Factor VIII Mimics in Treatment of Hemophilia A

The reader is directed to an excellent review of this area (Shetty, S. and Ghosh, V., Novel therapeutic approaches for haemophilia, Haemophilia 21, 152-161, 2015)


               I have sent the 1200+ page manuscript for the fourth edition of Chemical Reagents for Protein Modification and, after trying to organize the office  (an extremely generous description of my writing space) and I have plunged into the next book which concerns extra vascular fluids.   In the course of working on the introductory chapter,  I became interested in the subcutaneous administration of protein biologics.  While most work has focused on the subcutaneous administration of immunology therapeutics(1), I did find two application to coagulation factors, one on a glycoengineered factor IX (2) and other on a novel bi specific antibody with factor VIII activity (3).  The latter product, a bi specific antibody with factor VIII activity, resulted in my consideration of the various factor VIII substitutes which I have come across in the past fifty years which has, in turn, led to the current note.    At the onset, it is important to note that  there are materials such as peanuts and peanut flour which were suggested to have an in vivo hemostat ic effect in hemophilia A (4) but were subsequently shown have an effect on fibrinolysis (5) similar to that seen with tranexamic acid (6).  The effect of peanuts is inhibition of fibrinolysis which stabilizes fibrin clot formation with improved hemostasis in individuals with hemophilia.
               As I have previously noted, I met factor VIII as a young graduate student at the University of Washington in 1962.  My thesis research was focused on function of factor VIII in blood coagulation and resulted in several publications (7,8).   While later  work by other investigators showed that the conclusions in this study were incorrect in that factor VIII is not a proenzyme which is converted to an active enzyme by factor IXa but rather functions as cofactor (9) in the what become known as the  'tenase' complex (10).    The formation of the 'tenase' complex  results in an increase in the catalytic efficiency (kcat/Km) of factor IXa in the activation of factor X by a factor of 106.  As an aside, this increase in catalytic efficiency is somewhat larger than that observed on the activation of chymotrypsinogen to chymotrypsin as measured with low molecular weight substrates (11).   The work by van Dieijen and coworkers (9) established the platelet surface effectively concentrates the enzyme (factor IXa) and the substrate (factor X) thus lowering Km while factor VIII increases the catalytic efficiency of the reaction.  The mechanism by which catalytic efficiency is increased is poorly understood; however, progress is being made in this area (12,13) most likely involving a conformational change occurring on the specific  binding of factor IXa to factor VIIIa (14).   The function of factor VIII is closely regulated with activation by thrombin required for function (15) with inactivation occurring either by dissociation of factor VIIIa or inactivation by activated protein C (16).   In addition, platelets need to be activated to participate in in vivo 'tenase' complex (17).  While a variant of factor VIII analogous to factor V Leiden has not been observed (18), increased levels of factor VIII (by functional assay) is considered to be a thrombotic risk (19,20).  Before moving on,  I want to emphasize that the above synopsis of a large body of work emphasizes that the acceleration of factor IXa activation of factor X  by factor VIII is highly regulated process dependent on the structure of factor VIII and failure of inactivation could result in thrombosis.   
               There have been a number of studies over the past several decades to identify materials that would substitute for factor VIII with the goal of either increasing understanding of factor VIIII function.  Early work from several laboratories suggested that factor VIII activity could be obtained from sources other than blood plasma.  A group at Harvard Medical School published a study in 1936 (21) demonstrating  the therapeutic effectiveness of orally administered extract of human placenta (presumably crude human tissue factor) in children with hemophilia.  A study on the oral administration of bovine serum  by van Crevald and associates in Holland was somewhat less successful (22).    The experiments with human placenta (21) might reflect the activity of tissue factor  (23) while the bovine serum experiments might reflect the activity of a mixture of activated coagulations factors such as those found in Autoplex® (24).   The oral administration of enzymes such as kallikrein has been reported (25, 26) with the effect demonstrated by physiological effect, not necessarily by physical presence.
               The early work on factor VIII was complicated by the low level of the protein in plasma and one distinguished investigator at the time questioned as to whether factor VIII was a protein (27).   As a personal aside, Don Hanahan who was the lipid chemist on the faculty asked me whether factor VIII was a protein as the first question during my oral examination - needless to say with a quantum mechanic chemist as the grad school wild card it was a very long afternoon.  The committee also included a future Nobel Laureate and a couple of future NAS (NOT NSA) members.   The poorly defined (a phrase used by biochemists to suggest that we don't have a clue) nature of factor VIII at that point in time provided the basis for diverse research projects.    Emily Barrow and John Graham at the University of North Carolina isolated a low molecular protein (25-28 kDa) from kidney which had factor VIII activity (28,29).   This material appeared to related to leucine aminopeptidase and required preincubation at 40oC in the presence of manganese ions prior to assay; magnesium was required during the process of purification.    The specific activity of the purified protein is modest, 117 factor VIII units per mg of a protein (28).   Later work by Barrow and Graham (29) showed that the protein with factor VIII activity could be isolated from either a normal canine kidney or a kidney obtained from a dog with hemophilia A.   This observation and the failure of a transplanted normal kidney to restore hemostasis in a hemophilic dog suggest that the kidney is not a source of physiologically significant factor VIII activity in the vascular system.    Barrow and Graham (29) did observe the kidney factor VIII was neutralized by a factor VIII inhibitor.   The two investigators later showed that factor VIII could be produced by the succinylation of plasma (30) and albumin (31) and observed that polyglutamic acid and polyaspartic acid possessed factor VIII activity (32).   These studies were performed by colleagues of mine at the University of North Carolina and I have no question about the validity of the data.  It is most useful for today's investigators to clearly understand the morass of factor VIII research now some 50 years ago.   I commend the reader to an excellent vignette by Whyte Owen (33) on "big piece, little piece."   Somewhat later, Lundblad and Roberts also at the University of North Carolina (34) showed the polylysine could function as a cofactor in activation of factor X by factor IXa.  The effect of polylysine was inhibited by the presence of calcium ions.   Jumping ahead a bit in time, a sulfated polysaccharide was advanced as therapeutic for hemophilia A or hemophilia B (deficiency of factor IX) (35,36).  It is suggested that the therapeutic effect is based on the inhibition of tissue factor pathway inhibitor (TFPI).  The sulfated polysaccharide did show effectiveness in vivo while polylysine was a potent inhibitor of the partial thromboplastin time most likely through inhibition of fibrin polymerization.   As the sulfated polysaccharide is a polyanion, it would be of interest to know if the earlier observations of Barrow and Graham on the effect of succinylated albumin (31) is due to TFPI inhibition.    A recent report (37) ascribed factor VIII activity to thioredoxin and suggested that thiol-disulfide exchange might involved in factor VIII function.   The sulfhydryl groups in factor VIII continue to be  poorly understood.  Finally, I would be remiss if I did not mention the potentiating of factor IXa activity in the hydrolysis of p-nitroanilide substrates (38).   There is also a report that the inclusion of ethylene glycol resulted in a 3-fold enhancement of the rate of factor X activation by factor IXa in the presence of phospholipid (39).  It is generally accepted that factor IXa does undergo a conformational transition with incorporation into the 'tenase' complex (40).  There has been one report that a small peptide enhanced the rate of factor X activation by factor IXa (41) and several reports on small peptide inhibitors of factor VIII  binding to vWF(42) and binding to phospholipid (phosphatidyl serine) surfaces (43).
               There has been some interesting work using antibody technology which is of some interest.   Antibodies which are agonists in activating receptors have been known for some time (44) and the reader is directed to a current example with the activation of fibroblast growth factor receptor 1 by a monoclonal antibody (45).  Kerschbaumer  and coworkers (46) described an antibody which bound to factor IXa and increased the rate of factor X activation in the presence of factor VIII, phospholipid vesicles and calcium ions; the antibody alone had no factor VIII activity.  Further work  suggested that the antibody could increase kcat for the reaction with little effect on Km for the reaction.   The antibody also enhanced the activity of factor VIII in promoting thrombin generation in factor VIII-depleted plasma (presented as a "booster effect').    Takeyama and coworkers (47) described a monoclonal antibody to factor VIII that enhanced the rate of thrombin activation of factor VIII; the antibody also enhanced the thermal stability of factor VIII.   Subsequently Scheiflinger and coworkers (48)described an antibody to factor IX which could replace factor VIII in the activation of factor X by factor IXa in the presence of calcium ions and phospholipid.  This antibody also supplied factor VIII activity in as plasma-based assay system.  These investigators also suggested that this antibody could be useful in the treatment of factor VIII inhibitors.   The sum of the data suggest that the antibody increases the catalytic efficiency of factor IXa.   More recent work (49,50) from Japanese workers describe the development of a bi specific antibody (factor IXa, factor X) which supplied factor VIII activity for the factor IXa-catalyzed activation of factor X.  This material can be formulated for subcutaneous delivery and represents an exciting advance with potential for the treatment of factor VIII inhibitor patients. I am less optimistic about its use as factor VIII product; however,  I could be wrong.  As a former football official, I do know that I can make mistakes and can likely find the films to prove such an assertion.
Conclusions
1.  There are a variety of substances which can substitute for factor VIII in the factor IXa-catalyzed activation of factor X.
2.  A material which has in vitro activity does not necessarily have in vivo function
3. There are some substances which may show in vivo activity in correcting bleeding in hemophilia but do not have factor VIII activity in in vitro systems.
4.  It is not clear that the in vivo function of those substances which have in vitro activity is dependent on the in vivo regulatory elements which control factor VIII function and thus pose a potential thrombotic risk.
3. The bi specific antibody has significant potential for the treatment of factor VIII inhibitors.
©Roger L. Lundblad, October, 2013

 

 


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