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The molecular basis ofhemophilia 223
1%
ARE
(A of ATG)
https://t.me/med1917
to control. Inhibitory antibodies interfere with the normal
function of factor VIII in a number of different ways. The
most frequent site of inhibitor binding occurs within the A2
and C2 domains. Inhibitors may thereby block the ability of
activated factor VIIIa to bind and activate factor IX, or
inhibit the binding of factor VIII to VWF or negatively
charged phospholipid surfaces. Inhibitors may also hinder
the activation of factor VIII by thrombin, or the subsequent
release of factor VIII from VWF. Proteolysis of factor VIII
has recently been identified as a novel additional mechanism
of inactivation in some cases.
Activated prothrombin complex concentrates (e.g.
FEIBA®) and recombinant activated factor VIIa (NovoSeven®)
Missense/nonsense
64%
Small indels
18%
Complex
rearrangements
Figure16.8 Relative frequency of different variant types associated
with hemophilia B. Like most monogenic disorders, single nucleotide
variants (shown in various shades of gray) are responsible for the
majority of cases (75%). Gross abnormalities, such as copy number
variants, are less common and are responsible for a quarter of cases.
Large
indels
6%
Splicing
9%
Regulatory
2%
are valuable therapeutic materials in controlling bleeding in
those patients with inhibitory antibodies. Recombinant porcine factor VIII (susoctocog alpha, Obizur®) has also been
developed and is emerging as an additional, licensed therapeutic agent, but only in acquired hemophilia A currently.
The rationale is that the porcine molecule is sufficiently similar (homologous) to human factor VIII to exert a hemostatic
effect but at the same time sufficiently different to avoid
rapid neutralization by antibodies. An alternative novel therapeutic has harnessed a biphenotypic monoclonal antibody
(Emicizumab, Hemlibra®) to mimic the function of the FVIII
molecule, bringing activated FIX in proximity to FX to activate it. While this was initially used as a treatment for hemophilia A patients with inhibitors, it is now also used in
patients without inhibitors because it can be administered
subcutaneously at intervals of 2–4 weeks. This compares very
favorably with factor VIII concentrate which is infused
intravenously every 2–3 days. However, additional factor
replacement is required to treat breakthrough bleeds or to
cover surgery. For patients with inhibitors, the choice of
additional bypassing agents to treat bleeds has to take
account of potential thrombotic complications.
Another important strategy in the management of these
patients who develop inhibitory antibodies is immune tolerance, which involves the daily administration of conventional coagulation factor concentrate over a period of some
months. This usually results in the eventual disappearance of
the antibody, as the body becomes tolerant of the protein and
inhibitor formation is suppressed.
Following the elimination of the risk of transmission of
viruses by coagulation factor concentrates, the risk of
inhibitor development remains the principal danger faced
by people with hemophilia nowadays. Data from the UK
registry suggest that approximately 30% of all patients with
severe hemophilia A will develop antibodies at some time,
half of which are transient and low- titer inhibitors. The
majority of inhibitors in severe hemophilia A occur in
previously untreated patients (PUPs) early in life, during
the first 50 exposure days (ED) to the therapeutic CFC
Figure16.9 F9 variants resulting in hemophilia B Leyden. Map of the F9 promoter showing the position of selected transcription factor binding
sites. ARE, androgen response element; LF- A1/HNF4, liver factor- A1/hepatocyte nuclear factor 4; C/EBP, CCAAT/enhancer binding protein. The
dashed lines indicate the nucleotide number upstream of the transcription start site. White triangles indicate the positions of the most common
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variants associated with the Leyden phenotype. Note that these do not involve the ARE. The black triangle indicates the position of a variant that
causes hemophilia B with stable levels throughout life.
LF-A1/HNF4
–51 –44 –29 –12 +1
C/EBP
Factor IX coding sequence
224 Molecular Hematology
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Median occurrence is at approximately 10–15 days in severe
hemophilia A. However, in non- severe hemophilia A, the risk
is now thought to be lifelong and persists after 50 exposure
days. In this context, the alloantibody has the potential to
cross- react with the individual’s endogenous FVIII, decreasing
their baseline to potentially severe levels (<1%) with a resulting deterioration in bleed phenotype. There is also a low- level
occurrence of inhibitor detection in previously treated patients
(PTPs) with severe hemophilia A, despite years, often decades
of regular exposure to FVIII CFC.
It is now clear that the major factor that determines the
predisposition to inhibitor development is the underlying
genetic variant. Certain types of gene defects in hemophilia
are undoubtedly associated with a significantly increased
risk of inhibitor development (Figure 16.10). The risk of
inhibitor development in patients with severe molecular
defects, such as large deletions, nonsense variants, and the
intron- 22 inversion, is 7–10 times higher than in patients
with other defects such as missense variants, small deletions,
and splice- site variants. The overall risk of inhibitor development in patients with the common intron- 22 inversion is
approximately 30%.
There is evidence from family and twin studies that other
subtle genetic factors play a role, although no associations
with specific human leukocyte antigens (HLA) or other
linkages have been conclusively identified. Race may also
influence the risk of inhibitor development, and several studies have shown that people of Afro- Caribbean origin are
more susceptible to inhibitor formation. Links between
variants in immune response genes have been reported. Such
variants might have an impact on inhibitor development by
increasing the production and secretion of chemicals that
ultimately enhance the production of antibodies directed
against factor VIII. In the case of interleukin (IL)- 10, one
particular apparently benign variant (134- bp variant of a CA
repeat microsatellite in the promoter region of the IL10 gene)
was found to be associated with a 4.4- fold increased risk of
inhibitor development. This same variant has already been
shown to influence the level of antibody production in such
diverse diseases as myasthenia gravis, multiple myeloma,
and systemic lupus erythematosus. More recently, the same
group reported that a particular variant (C/T at −318) in the
cytotoxic T- lymphocyte- associated protein (CTLA)- 4 receptor
of certain immune cells actually confers protection against
inhibitor development. Other than knowledge of a family
history of inhibitor formation, which confers increased risk,
it continues to be challenging to risk- stratify inhibitor risk by
other genetic markers. It has become clear that CFC product
choice may be contributory to inhibitor risk in previously
untreated patients (PUPs), either between recombinant
products or between a recombinant (rFVIII) and plasmaderived, von Willebrand factor (VWF) containing product
(pdFVIII).
Inhibitor development in hemophilia B is a rare event,
occurring in probably less than 3% of patients, and these
patients often have an underlying large gene deletion. In contrast with the immunoglobulin inhibitors in patients with
hemophilia A, inhibitory antibodies in patients with hemophilia B are often capable of fixing complement proteins. The
administration of coagulation factor concentrate in such
cases may therefore trigger severe, often anaphylactic, allergic reactions. The development of nephrotic syndrome has
also been reported in patients with hemophilia B and inhibitors undergoing immune tolerance induction treatment with
factor IX concentrates.
Therapeutic applications ofmolecular
biology topatient care
Carrier testing
High risk
75%0%Multi domain
Large
deletions
Single domain
Low risk
Figure16.10 Variant types and risk of inhibitor development in
hemophilia A.
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Light chain
Nonsense
mutations
Heavy chain
Intron 22
Inversions
Non A-run
Small
deletions
A-run
C1–C2 junction
Missense
Non C1–C2 junction
Splice site
mutations
Ideally, carriers of hemophilia should be identified before a
pregnancy, and offered counseling. The inheritance of hemophilia is sex-
linked, as with other disorders such as color
blindness and Duchenne muscular dystrophy. The daughters
of men with hemophilia are thus obligate carriers of the condition, with a 50:50 chance of passing on the condition to a
son, and there is a similar chance that a daughter of a carrier
will also herself be a carrier of the condition (Figure16.11).
The genes for factors VIII and IX are both encoded on the
X chromosome, and inheritance is thus sex- linked. The
daughter of a man with hemophilia is an obligate carrier
except in rare cases of mosaicism, but an affected man cannot pass the variant gene onto his sons. Hemophilia may thus
be transmitted to a grandson via a carrier daughter. Female
carriers have a 50% chance of passing the affected allele on to
each child. Thus, any daughters will have a 50% chance of
being carriers and any sons will have a 50% chance of being
The molecular basis ofhemophilia 225
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Affected father
Unaffected son
Unaffected
Carrier
Affected
Figure16.11 The inheritance of hemophilia.
Unaffected mother
affected. Some female carriers have reduced factor levels and
have mild hemophilia.
Note that while a positive genetic test confirms carriership
of hemophilia, a negative test does not necessarily exclude it.
If the familial variant is known, a negative test excludes
inheritance of that variant and so the risk of being a carrier
defaults to that of the general population. If the familial variant is not known, then a negative test is not helpful in excluding carriership. For this reason, genetic studies should start
with an affected male or obligate carrier where possible.
The phenotype of hemophilia remains constant within a
family, so that the daughter of a man with only mild hemophilia may be reassured that she can only transmit a similarly
mild form of the condition. However, a more common problem is to be confronted with a woman with only a vague history of a bleeding disorder in a distant relative. National
patient registers can be very helpful in establishing the type
and severity of bleeding disorder of an affected relative as a
first step in determining which tests need to be carried out. It
may seem logical to initiate carrier testing to determine carrier status as soon as possible in girls with a family history of
the condition, as this would facilitate management in the
case of an early and possibly unexpected pregnancy. There
are significant differences in legislation as well as clinical
practice among healthcare professionals in various countries
with regard to the timing of testing of children for genetic
disorders. Some take the view that it is unethical to test very
young children in order to determine carrier status for inherited disorders for conditions that have no immediate implications for their own health. In the United Kingdom, most
hematologists would generally be prepared to offer genetic
confirmatory carrier testing to girls in their early teens, with
the proviso that the issues must be discussed with the family
Unaffected father
Unaffected son Unaffected daughterUnaffected sonCarrier daughter Carrier daughter Carrier daughter
Carrier mother
Affected son
and the child deemed able to understand the implications of
such testing.
It is important to check FVIII or FIX plasma activity levels
in obligate and possible carriers soon after birth. It should be
emphasized that these plasma levels should not be used to
determine whether a female is a carrier of hemophilia and
that only DNA- based tests should be used. Indirect testing of
carrier status using the tracking of restriction fragment
length polymorphisms (RFLPs) has now been superseded by
direct identification of the underlying causative genetic
abnormality. Once the molecular defect has been identified
in an individual with either hemophilia A or B, direct screening for that defect could be applied in subsequent generations for both carrier testing and also antenatal diagnosis. It
is recommended that hemophilia centers collate information
on the family pedigree (“family tree”) to identify potential
carriers and facilitate screening.
Germline and somatic mosaicism may complicate the picture. This needs particular consideration in cases of sporadic
hemophilia where the mother of a child with hemophilia
does not appear to carry the variant in her own leukocyte
DNA. For this reason, negative reports stating that the familial variant has not been identified should acknowledge that
mosaicism has not been excluded.
Antenatal diagnosis ofhemophilia
It is possible to perform antenatal procedures to determine
whether or not a male fetus has hemophilia when a termination is being contemplated or when the result will affect
obstetric management. The currently available techniques
are amniocentesis and chorionic villous biopsy, which carry
a small risk of miscarriage. Quantification of the variant
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226 Molecular Hematology
Uterine wall
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using free fetal DNA has shown promise as a non- invasive
option but currently lacks sensitivity in the first trimester.
For these reasons, antenatal diagnosis is rarely utilized. This
also reflects the fact that many women with affected relatives
appreciate the major treatment advances in recent years, such
as recombinant products for lifelong prophylaxis, the expectation of an essentially normal life quality and life expectancy
for the younger generation of people with hemophilia and
the imminence of potentially curative gene therapy.
Amniocentesis was the first technique employed for antenatal diagnosis of hemophilia and other X- linked disorders
such as muscular dystrophy. While amniocentesis is both
technically simple and safe, an important limitation is the
fact that it may only be employed in the second trimester
ofpregnancy, at approximately 15 weeks’ gestation. Chorionic
villus sampling (CVS) was first applied to antenatal diagnosis
of a number of genetic disorders in the early 1980s, but is
now the principal method used for antenatal diagnosis of
hemophilia and several other single gene disorders. The
main advantage is that the method may be applied for antenatal diagnosis during the first trimester, so that if termination of the pregnancy is required this is easier to carry out.
Furthermore, the results of the test are often available within
only a few days of the procedure as (in contrast to amniocentesis) there is no need to culture cells before genetic analysis.
A sample is obtained by either a transabdominal or transvaginal route, under ultrasound guidance (Figure 16.12).
CVS should not be undertaken before 11 weeks of pregnancy
in order to minimize the risk of inducing congenital limb
abnormalities. It is now common for non- invasive, prenatal
determination of the fetal gender early in the first trimester
(as early as 5 weeks gestation) by free- fetal DNA analysis
(detecting a Y chromosome in fetal cells identified in the
maternal circulation). This result then contributes to the discussion of whether to pursue more invasive confirmatory
testing for the presence of the mutated F8 or F9 gene and
generally obviates the need for CVS in the presence of a
female fetus. An imminent extension to this non- invasive
assay will be for full antenatal diagnostics.
Direct fetal blood sampling may also be used for antenatal
diagnosis of hemophilia, but this method is usually only
offered as a last resort, either because it was not possible to
carry out DNA- based family studies in time or because such
studies were carried out but did not yield results. In this technique, fetal blood is taken from fetal umbilical vessels under
ultrasound guidance. The procedure requires considerable
expertise and will thus not be available in all hospitals. It is
usually carried out at a minimum of 18 weeks’ gestation. The
levels of factor VIII and IX in a normal fetus at around
19 weeks’ gestation are significantly lower than those in an
adult, approximately 40 and 10 IU/dL, respectively.
The need to abort an implanted fetus is a formidable
ethical concern for many female carriers of hemophilia.
Preimplantation diagnosis is another technique that has been
developed, and this may prove to be particularly attractive to
women who would not be prepared to undergo conventional
termination of a well- established pregnancy. The female carrier must undergo an invitro fertilization cycle and the fertilized ova are then biopsied. Embryos shown to be unaffected
by genetic testing can then be transferred to the uterus.
Amniotic uid
Placenta
Chorionic villi
Figure16.12 Chorionic villus sampling (CVS). A sample of trophoblastic
tissue from the placental area is aspirated with a fine needle under
general anesthesia. The procedure is usually carried out after 11 weeks’
gestation. DNA isolated from the fetal tissue can then be analyzed to
determine fetal sex and status with regard to hemophilia.
Recombinant factor concentrates
The development of plasma- derived coagulation factor
concentrates in the early 1970s dramatically improved both
the longevity and quality of life of patients with hemophilia,
and the demand for factors VIII and IX has risen steadily.
The burgeoning global demand for factor VIII can no longer
be met by products derived from volunteer blood donors.
The manufacture of recombinant coagulation factor proteins
can overcome these supply issues. However, manufacturing
processes still need to meet quality standards that increase
costs and are a barrier to increasing production on a global
scale. The most important advantage of recombinant products is safety by eliminating the risk of transmission of
human pathogens associated with plasma-
derived products.
Many patients with hemophilia were infected with HIV and/
or hepatitis C before the introduction of physical methods of
viral inactivation of plasma- derived coagulation factor concentrates in the 1980s. In the 1990s, there was concern about
the possibility of transmission of variant Creutzfeldt–Jakob
disease (vCJD) via blood products, as the prions believed to
be the cause of this neurological disorder are extremely
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The molecular basis ofhemophilia 227
FVIII
Vial of cells is “cultured” in
Ab column and ion-exchange columns
(E)
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resistant to the usual viral inactivation procedures such as
heat treatment and exposure to a solvent/detergent mixture.
Many clinicians now regard recombinant products as the
treatment of choice for all patients with hemophilia as they
offer the best possible protection from transmission of
blood- borne pathogens. However, high cost and limited production restricts the availability of these products in many
parts of the world.
Standard half- life recombinant coagulation factor concentrates are manufactured by insertion of the wild- type human
F8 sequence into mammalian cell lines (such as Chinese
hamster ovary (CHO) cells or baby hamster kidney (BHK)
cells), which are then grown in culture on an industrial scale.
(Circle of bacterial DNA that replicates)
Plasmid vector
(A) (B)
FVIII
Nucleus
vWF
Factor VIII (or IX) is then secreted into the growth medium,
from which it is subsequently extracted by monoclonal or
other immunoaffinity chromatography (Figure16.13). The
original recombinant factor VIII products all contained
added human albumin as a stabilizer. However, third- and
fourth- generation products are now available in which alternative stabilizers are used. Human albumin and all bovine
proteins have also been eliminated from the culture media of
these modern products and the incorporation of specific viral
elimination/inactivation steps further increases the margin
ofsafety. Standard half- life recombinant factor VIII has an
essentially identical amino acid structure to natural
plasma factor VIII, although increasingly, the apparently
vWF
Isolate human gene sequence and
splice into a suitable vector
Cells in
nutrient media
CHO cell
Chinese hamster ovary (CHO) cell (D)(C) Bioreactor
FVIII is puried from media using a monoclonal
Figure16.13 Manufacture of recombinant factor VIII. The gene for human factor VIII is incorporated into a bacterial vector (A, B). Inclusion of
the von Willebrand factor gene also enhances production of factor VIII. The vector is then inserted into a mammalian host cell (C). The cells grow
and multiply in a nutrient medium, and then secrete factor VIII (D). Factor VIII can be extracted and purified by immunoaffinity chromatography
(E). The final product also contains no von Willebrand factor.
Insert recombinant
plasmid into host cell
Impurities pass
through column
and rFVIII binds
to antibodies
Impurities
Column is washed
and rFVIII protein
is released
media, cells multiply into
millions of cells, each
expressing FVIII into the
media
FVIII
Impurities
rFVIII
Protein
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228 Molecular Hematology
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functionlessB domain of the F8 gene is deleted or truncated to
optimize production efficiency. Human cell (HEK293) derived
recombinant factor VIII has now been introduced. Evidence
exists to demonstrate different protein post- translational
modifications between human and CHO or BHK- derived
CFC products. It remains unclear whether this translates into
human- cell- derived rFVIII products having a reduced immunogenicity compared to CHO or BHK- derived products.
The main problem with standard recombinant factor concentrates is the relatively short half- life of about 12 h for factor
VIII and 18 h for factor IX. Fusion of the factor IX molecule
to the Fc portion of human IgG, albumin, or polyethylene glycol (PEG) has resulted in clinically significant increases in
half- life to 80–100 h. This has allowed some patients to
increase the injection interval from once every 3–4 days to
once a week or once a fortnight. Some concerns remain about
the accumulation of PEG in the body over many years of use,
particularly if treatment is started in childhood. However,
experience of using PEGylated drugs in other diseases has not
shown any adverse effects due to PEG accumulation.
The same recombinant engineering techniques have been
tried with FVIII but with much more modest improvements
in half- life to 14–20 h. The reason for this lack of effect is that
FVIII is bound to VWF in the circulation. This interaction
chaperones FVIII as demonstrated by the fact that in type 3
von Willebrand disease, when VWF is completely absent, the
half- life of FVIII is less than 2h. This knowledge has led to
the development of efanesoctocog alfa, a product that fuses
the B- domain- deleted recombinant FVIII protein to the
VWF D’D3 domain and two XTEN polypeptides. The XTEN
polypeptides are short molecules of six amino acids that further shield FVIII from degradation. This results in half- life
extension to 40–50 h.
Recombinant factor IX is also available. Recombinant factor IX is identical in amino acid sequence to the Ala148 (as
opposed to the less common Thr148) human polymorphic
variant. Plasma factor IX is synthesized in the liver and
undergoes post-
translational glycosylation of a number of
glutamic acid residues. Vitamin K is a vital cofactor in this
process, which is essential for its activity, but recombinant
factor IX is not as effectively carboxylated. The post- infusion
recovery does appear to be reduced when compared with
plasma- derived products, although the plasma half- life is
identical. It is a smaller molecule than factor VIII and requires
no albumin, or other material, to be added to the final product as a stabilizer. The cell line is grown in media that contain
no animal or human- derived proteins, but the product is subjected to nanofiltration to enhance its safety profile. There is
no suggestion of an increased risk of inhibitor development
associated with the use of recombinant factor IX.
Factor IX has more complex pharmacokinetics than
FVIII, as a smaller protein that is distributed extravascularly
and also binds to collagen IV. There is ongoing debate on the
true half-
life ofFIX, but it is likely longer than the previously
estimated 18 h due to this complex, multi- compartmental
pharmacokinetics. Strategies to prolong FIX half- life in the
intravascular space have proved successful with either glycopegylation, fusion to albumin, or the immunoglobulin Fc
molecule. Although prolonging detectable FIX activity in the
intravascular space three- to fivefold, the impact of each of
these novel technologies on extravascular distribution of FIX
is incompletely understood.
Another useful recombinant product is recombinant
activated factor VII (NovoSeven®; Novo Nordisk). It is now
recognized that factor VII plays a key role in the initiation of
the coagulation cascade through contact with tissue factor
released from damaged tissues, to form activated factor VII
(see also Chapter17). Recombinant activated factor VII is
very useful in the clinical management of patients with either
hemophilia A or B and inhibitory antibodies, as well as those
with acquired hemophilia.
Non- factor therapies
Emicizumab, the first FVIII mimetic, has already been
described in the section above on treating inhibitors. There
are other FVIII mimetics in development.
It has long been considered that hemophilia could be
treated by redressing the balance between pro- coagulant and
anticoagulant processes. As hemophilia is a defect in procoagulant function then hemostasis could be rebalanced by
inhibiting anticoagulant function. The key is to get the rebalancing right so that the reduction in bleeding does not come
at the cost of an increase in thrombosis. An advantage of this
approach is that it can be used in both hemophilia A and B,
whereas a FVIII mimetic will only work where there is a
deficiency of FVIII. Rebalancing therapies have focused
on reducing the activity of natural anticoagulants such as
antithrombin, tissue factor pathway inhibitor (TFPI), and
activated protein C. This can be achieved by monoclonal
antibodies that bind and remove the anticoagulant protein or
small RNA molecules that reduce transcription of the gene
for the anticoagulant protein. There have been thrombotic
complications in early studies, which is not unexpected, and
further development is required to determine if this can be
an effective and safe approach.
Gene therapy for hemophilia
(see also Chapter25)
Gene therapy poses a number of ethical problems, particularly
since effective and safe treatment with recombinant coagulation factors is already available for patients with both hemophilia A and B. However, gene therapy offers the prospect of a
permanent cure for hemophilia, but it must be emphasized
that although clinical trials in both hemophilia A and B have
demonstrated correction of factor levels lasting for many
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The molecular basis ofhemophilia 229
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years, it is not yet certain that these levels will remain high
long- term. Furthermore, surveillance for potential side effects
is required. Trials are all currently in adults and it is not known
There are two basic approaches for gene delivery into cells.
The first technique involves the direct injection of transducing vector into the bloodstream or target tissue, with
subsequent invivo transformation of the cells that take up
the gene, namely hepatocytes in hemophilia. Alternatively,
target cells may be modified by removal of cells from a
patient, with subsequent modification exvivo of these cells
followed by reinfusion. The first strategy has been favored in
hemophilia trials, utilizing non- integrating, non- replicative,
hepatotropic adeno- associated viruses (AAV). Wild- type
AAVs are communally acquired, intercurrent viral infections
of little consequence to an immunocompetent host. AAV
exists in multiple serotypes, some more homologous than
others, and are less immunogenic than adenovirus vectors
inthe gene therapy setting. Patients may have pre- existing
immunity to a given AAV serotype, currently making them
ineligible for gene therapy. A dose of AAV gene therapy does
generate high titer antibodies against the specific AAV serotype vector used, which may cross- react with other serotypes. This may prevent further doses of AAV gene therapy
with another serotype vector. Some, but not all, serotypes
trialed to date provoke a viral capsid- specific T cell response
thought, at least in part, to contribute to a complicating rise
in liver enzymes requiring intervention with steroids.
Despite the recent success of non- integrative AAV strategies, other gene therapy, gene editing, or base editing platforms are also undergoing evaluation. Integration events
provoke concerns of oncogenesis. However, “safe- haven”
gene editing may circumvent this issue, as is now being
trialed in hemophilia B, precisely inserting the F9 gene
downstream of the albumin promoter in hepatocytes using
zinc finger nuclease (ZFN).
The smaller F9 gene made it easier to package into the
modified AAV vectors for early study, yielding durable low
level, yet effective FIX expression in humans. Implementation
of the aforementioned R338L gain of function F9 Padua variant has further improved expression in subsequent hemophilia B trials. As expected, the larger F8 gene presented
problems of vector packaging. These have been overcome to
some extent and hemophilia A gene therapy trials have normalized FVIII activity levels in some participants. Curative
gene therapy does now seem to be within reach.
Conclusions
Hemophilia is an inherited disorder of coagulation, associated with congenital deficiency of factor VIII or IX. The
mode of inheritance is X- linked recessive, so that males are
predominantly affected, but some female carriers will also
have low levels and bleeding symptoms. These female carriers should be considered to have mild hemophilia.
Approximately half of cases arise in families with no previous
family history, and represent new variants. The typical features of severe hemophilia include spontaneous bleeding
into joints and resultant disability, but in the absence of treatment, more serious complications (such as intracranial hemorrhage) will lead to early death.
The first products used for the treatment of hemophilia
were derived from human plasma, but unfortunately, the use
of pooled plasma products before 1985 resulted in the transmission of serious viral infections such as HIV and hepatitis
C to many patients. The development of recombinant blood
products has eliminated the risk of transmission of these
infections, and also offers the prospect of a secure supply
chain and extended half- life products. The life expectancy of
the younger generation of hemophiliacs now approaches
that of the normal population.
The commonest molecular defect in severe hemophilia
A is an inversion in intron 22 of the factor VIII gene on
theX chromosome, which accounts for approximately half
of all cases. Genetic testing documents the genetic defect
in each family, and is required for identification of female
carriers. Antenatal diagnosis is available, although in
countries with sustainable hemophilia care, recourse to
termination of an affected pregnancy is rare. Prenatal
diagnosis is another option in some healthcare systems
forpreventing transmission of the condition. The development of extended half- life concentrates, non- factor therapies, and gene therapy have massively expanded the
treatment options for hemophilia.
Hemophilia resources onthe internet
EAHAD Coagulation Factor Variant Databases. http://dbs.eahad.org
World Federation of Haemophilia. http://wfh.org
United Kingdom Haemophilia Centre Doctors’ Organisation (UKHCDO).
http://ukhcdo.org
European Association for Haemophilia and Allied Disorders (EAHAD).
http://eahad.org
Further reading
Introduction
Darby, S.C., Kan, S.W., Spooner, R.J. et al. (2007). Mortality rates, life
expectancy, and causes of death in people with hemophilia A or B in
the United Kingdom who were not infected with HIV. Blood 110:
815–825.
Gomez, K. (2021). Genomic analysis for the detection of bleeding and
thrombotic disorders. Semin. Thromb. Hemost. 47 (2): 174–182.
Lee, C.A., Berntorp, E., and Hoots, W.K. (eds.) (2010). Textbook of
Hemophilia, 2e. Oxford: Blackwell Publishing.
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230 Molecular Hematology
https://t.me/med1917
Hemophilia A
Antonarakis, S.E., Rossiter, J.P., Young, M. etal. (1995). Factor VIII gene
inversions in severe hemophilia A: results of an international consortium study. Blood 86: 2206–2212.
Bagnall, R.D., Waseem, N., Green, P.M., and Giannelli, F. (2002).
Recurrent inversion breaking intron 1 of the factor VIIII gene is a
frequent cause of severe hemophilia A. Blood 99: 168–174.
Davidson, C.J., Tuddenham, E.G., and McVey, J.H. (2003). 450million
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Chapter17
https://t.me/med1917
The molecular basis ofvon
Willebrand disease
Omid Seidizadeh
1
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Foundation IRCCS Ca’Granda Ospedale Maggiore Policlinico, Milan, Italy
2
Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy
Introduction, 231
Function of von Willebrand factor in primary hemostasis, 231
Function of von Willebrand factor in intrinsic coagulation, 232
Gene organization, synthesis, and multimeric structure of von
Willebrandfactor, 233
1,2
and Luciano Baronciani
Introduction
von Willebrand disease (VWD) is a common inherited
bleeding disorder. Precise data regarding its prevalence are
not available due to the extreme variability in clinical symptoms of mild VWD. However, population- based studies give
an estimate of clinically significant VWD with a prevalence
of at least 100 per million. VWD is caused by the deficiency
or dysfunction of a multimeric plasma glycoprotein, von
Willebrand factor (VWF). Because of its ability to bind to
several ligands, VWF is involved in hemostasis through different mechanisms; however, it is best known for having two
main roles in primary hemostasis and intrinsic blood coagulation. VWF is directly involved in platelet binding to the
subendothelium and in platelet–platelet interactions and
acts as the carrier of procoagulant factor VIII (FVIII).
Mutations at the VWF locus can affect VWF synthesis, its
complex biosynthetic assembly, stability in the circulation,
and its binding interactions with specific ligands.
Function ofvon Willebrand factor
inprimary hemostasis
Under high shear stress conditions, VWF is essential for
both the recruitment and activation of platelets at the site of
vessel damage. During a hemostatic challenge, VWF acts as
a bridge between platelets and the subendothelium of blood
vessels and is involved in the formation of the platelet plug.
The role of platelets in hemostasis is to become irreversibly
attached to the sites of injury and cluster together. The primary physical factor that affects platelet binding to the vessel
1
von Willebrand disease and its classification, 235
Genetic defects in von Willebrand disease, 235
Treatment of von Willebrand disease, 243
New therapies for von Willebrand disease, 245
Further reading, 246
wall is the rate of blood flow in the vessel, being faster at the
center and slower close to the vessel wall. These variations in
velocity create a shearing effect, or shear stress, between layers of fluid. Under physiological conditions, VWF circulates
in a folded conformation, which prevents its unnecessary
interaction with platelets (Figure17.1A). Disruption of the
vascular endothelial surface leads to exposure of the subendothelium, with collagen fibrils and matrix VWF, and results
in an alteration of blood flow rate, thus increasing shear
stress. Plasma VWF binds rapidly and tightly, via its collagen
binding sites (A1 and A3 domains), to the exposed subendothelial matrix. Then, the immobilized VWF is activated
through conformational changes. This happens by flow shear
forces, which enable the VWF A1 domain to bind the platelet
receptor glycoprotein (GPIbα), a subunit of the platelet
GPIb- IX- V complex. However, the VWF- GPIbα interaction
does not provide irreversible platelets adhesion because of
the fast dissociation rate, and platelets tethered to the vessel
wall still move constantly in the direction of the flow (rolling), but at a much slower rate (Figure17.1B). At this stage,
the platelet receptor GPVI and the integrin α2β1 contribute,
by interacting directly with the exposed subendothelial collagen fibrils, to reach platelets adhesion. Both the GPIbα
binding to the immobilized VWF and GPVI binding to collagen fibrils induce downstream intracellular platelet signaling, which results in platelet activation. This event induces
an allosteric modification on the platelet surface integrin
GPIIb-
IIIa (αIIbβ3) which is required to obtain a firm platelet adhesion. αIIbβ3 does not appear to be involved in the
first events of platelet adhesion probably because its rate of
binding to the C4 domain (RGDS) of VWF is too slow to
mediate the initial platelet attachment to the vessel wall
Molecular Hematology, Fifth Edition. Edited by Drew Provan and Hillard M. Lazarus.
© 2024 John Wiley & Sons Ltd. Published 2024 by John Wiley & Sons Ltd.
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231
232 Molecular Hematology
(A) (B) (C)(C)
cellular
https://t.me/med1917
Intact vessel wall
Collagen
brils
Matrix
VWF
Extra
matrix
Figure17.1 The role of von Willebrand factor (VWF) in platelet- plug formation. Under conditions of physiologic blood flow, with an intact
vessel wall (panel A), plasma VWF is present in a coiled conformation, preventing its interaction with the platelet receptor glycoprotein Ibα
(GPIbα). The intact layer of endothelial cells also prevents platelets membrane glycoproteins GPIbα, nonactivated αIIbβ3, collagen receptors GPVI
and α2β1 to interact with VWF and collagen fibrils localized in the subendothelial matrix. At the site of vascular injury (panel B) with endothelial
denudation collagen fibrils and matrix VWF became exposed to flowing blood and shear forces. Plasma VWF rapidly binds to collagen fibrils and
then the immobilized VWF is uncoiled by flow shear forces, which enable its binding to the GPIbα. At first, VWF interacts only with the GPIbα and
platelet tethering occurs. This interaction has a rapid dissociation rate and platelets, due to the torque imposed by the flowing fluid, began to roll
in the direction of the flow, but at a much slower rate. At this time, GPVI and α2β1 bind to collagen (not shown) and promote platelet adhesion
and activation in synergy with the VWF–GPIbα interactions. As a consequence of platelet activation, integrin αIIbβ3 enhances its affinity for VWF.
This occurrence, along with the slow platelets rolling due to the VWF–GPIbα interaction, allows αIIbβ3 to firmly bind platelets at the site of
vascular injury (panel C). Platelet- to- platelet interactions, that finally lead to platelet- plug formation, is also mediated by the interaction of
αIIbβ3with VWF and, at low flow rate, with fibrinogen (not shown). Source: Adapted from Mannucci P.M. (2004). Treatment of von Willebrand’s
Disease. New England Journal of Medicine. 351: 683- 94.
Endothelial
cell
Platelet
Plasma
VWF
GPIbα
Non activated
αIIbβ3
Damaged vessel wall Platelet plug formation
Torque
Initial
platelet
tethering
Platelet
rolling
Platelet
activation
and adhesion
Activated
αIIbβ3
under high- flow conditions. However, when platelets
become activated, αIIbβ3increases its affinity for VWF. This
event, together with the slow motion of platelets due to the
VWF- GPIbα and the GPVI- collagen interactions, allows
αIIbβ3 to irreversibly bind platelets to the vessel wall. αIIbβ3
is also responsible for the platelet–platelet interaction, which
is mediated by VWF and, under slow- flow conditions, by
fibrinogen (Figure17.1C).
proteases (e.g. protein C) and avoiding its binding to the surface of activated platelets and endothelial cells (ECs). This
interaction with VWF is critical in prolonging FVIII halflife. When patients with severe VWD such as type 3 are
treated with purified FVIII, this is cleared with a half- life of
less than 2h, whereas the VWF- FVIII complex infused in
the same patient has a half- life of about 12–14 h. FVIII binding to VWF has also the effect of conveying FVIII at the sites
of vascular injury, thus increasing its low plasma concentration (1 nM), where it is mostly needed.
Function ofvon Willebrand factor
inintrinsic coagulation
VWF binds to FVIII via regions within the first 272 amino
acid residues of the mature subunit, in the D′ and D3
domains of VWF. Available data suggest that approximately
VWF and FVIII circulate in plasma as a noncovalent complex. VWF binding to FVIII is required to stabilize FVIII in
the circulation, preventing its premature cleavage by serine
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95–98% of FVIII is in dynamic equilibrium complexed with
VWF and that this association is constant with a 50:1molar
ratio of VWF (subunits) to FVIII.
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