stabilize the structure of a protein or to function as a cofactor in enzyme action.100 As an ion which can exist in several valence states, copper is a redox system.  While it seems unlikely that a redox system is involved in factor VIII function, the redox action of copper may be involved in the stability of factor VIII.  Factor VIII does contain three free cysteine residues and the anaerobic101 oxidation of cysteine to cystine by cupric ions and the cupric ion-catalyzed aerobic oxidation of cysteine102 has been reported.   The copper-catalyzed auto-oxidation of cysteine is suggested to result in the production of hydrogen peroxide.103
There are then the following questions about copper and factor VIII.

We now move to a consideration of an issue related to copper, the three free cysteine residues.  In general, proteins that are secreted from the cell do not contain free sulfhydryl groups.  There are, of course exceptions to this dictum with the single free cysteine residue in albumin likely best known example in blood.105 This cysteine residue is thought to be involved in the thioesterase activity in the degradation of disulfiram but other residues including lysine and tyrosine are involved in the hydrolysis of other esters by albumin.  In the case of factor VIII, no catalytic activity has been ascribed to this protein but this author knows of no serious attempt to investigate this possibility.
Austen106 published the first systematic work on the modification of factor VIII with reagents considered to be reasonably specific for sulfydryl groups in proteins.  Inactivation of factor VIII activity (bovine factor VIII measured with the classical two-stage assay) was observed with iodine, hydrogen peroxide, iodoacetamide, and p-chloromercuribenzoate.   Iodoacetamide and p-chloromercuribenzoate can be considered to be reasonably specific for sulfydryl groups in proteins and, in the case of p-chloromercuribenzoate, reactivation was observed with the addition of cysteine.   It is of interest that the alkylation of copper-thionein with iodoacetamide results in the loss of Cu(I).107 In the case of factor VIII,  alkylation of a cysteine residue or residues might result in the loss of copper with resultant loss of activity.  The lost of activity would be a result of the loss of copper and not the direct result of cysteine modification.   Manning and coworkers108 studied the effect of various reagents on recombinant human factor VIII.   Reaction with bifunctional maleimide reagents such as bis-(maleimido)-methyl ether did not result in significant loss of activity; assay of free sulfydryl groups with Ellman’s reagent demonstrated 60% loss of sulfhydryl groups.   
What can be concluded about the importance of the free cysteine residues in factor VIII?

The above is intended to indicate some basic issues with factor VIII which need to be resolved for progress in the development of new therapeutic strategies.  Answers to these questions will come from the application of classical protein chemistry to the study of factor VIII.

a There may be more companies joining the hemophilia A market in the near future.
b The work was sponsored by the Office of Science Research and Development which was a precursor agency to DARPA.  As such, all of this work was classified.  However, even the papers cited are sparse on experimental detail but such detail can be obtained by examined the reports submitted by Harvard University during this period. These reports are in the Library of Congress in Washington, DC.



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