Coagulation: In medicine, the clotting of blood. The process by which the blood clots to form solid masses, or clots. More than 30 types of cells and substances in blood affect clotting. The process is initiated by blood platelets. Platelets produce a substance that combines with calcium ions in the blood to form thromboplastin, which in turn converts the protein prothrombin into thrombin in a complex series of reactions. Thrombin, a proteolytic enzyme, converts fibrinogen, a protein substance, into fibrin, an insoluble protein that forms an intricate network of minute threadlike structures called fibrils and causes the blood plasma to gel. The blood cells and plasma are enmeshed in the network of fibrils to form the clot.

1. Lecture
6
Congenital
and
Acquired
Disorders
Secondary
Hemostasis

2. Groups
of
Coagula<on
Factors
• Fibrinogen
group
– Fibrinogen,
V,
VIII,
XIIII
– All
are
acted
upon
by
thrombin
– All
are
consumed
during
coagula<on
(not
present
in
serum)
– FV
and
FVIII
à
Labile
– Fibrinogen
and
FVIII
à
acute
phase
reactants
• Increase
during
inflamma<on,
pregnancy,
estrogen
therapy,
stress
• Prothrombin
group
– II,
VII,
IX,
X
,
C,
S,
Z
– Depend
on
vitamin
K
during
their
synthesis
– Have
a
GLA
domain
at
the
N-­‐terminus
–
consis<ng
of
10-­‐12
glutamic
acid
(GLA)
residues
– Vitamin
K
catalyzes
the
carboxyla<on
of
the
y-­‐carbon
of
the
glutamic
acids
à
addi<on
of
a
second
carboxyl
group
– These
groups
are
nega<vely
charged
à
binding
Ca2+
ions
–
necessary
for
binding
to
PF3
• Contact
group
– PK,
HMWK,
XII,
XI
– Involved
in
ac<va<on
of
the
intrinsic
pathway
of
the
plasma-­‐based
coagula<on
model
– Moderately
stable
– NOT
consumed
during
cloZng
(found
in
serum)

3. Vitamin
K
Deficiency
• Found
in
leafy
green
plants
as
phylloquinone
and
in
bacteria
as
menaquinone
• Required
for
the
a]achment
of
gamma-­‐carboxyglutamic
acid
(GLA)
residues
to
the
VKDFs
• Factors
produced
in
the
absence
of
VK
lack
the
required
number
of
GLA
residues
and
are
func<onally
inac<ve
à
PIVKAs
• GLA
residues
facilitate
the
a]achment
of
the
factors
to
PF3
through
calcium
binding
• VK
deficiency
seen
in
1. Absence
of
bile
salts
in
GI
tract
• VK
is
fat
soluble
à
bile
salts
are
required
for
adsorp<on
2. Malabsorp<on
syndromes
• VK
is
absorbed
primarily
through
the
GI
tract
3. Dietary
lack
of
phylloquinone
• Due
to
lack
of
green
leafy
vegetables
in
the
diet
4. An<bio<c
therapy
• Kills
the
normal
flora
of
the
GI
tract—responsible
for
menaquinone
5. Bowel
surgery
• Combina<on
of
loss
of
phylloquinone
and
menaquinone
6. Newborn
infants
• Deficient
in
vitamin
K
at
birth
3

4. What
is
Vitamin
K?
• Fat
soluble
compound
– Necessary
for
the
synthesis
of
several
proteins
required
for
blood
cloZng
1) Vit
K
1
(Phylloquinone)
-­‐
Natural
form
-­‐
Found
in
plants
-­‐
Provides
the
primary
source
of
vitamin
K
to
humans
through
dietary
consump<on
2)
Vitamin
K2
compounds
(Menaquinones)
-­‐
Made
by
bacteria
in
the
human
gut
-­‐
Provide
a
smaller
amount
of
the
human
vitamin
K
requirement
Required
for:
1)
Coagula<on
2)
Bone
Mineraliza<on
3)
Cell
growth

5. Vitamin
K
-­‐
Carboxyglutamate
FII,
FV,
FVII,
FX,
PC,
PS
ACTIVE
FII,
FVII,
FIX,
FX,
PC,
PS
INACTIVE
reduced
oxidized

6. The
Vitamin
K
Cycle
• Dietary
vitamin
K
à
reduced
by
vitamin
K
reductase
to
generate
vitamin
K
hydroquinone
• Vitamin
K
hydroquinone
– Serves
as
a
cofactor
for
the
vitamin
K-­‐
dependent
carboxylase
– Converts
glutamic
acid
residues
at
the
N-­‐
termini
of
the
vitamin
K-­‐dependent
precursors
to
carboxyglutamic
acid
residues
– Creates
the
so-­‐called
Gla-­‐domain
• Gla-­‐domain
is
cri<cal
for
the
interac<on
of
the
vitamin
K-­‐dependent
cloZng
factors
with
nega<vely
charged
phospholipid
membranes
calcium
bridging
• During
vitamin
K-­‐dependent
carboxyla<on
1. Vitamin
K
is
oxidized
to
vitamin
K
epoxide
2. Vitamin
K
epoxide
is
then
converted
to
vitamin
K
by
vitamin
K
epoxide
reductase
6

7. Vitamin
K
Deficiency
• Diagnosis
– Prolonged
PT
and
aPTT,
normal
BT
and
TT
– In
mild
VKD
–
aPTT
will
be
normal
because
only
FVII
will
be
decreased
(FVII
has
shortest
½
life)
– Factor
assays
for
II,
VII,
IX,
and
X
• Note
1. FV
is
used
to
differen<ate
between
LD
and
VKD
2. FV
is
not
VK-­‐dependent,
but
is
synthesized
in
the
liver
3. TT
is
normal
to
prolonged
in
LD
4. TT
is
normal
in
VKD
• Treatment
– Aquamephyton
—
colloidal
solu<on
of
vitamin
K
for
parenteral
injec<on
• Given
intramuscularly
and
a
normaliza<on
of
the
PT
is
seen
in
12-­‐14
hours
• In
life-­‐threatening
situa<on—FFP
to
supply
the
missing
factors
7

8. Renal
Dysfunc<on
• Recognize
>
200
years
ago
• Underlying
pathophysiology
– Impaired
platelet
func<on
à
one
of
the
main
determinants
of
uremic
bleeding
– Mul<factorial
• Intrinsic
platelet
defects
• Abnormal
platelet
–endothelial
interac<on
• Uremic
toxins
and
anemia
also
contribute
• Levels
of
circula<ng
coagula<on
factors
are
normal
– Normal
PT/aPTT
– Unless
there
is
a
coexis<ng
coagulopathy
8

9. Renal
Disease
• Bleeding
from
uremia
is
major
cause
of
morbidity
in
pa<ents
with
end-­‐stage
renal
disease
• Focus
a. Platelet
dysfunc<on
b. Abnormal
platelet-­‐vessel
wall
interac<ons
c. Reten<on
of
uremic
toxins
d. Increased
levels
of
nitrous
oxide
• Correc<on
of
the
anemia
with
RBC
transfusions
or
rEPO
à
improve
the
bleeding
tendency
• Hemodialysis
par<ally
corrects
the
BT
9

10. Pathophysiology
of
Renal
Disease
• Platelet
dysfunc<on
is
the
most
important
– Decreased
platelet
aggrega<on
and
impaired
adhesiveness
• Impaired
IIb/IIIa
gp
receptor
• Altered
release
of
ADP
and
serotonin
from
α-­‐granules
• Decreased
TXA2
genera<on
• Abnormal
platelet
cytoskeletal
assembly
– Uremic
toxins
• Uremic
platelets
mixed
with
normal
plasma
func<on
normally
• Uremic
plasma
with
normal
platelets
à
impaired
func<on
• Guanidinosuccinic
acid
and
methylguanidine
may
be
poten<al
contributors
– Urea
does
not
appear
to
be
– No
correla<on
with
azotemia
(BUN)
and
platelet
dysfunc<on
10

11. Pathophysiology
of
Renal
Disease
• Anemia
– Common
finding
in
chronic
kidney
disease
– Due
to
decreased
produc<on
of
erythropoie<n
– Rheologic
factors
play
an
important
role
• HCT
of
30%
à
RBCs
primarily
occupy
the
center
of
the
vessel
• Where
platelets
are
in
a
skimming
layer
at
the
endothelial
surface
• Close
proximity
of
platelets
to
the
endothelium
promotes
adherence
and
platelet
plug
forma<on
• HCT
less
than
30%
platelets
are
more
dispersed
à
impaired
adherence
to
the
endothelium
– Nitric
Oxide
• NO
synthesis
is
increased
in
uremic
pa<ents
à
inhibitor
of
aggrega<on
• Increased
NO
synthesis
may
be
due
to
guanidinosuccinic
acid
(a
uremic
toxin)
11

12. Congenital
Disorders
of
Secondary
Hemostasis
Factor
Deficiency
½
Life
Hours
Lab
Finding
Clinical
Finding
I
1. Afibrinogenemia
No
clot,
Prolonged
PT,
aPTT,
TT,
No
Fibrinogen
Umbilical
stump
bleeding,
easy
bruising,
ecchymoses,
oozing,
poor
wound
healing,
hematuria
2. Hypofibrinogenemia
Prolonged
PT,
aPTT,
TT,
Low
Fibrinogen
Mild
bleeding
3. Dysfibrinogenemia
Normal
Fib
an<gen
with
low
ac<vity
(clot)
Possible
hemorrhage/thrombosis
Possibly
asymptoma<c
II
Hypoprothrombinemia
100
Prolonged
PT,
aPTT
Postopera<ve
bleeding,
epistaxis,
menorrhagia,
easy
bruising
V
Parahemophilia
25
Prolonged
PT,
aPTT,
BT
Epistaxis,
menorrhagia,
easy
bruising
VII
Hypoproconver<nemia
5
Prolonged
PT,
aPTT
Epistaxis,
menorrhagia,
cerebral
hemorrhage
VIII
Hemophilia
A
8-­‐12
Prolonged
aPTT,
normal
PT,
BT
Mild,
moderate,
severe
vWF
16-­‐24
Variable
aPTT
and
BT,
normal
PT
Mild,
moderate,
severe
IX
Hemophilia
B
(Christmas
Disease)
20
Prolonged
aPTT,
normal
PT
Mild,
moderate,
severe
X
Stuart-­‐Prower
Deficiency
65
Prolonged
aPTT,
normal
PT
Menorrhagia,
bruising,
epistaxis,
CNS
bleeding
XI
(Hemophilia
C)
65
Prolonged
aPTT,
normal
PT
Mild
bleeding,
bruising,
epistaxis
XII
Hageman
Trait
60
Prolonged
aPTT,
normal
PT
Thrombo<c
tendency,
NO
bleeding
XIII
Factor
XIII
Deficiency
150
Normal
aPTT
and
PT,
abnormal
5M
Urea
Solubility
Assay
Umbilical
stump
bleeding,
poor
wound
healing,
excessive
fibrinolysis,
male
sterility,
difficulty
conceiving,
intracranial
hemorrhage
PK
Prekallikrein
(Flecther
Factor)
35
Normal
aPTT
and
PT
Thrombo<c
tendency,
NO
bleeding
HMWK
Fitzgerald
Factor
156
Normal
aPTT
and
PT
Thrombo<c
tendency,
NO
bleeding
12

13. Bleeding
disorders
have
been
recognized
since
ancient
<mes…
The
Talmud
(2nd
century
AD)
states
that
male
babies
do
not
have
to
be
circumcised
if
two
brothers
have
died
from
the
procedure
In
12th
century
Albucasis,
an
Arab
physician,
wrote
about
a
family
in
which
males
died
of
excessive
bleeding
from
minor
injuries
In
1803,
Dr.
John
O]o,
Philadelphia,
wrote
about
an
inherited
hemorrhagic
disposi<on
affec<ng
males
In
1828
at
the
University
of
Zurich,
“hemophilia"
was
first
used
to
describe
a
bleeding
disorder

14. Congenital
Factor
Deficiencies
• Most
common
– Hemophilia
A
–
deficiency
of
FVIII
– Hemophilia
B
–
deficiency
of
FIX
Occur
very
early
in
life
• Characterized:
1. Sow
<ssue
bleeds
2. Joint
bleeds
3. Bleeding
into
body
cavi<es
4. Bleeding
into
CNS
• Manifest:
– Awer
minor
trauma,
surgery,
tooth
extrac<ons
– May
be
spontaneous
• Physical
Exam:
– Petechiae,
ecchymoses,
hematomas,
joint
deformi<es
• Lab
Exam:
– CBC
including
platelet
count,
PT,
aPTT,
Fibrinogen,
Thrombin
Time

15. Hemophilia
• The
hemophilias
are
a
group
of
related
bleeding
disorders
that
most
commonly
are
inherited
• “Hemophilia"
is
used,
it
most
owen
refers
to
the
following
two
disorders
– Factor
VIII
deficiency
(Hemophilia
A)
– Factor
IX
deficiency
(Hemophilia
B
à
Christmas
disease)
• Hemophilia
A
and
B
are
X-­‐linked
recessive
diseases
• They
exhibit
a
range
of
clinical
severity
that
correlates
well
with
assayed
factor
levels

16. Disorders
of
Secondary
Hemostasis
• Hemophilia
A
and
B
– Sex-­‐linked
recessive
disorders
first
described
in
the
Talmud
in
the
5th
century
– By
the
end
of
the
19th
century
the
cloZng
<mes
of
plasma
from
persons
with
hemophilia
were
found
to
be
greatly
prolonged
compared
with
the
cloZng
<mes
in
nonbleeders
– By
1947
hemophilia
was
a]ributed
to
a
single
protein
deficiency
– Pavlovsky
showed
that
plasma
of
some
hemophilic
pa<ents
could
correct
the
in
vitro
or
in
vivo
defects
of
other
pa<ents
with
clinically
iden<cal
bleeding
disorders
à
led
to
recogni<on
of
mulLple
types
of
hemophilia
– Hemophilias
A
and
B
together
occur
in
about
1/5,000
of
the
general
popula<on
– Hemophilia
A
is
about
4-­‐6x
more
common
than
Hemophilia
B
– Defect
in
hemophilia
is
due
to
a
muta<on
located
on
the
“X”
chromosome
• Females
can
be
carriers
– One
normal
+
one
defecLve
“X”
chromosome
• Females
are
asymptoma<c
1. Transmit
one
abnormal
X
chromosome
to
each
male
offspring
2. Male
offspring
would
have
hemophilia
16

17. Disorders
of
Secondary
Hemostasis
• Hemophilia
A
and
B
– Sex-­‐linked
recessive
disorders
first
described
in
the
Talmud
in
the
5th
century
– By
the
end
of
the
19th
century
the
cloZng
<mes
of
plasma
from
persons
with
hemophilia
were
found
to
be
greatly
prolonged
compared
with
the
cloZng
<mes
in
nonbleeders
– By
1947
hemophilia
was
a]ributed
to
a
single
protein
deficiency
– Pavlovsky
showed
that
plasma
of
some
hemophilic
pa<ents
could
correct
the
in
vitro
or
in
vivo
defects
of
other
pa<ents
with
clinically
iden<cal
bleeding
disorders
à
led
to
recogni<on
of
mulLple
types
of
hemophilia
– Hemophilias
A
and
B
together
occur
in
about
1/5,000
of
the
general
popula<on
– Hemophilia
A
is
about
4-­‐6x
more
common
than
Hemophilia
B
– Defect
in
hemophilia
is
due
to
a
muta<on
located
on
the
“X”
chromosome
• Females
can
be
carriers
– One
normal
+
one
defecLve
“X”
chromosome
• Females
are
asymptoma<c
1. Transmit
one
abnormal
X
chromosome
to
each
male
offspring
2. Male
offspring
would
have
hemophilia
17

18. Hemophilia
• Hemophilia
A
and
hemophilia
B
are
clinically
idenLcal
and
must
be
dis<nguished
from
von
Willebrand
disease
– Hemophilia
A
demonstrates
sex-­‐linked
inheritance
• Muta<on
occurs
on
the
FVIII
gene
located
on
Xq28
– Hemophilia
B
(Christmas
disease)
demonstrates
sex-­‐linked
inheritance
• Muta<on
occurs
on
the
FIX
gene
located
on
Xq27
• Primarily
a
disease
of
males—females
carry
the
defec<ve
gene
(asymptoma3c)
• Hemophilic
females
are
exceedingly
rare
• Carriers
possess
~
50%
factor
levels
–
protec<ve
against
bleeding
– Hemophilic
females
1. Doubly
heterozygotes
–
affected
inherited
from
a
carrier
mother
and
an
affected
father
2. Carriers
with
a
defec<ve
allele
on
one
X
chromosome
and
the
normal
allele
on
the
other
X
chromosome
undergoes
inacLvaLon
(lyoniza3on)
3. Turner’s
Syndrome
–
loss
of
one
X
chromosome
18

19. X-­‐Linked
Recessive
Inheritance
Carrier
female
Affected
male
Normal
male
• Affected males (XY):
– sons unaffected (no male to male
transmission)
– daughters obligate carriers
• Carrier female (XX):
– ½ sons affected; ½ daughters carriers
• Affected females: very rare.
New
muta<on
in
germ
cell
New
muta<on
in
maternal
or
paternal
germ
cell

20. 20
What’s
wrong
with
this
picture?
X-­‐linked
recessive
inheritance
of
hemophilia.
Asterisk
(*)
designates
affected
chromosome
XX

21. Thrombin
Genera<on
in
Normal
Individuals
• Normal
individuals
1. Forma<on
of
TF/VIIa
complex
following
vascular
injury
2. Extrinsic
pathway
ac<va<on
of
FX
via
Extrinsic
Tenase
complex
[TF:FVIIa:PF3:Ca2+]
3. Ini<al
burst
of
thrombin
4. TFPI
is
released
from
endothelial
cells
and
down-­‐regulates
the
[TF:VIIa:FXa]
complex
à
turns
off
the
extrinsic
genera<on
of
thrombin
5. Thrombin
generated
from
the
Extrinsic
Pathway
à
thrombin
genera<on
6. Thrombin
converts
FVIII
à
FVIIIa
7. FVIIIa
is
a
cofactor
for
the
forma<on
of
the
Intrinsic
Tenase
complex
[VIIIa:IXa:PF3:Ca2+]
8. Intrinsic
tenase
complex
is
responsible
for
con$nued
thrombin
genera<on

22. Pathophysiology
Hemophilia
A
• Insufficient
genera<on
of
thrombin
by
– FIXa/VIIIa
complex
through
the
intrinsic
pathway
of
coagula<on
cascade
– Bleeding
severity
complicated
by
excessive
fibrinolysis
1. IIa
cannot
feedback
to
ac<vate
VIII
à
VIIIa—VIII
is
defec3ve
[Hemophilia
A]
2. As
a
result
—VIIIa
cannot
bind
to
FIX
(FIX
is
normal
but
nonfunc3onal)
3. Due
to
lack
of
thrombin
ac<va<on
of
TAFI
– IIa
à
genera<on
of
TAFI
– In
normal
TAFI
turns
OFF
fibrinolysis
– In
hemophilia
there
is
a
decrease
in
TAFI
so
TAFI
cannot
turn
off
fibrinolysis
» Decreased
cloZng
due
to
decreased
FVIII/FIX
» Increase
in
fibrinolysis

23. Pathophysiology
Hemophilia
B
• Insufficient
genera<on
of
thrombin
by
– FIXa/VIIIa
complex
through
the
intrinsic
pathway
of
coagula<on
cascade
– Bleeding
severity
complicated
by
excessive
fibrinolysis
1. IIa
feedbacks
to
ac<vate
VIII
à
VIIIa
—FVIIIa
serves
as
a
cofactor
to
orient
FIXa
in
forming
the
intrinsic
tenase
complex
2. As
a
result
—FIX
cannot
bind
form
the
intrinsic
tenase
complex
to
ac3vate
FX
[FIX
is
defec3ve]
– In
hemophilia
TAFI
cannot
turnoff
fibrinolysis
» Decreased
cloZng
due
to
decreased
FVIII/FIX
» Increase
in
fibrinolysis

24. Hemophilia
• Pathophysiology
of
hemophilia
A
and
hemophilia
B
is
based
on
– Insufficient
generaLon
of
thrombin
by
the
FIXa/FVIIIa
complex
in
the
intrinsic
pathway
of
the
coagula<on
cascade
–
defec3ve
intrinsic
tenase
complex
forma3on
24
Hemophilia
B
DefecLve
FIX
Hemophilia
A
Defec$ve
FVIII
Intrinsic
Tenase
Defec$ve
Extrinsic
Tenase
Normal

25. Disorders
of
Secondary
Hemostasis
• Clinical
Symptoms
of
Hemophilia
A
and
B
– Clinical
symptoms
are
iden$cal
1. Deep
muscle
hematomas
2. Hemarthroses
3. Intracranial
bleeding
4. Delayed
bleeding
5. Prolonged
oozing
awer
injuries
and
tooth
extrac<on
???
6. Superficial
ecchymoses
• Hemophilia
A
and
B
can
be
divided
into
3
groups
25
Severe
cases
ClassificaLon
ConcentraLon
of
factor
Symptoms
Age
at
diagnosis
Mild
6-­‐30%
(FVIII)
4-­‐50%
(FIX)
1. Bleeding
awer
major
trauma,
surgery,
dental
extrac<on
2. No
spontaneous
bleeding
seen
Owen
in
adulthood
Moderate
1-­‐5%
1. Muscle
and
joint
bleeding
awer
minor
trauma
2. Excessive
bleeding
awer
minor
surgery
and
dental
extrac<ons
3. Occasional
spontaneous
bleeding
may
occur
<5-­‐6
yrs
Severe
≤1%
1. Frequent
spontaneous
bleeding
2. Deep
muscle
bleeds,
hemarthroses,
intracranial
bleeds
3. Profuse
bleeding
awer
trauma,
minor
surgery,
dental
extrac<ons

26. Factor
VIII
Deficiency
(An<hemophilic
Factor)
• Deficiency
of
the
FVIII:C
por<on
of
the
circula<on
FVIII:vWF
complex
– In
Hemophilia
A—the
FVIII
component
is
missing
or
defec$ve
while
the
vWF
component
is
normal
– In
vWD—the
vWF
component
is
defecLve
while
the
FVIII
component
is
NORMAL
• FVIII:C
may
be
decreased
since
vWF
is
not
protec<ng
the
circula<ng
FVIII
• Defect
in
secondary
hemostasis
à
unable
to
form
stable
fibrin
clot
– Primary
hemostasis
is
normal
– Abnormal
bleeding
is
due
to
delayed
fibrin
forma<on
and
results
in
inadequate
fibrin
forma<on
• Factor
VIII
has
molecular
weight
of
330,000
D
– Gene
was
first
characterized
in
1980
and
located
near
the
<p
of
the
long
arm
of
the
X
chromosome
– Glycoprotein
that
par<cipates
in
the
middle
phase
of
the
intrinsic
pathway
– Synthesized
in
the
liver
(and
endothelium)
and
secreted
into
plasma
where
it
complexes
with
vWF
26

27. Structure
FVIII
• Factor
VIII
gene
is
located
on
the
X
chromosome
–
Xq28
• One
of
the
largest
known
genes
• Divided
into
26
exons
that
span
186,000
base
pairs
• Synthesized
as
a
single
chain
polypep<de
of
2351
amino
acids
• A
19-­‐amino
acid
signal
pep<de
is
cleaved
by
a
protease
shortly
awer
synthesis
so
that
circula<ng
plasma
factor
VIII
is
a
heterodimer
• FVIII
circulates
in
plasma
in
a
noncovalent
complex
with
von
Willebrand
factor

28. • The
func<ons
of
factor
VIII
reflect
binding
at
specific
sites
within
the
molecule
• Factor
VIII
consists
of
– A
heavy
chain
with
A1
and
A2
domains
• A2
domain
is
a
site
of
factor
IXa
binding,
the
ac<ve
enzyme
in
the
X-­‐
ase
pathway
– A
connec3ng
region
with
a
B
domain
• Connec<ng
region
that
separates
the
second
and
the
third
A
domains
but
is
not
required
for
cloZng
ac<vity
– A
light
chain
with
A3,
C1,
and
C2
domains
• C2
domain
binds
to
the
procoagulant
phospholipid
phospha<dylserine
on
ac<vated
platelets
and
endothelial
cells
and
to
von
Willebrand
factor
www.tankonyvtar.hu/hu/tartalom/tamop425/0011_1A_Molekularis_te

29. FVIII
Deficiency
• Most
muta<ons
occur
in
intron
22
1. Most
defects
are
point
muta<ons
2. Dele<ons
and
nonsense
muta<ons
lead
to
truncated
molecules
of
FVIII
– High
frequency
of
intron
22
inversions
may
relate
in
part
to
the
flexibility
of
the
telomeric
end
of
the
long
arm
of
the
X
chromosome—called
flip-­‐Lp
mutaLon
– Intron
22
inversions
are
responsible
for
~43%
of
severe
hemophilia
A
cases
29

30. Clinical
Manifesta<ons
• Hallmark
of
hemophilia
is
hemorrhage
into
the
joints
– Resul<ng
in
• Permanent
deformi<es
– Painful
and
lead
to
long-­‐term
inflamma<on
and
deteriora<on
of
the
joint
1. Misalignment
2. Loss
of
mobility
3. Extremi<es
of
unequal
lengths
– Intracranial
hemorrhage
– Hemorrhage
into
sow
<ssue
around
vital
areas
• Pathophysiology
– Bleeding
probably
starts
from
synovial
vessels
into
the
synovial
space
– Reabsorp<on
of
blood
is
owen
incomplete
à
chronic
prolifera<ve
synovi<s
à
thickening
of
the
snynovium
crea<ng
a
“target
joint”
with
recurrence
of
bleeding
– Destruc<on
of
surrounding
structures
and
bone
necrosis
with
cyst
formaLon
and
osteophytes

31. Clinical
manifesta<ons
Intracranial
hemorrhage
• Leading
cause
of
death
of
hemophiliacs
• Spontaneous
or
following
trauma
• May
be
subdural,
epidural
or
intracerebral
• Suspect
always
in
hemophilic
pa<ent
that
presents
with
unusual
headache
• If
suspected-­‐
FIRST
TREAT,
then
pursue
diagnos<c
workup
• LP
only
when
fVIII
has
been
replaced
to
more
than
50%

32. Clinical
manifesta<ons
Pseudotumors
• Dangerous
and
rare
complica<on
• Blood
filled
cysts
that
are
gradually
expanding
• Occur
in
sow
<ssues
or
bones.
• Most
commonly
in
the
thigh
• As
they
increase
in
size
they
erode
con<guous
structures.
• May
require
radical
surgeries
or
amputa<on,
and
surgery
is
owen
complicated
with
infec<on
A
pseudotumor
is
deforming
the
cortex
of
the
femur
(arrow).
Other
ossified
masses
in
the
sow
<ssues
(arrowheads)
are
probably
sow-­‐<ssue
pseudotumors.

33. • Queen
Victoria
was
a
carrier
– Queen
of
England
(1837-­‐1901)
– Spontaneous
muta3on
• Her
father
(Duke
of
Kent)
was
not
affected
• Her
mother
did
not
have
any
affected
children
from
the
previous
marriage
• Leopold
(her
8th
child)
had
hemophilia
– Died
brain
hemorrhage
(age
31)
– Had
children
–
Alice
(carrier)
• Beatrice
(QV
youngest
child)
had
2
hemophilic
sons
and
a
daughter
(Victoria
Eugene)
who
was
a
carrier)
– Victoria
Eugene
introduced
hemophilia
into
the
Spanish
royal
family
by
marrying
king
Alfonso
XIII
• Alexandra
(QV
granddaughter)
married
Nicholas
–
Tsar
of
Russia
– Alexandra
was
a
carrier
–
her
1st
son
Alexei
had
hemophilia
• Raspu<n
(monk)
used
hypnosis
to
relieve
Alexei’s
pain

34. Complica<ons
of
Treatment
• Inhibitors/an<body
development
– Defini<on
• IgG
an<body
to
infused
factor
VIII
or
IX
concentrates,
which
occurs
awer
exposure
to
the
extraneous
VIII
or
IX
protein
– Prevalence
• 20-­‐30%
of
pa<ents
with
severe
hemophilia
A
• 1-­‐4%
of
pa<ents
with
severe
hemophilia
B
• Hepa<<s
A
• Hepa<<s
B
• Hepa<<s
C
• HIV

35. Inhibitors
• Inhibitors
are
alloan<bodies
directed
against
a
specific
factor
–
neutralizing
the
effect
of
replacement
therapy
• Directed
against
specific
epitopes
on
the
factor
VIII
molecule
•
FVIII
Inhibitors
• Usually
IgG
–
IgG4
subclass
• Occur
in
~30-­‐40%
of
pa<ents
with
large
dele3ons
or
missense
muta3ons
• Lead
to
severe
deficiency
of
FVIII
• Overall
occurrence
in
all
types
of
FVIII
deficiencies
is
~20%
• Low
Responders
– <
5-­‐10
BU
– Titers
do
NOT
increase
in
response
to
exposure
to
FVIII
• High
Responders
– >
10
BU
– Titers
increase
with
exposure
to
FVIII
• FIX
Inhibitors
– Less
common
–
occurring
in
~3%
of
cases
Dived
these
into
two
groups

36. Inhibitors
• Inhibitors
are
iden<fied
by
performing
mixing
study
– Corrects
in
the
immediate
and
prolongs
in
the
incubated
• FVIII
inhibitors
are
IgG
–
warm-­‐reac<ng
an<bodies
–
$me
dependent
• FIX
inhibitors
are
IgG
–
usually
immediate-­‐
ac<ng
inhibitors
– Bethesda
Assay
is
used
to
determine
the
amount
of
inhibitor
present

37. Acquired
Hemophilia
• Rare,
poten<ally
life-­‐threatening
bleeding
disorder
• Development
of
autoan<bodies
directed
against
FVIII
–spontaneous
autoimmune
disorder
–
FIX
autoan<bodies
are
less
common
– Alloan<bodies
in
congenital
hemophilia
– Autoan<bodies
in
acquired
hemophilia
• Type
II
kine<cs
–
complex
– Ini<al
rapid
inac<va<on
followed
by
a
slower
inac<va<on
curve
and
resul<ng
in
some
level
of
residual
FVIII
• Associated
with:
– Idiophathic,
pregnancy,
autoimmune
disorders
– Inflammatory
bowel
disease,
ulcera<ve
coli<s
– Rheumatoid
arthri<s,
systemic
lupus
mul<ple
sclerosis,
Graves
disease,
Sjogren
syndrome
– Drugs
– Some
hematologic
malignancies

38. Treatment
of
Hemophilia
• Replacement
therapy
– Plasma
• FFP
–
did
not
raise
FVIII
levels
too
high,
many
suffered
volume
overload,
pa<ents
spent
a
lot
of
<me
in
hospital
• Before
1985
all
plasma
derived
products
were
highly
contaminated
by
blood
borne
virus
such
as
HIV,
HBV
and
HCV
à
not
so
much
not
due
to
screening
of
donors
and
viral
inac<va<on
techniques
such
as
pasteuriza<on,
solvent
detergent
treatment
and
ultrafiltra<on
• Some
theore<cal
concern
about
non
lipid
coated
parvovirus,
HAV
and
prion
disease
such
as
Creutzfeld-­‐
Jakob

39. Treatment
of
Hemophilia
• Cryoprecipitate
– Contains
high
levels
of
FVIII,
Fibrinogen,
vWF,
and
FXIII
– 1
unit
of
FFP
prepared
by
cryoprecipitate
contains
50-­‐120
U
of
VIII
• Plasma
derived
FVIII
using
monoclonal
an<bodies
• Recombinant
FVIII
• First
genera<on
–
hamster
cell
culture
–
contains
albumin
for
stabiliza<on
–
possible
source
of
viral
contamina<on
• Second
Genera<on
–
mutated
FVIII
lacking
the
B
domain
(no
role
in
cloZng)
–
stabilized
by
sucrose
(albumin-­‐free)
• Porcine
FVIII

40. Treatment
of
Hemophilia
• Replacement
of
missing
cloZng
protein
– On
demand
– Prophylaxis
• Prophylac<c
transfusions
must
be
started
at
age
2
to
3
– Need
central
access,
risk
of
bacteriemia,
costly
– Humate-­‐P,
Alphanate,
Mononine
• DDAVP
/
S<mate
–
release
of
vWF
and
FVIII
• An<fibrinoly<c
Agents
– Amicar
• Suppor<ve
measures
– Icing,
immobiliza<on,
rest
• Prophylac<c
transfusions
must
be
started
at
age
2
to
3
– Need
central
access,
risk
of
bacteremia,
costly

41. Hemophilia
is
an
ideal
disease
for
gene
therapy:
•
caused
by
a
single
malfunc<oning
gene
•
just
small
increase
in
factor
level
will
provide
great
benefit:
raising
factor
by
2%
will
prevent
spontaneous
hemorrhages
into
joints,
brain
and
other
organs;
levels
greater
than
20%
to
30%
will
prevent
bleeding
in
most
injuries

42. Recombinant
FactorVIII:
Inser<on
of
human
factor
VIII
DNA
into
vector
system
allowing
incorpora<on
into
non-­‐human
mammalian
cell
lines
for
con<nued
propaga<on

43. Financial
&
Insurance
Issues
• >
70%
of
cloZng
factor
distribu<on
is
by
for-­‐profit
companies
average
cost/yr
for
human
plasma
derived
or
recombinant
factor
is
$50,000
-­‐
$100,000
•
Prophylaxis
requires
about
150,000
units/yr
for
a
65-­‐pound
child
cos<ng
$85,000
per
year
•
Prophylaxis
is
covered
by
insurance
on
a
case-­‐by-­‐case
basis.

44. Summary
Hemophilia
A
and
B
Hemophilia
A
-­‐
(Classic
Hemophilia)
Hemophilia
B
-­‐
(Christmas
Disease)
Factor
Deficiency
Factor
VIII
Factor
IX
Inheritance
X-­‐linked
recessive
X-­‐linked
recessive
Gene
1. FVIII
gene
on
X
chromosome
–cloned
1984
2. Large
gene—187kb,
26
exons
3. 98%
of
pa<ents
have
muta<on
–
on
locus
Xq28,
48%
of
individuals
with
severe
have
inversion
of
intron
22
1. FIX
gene
on
X
chromosome
–
cloned
1982
2. 34
kb,
8
exons
3. 99%
of
muta<ons
–
on
Xq27.1-­‐q27.2
Incidence
1/10,000
males
1/50,000
males
Severity
Related
to
Factor
Level
1. Severe
=
<1%
ac<vity
Bleeding
awer
major
trauma,
major
surgery,
dental
extrac<on;
no
spontaneous
bleeding
seen,
owen
seen
in
early
infancy
(<1
year)
2. Moderately
Severe
=
1-­‐5%
ac<vity
Muscle
and
joint
bleeding
awer
minor,
trauma;
excessive
bleeding
awer
minor,
surgery
and
dental
extrac<ons;
occasional
spontaneous
bleeding
occurs,
owen
seen
<5-­‐6
years
of
age
3. Mild
=
6-­‐30%
ac<vity
Bleeding
awer
major
trauma,
major
surgery,
dental
extrac<on;
no
spontaneous
bleeding
seen,
owen
seen
only
in
adulthood
Complica<ons
• Sow
<ssue
bleed,
Intramuscular
bleed,
Hemarthrosis,
Urinary
tract
bleeding,
CNS
(major
life
threatening
bleed)
44

45. Lab
Diagnosis
in
Hemophilia
A
and
B
• Laboratory
Diagnosis
1. Why
do
hemophiliacs
bleed?
2. Delayed
bleeding
(secondary
hemosta<c
defects)
3. Rapid
bleeding
(primary
hemosta<c
defects
4. Oozing
45
Hemophilia
A
Hemophilia
B
PT
Normal
Normal
aPTT
1. Prolonged
2. May
be
normal
in
mild
cases
1. Prolonged
2. May
be
normal
in
mild
cases
Platelet
Ct
Normal
Normal
PFA/BT
Normal
Normal
Mixing
Study
1. Corrects
immediately
and
awer
incuba<on
2. Time-­‐dependent
inhibitor
1. Corrects
immediately
and
awer
incuba<on
2. Immediate-­‐ac3ng
inhibitor
vWF
Normal
Normal
FVIII
Decreased
FIX
Decreased
How
do
these
differ?

46. Factor
XI
Deficiency
• Hemophilia
C
––
(Plasma
Thromboplas<n
Antecedent)
• Also
called
Rosenthal
Syndrome
(described
in
1953)
• Autosomal
dominant
or
recessive
à
occurs
in
males
and
females
– 2
common
muta<ons
(one
nonsense,
one
missense)
– Allele
frequency
as
high
as
10%,
0.1-­‐0.3%
homozygous
– Most
affected
pa<ents
compound
heterozygotes
with
low
but
measurable
levels
of
XI
ac<vity
– Different
from
hemophilias
A
and
B
which
are
sex-­‐liked
• Rare
in
the
general
popula<on
1
in
million
– More
common
in
the
Ashkenazi
Jewish
popula<on
1
in
450
• In
vivo
FXI
is
ac<vated
by
thrombin
• in
vitro
FXI
is
ac<vated
by
XIIa
• aPTT
is
abnormal
with
normal
PT,
FIX
levels
are
decreased
46

47. Factor
XI
Deficiency
• 50%
of
pa<ents
with
FXI
deficiency
bleed
and
50%
do
not
bleed
– Bleeding
is
associated
with
<ssues
high
in
fibrinoly<c
ac<vity
– Variable,
generally
mild
bleeding
tendency
• Bleeding
awer
trauma
&
surgery
• Spontaneous
bleeding
uncommon
• Bleeding
risk
does
not
correlate
well
with
XI
level
• Mucous
membranes,
oral
cavity
• FXI
is
a
nega3ve
regula<on
of
TAFI
–
this
may
explain
why
a
deficiency
leads
to
bleeding
in
some
pa<ents
• Treatment
• FFP,
cryoprecipitate,
FXI
concentrates,
and
an<fibrinoly<c
agents
47

48. Congenital
Deficiency
of
the
Contact
Factors
• FXII
Deficiency
– Markedly
prolonged
aPTT
– Pa<ents
do
NOT
exhibit
a
bleeding
tendency
– Pa<ents
have
thrombo<c
tendency
• Due
to
a
defect
in
contact
ac<va<on
of
the
fibrinoly<c
system
–
requires
FXII
and
PK
• Tendency
to
develop
thromboemboli
par<cularly
following
trauma
or
surgery
• Prekallikrein
Deficiency
(Fletcher
Trait)
– Prolonged
aPTT
– Pa<ents
do
NOT
exhibit
a
bleeding
tendency
– Pa<ents
have
a
thrombo<c
tendency
– Defect
in
contact
ac<va<on
of
the
fibrinoly<c
system
requiring
PK
– Prolonged
aPTT
will
normalize
by
increasing
the
incuba$on
$me
• High
Molecular
Weight
Kininogen
(Fitzgerald
Factor)
– Markedly
prolonged
aPTT
– Pa<ents
do
NOT
exhibit
a
bleeding
tendency
– Pa<ents
have
thrombo<c
tendency
• Due
to
a
defect
in
contact
ac<va<on
of
the
fibrinoly<c
system
–
requires
FXII
and
PK

49. FXIII
Deficiency
• FXIII
is
a
tetrameric
zymogen
that
is
converted
into
an
ac<ve
transglutaminase
by
thrombin
and
Ca2+
in
the
terminal
phase
of
the
cloZng
cascade
• Hallmarks
of
FXIII
deficiency
1. Umbilical
stump
bleeding
in
neonatal
period
2. Intracranial
hemorrhage
with
li]le
or
no
trauma
3. Recurrent
sow
<ssue
hemorrhage
4. Recurrent
spontaneous
abor<on
5. Impaired
wound
healing
and
spontaneous
abor<on
• Bleeding
– Usually
associated
with
trauma
– Bleeding
at
<me
of
surgery
is
not
excessive
• Delayed
bleeding
can
occur

50. Inherited
factor
XIII
deficiency
• Autosomal
recessive,
rare
(consanguineous
parents)
• Heterozygous
woman
may
have
higher
incidence
of
spontaneous
abor<on
• Most
have
absent
or
defec<ve
A
subunit
• F
XIII
ac<vity
<
1%
(1-­‐2%
is
adequate
for
hemostasis)
– Bleeding
begins
in
infancy
(umbilical
cord)
– Poor
wound
healing
– Intracranial
hemorrhage
– Oligospermia,
infer<lity
• Diagnosis:
– Urea
solubility
test
– Quan<ta<ve
measurement
of
XIII
ac<vity
– Rule
out
acquired
deficiency
due
to
autoan<body
• F
XIII
concentrates
available
(long
half
life,
can
administer
every
4-­‐6
weeks
as
prophylaxis)

51. Clinical Testing for Factor XIII
Urea
Clot
Solubility
Test
• Qualita<ve
assay
• Pa<ent
sample
is
clo]ed
and
then
clot
is
placed
in
5
M
urea
for
24
hours
at
room
temperature
– Clots
formed
by
normal
individuals
remain
stable
– Clots
from
factor
XIII
deficient
pa<ents
dissolve
• Detects
only
the
most
severely
affected
homozygous
pa<ents
with
1%
to
2%
factor
XIII
ac<vity
or
less
• Urea
solubility
assay
• Factor
XIII
forms
covalent
cross
links
between
fibrin
chains
• In
the
absence
of
Factor
XIII
à
the
fibrin
clot
will
be
dissolved
by
5
M
urea
which
disrupts
the
hydrogen
bonds
• This
assay
will
be
abnormal
only
if
the
factor
XIII
level
is
<2-­‐5%

52. FXIII
Deficiency
• Laboratory
results
– Normal
PT,
APTT,
TT,
BT
despite
history
of
bleeding
– Solubility
of
fibrin
clots
in
5
M
urea
or
1%
monochloroace<c
acid
– Minimal
ac<vity
(2-­‐5%)
needed
to
maintain
hemostasis
– Therapy
if
needed
• FFP,
cryoprecipitate,
FXIII
concentrates
are
available
in
Europe
• Acquired
factor
XIII
deficiency
– Autoan<body-­‐mediated
• Very
rare
• Most
pa<ents
elderly
• May
be
drug-­‐induced
(isoniazid,
other
an<bio<cs)
• Bleeding
may
be
severe
• Diagnosis:
– Urea
solubility
– F
XIII
ac<vity
– Mixing
study?

53. Factor
Assays
• Principle
– Ability
of
the
pa<ent’s
plasma
to
correct
a
prolonged
PT
or
APTT
of
a
known
factor
deficient
plasma
– Normal
ac<vity
range
is
50-­‐150%
or
50%
factor
ac<vity
• Determines
type
of
factor
deficiency
and
ac<vity
• Targets
either
–
PT:
Factors
VII,
X,V,
III
and
II
–
APTT:
Factors
XII,
XI,IX
and
VIII
• Methodology
– Factor
deficient
plasmas
are
used
that
contain
100%
of
all
factors
except
the
one
in
ques<on
–
1:10,
1:20,
1:40
dilu<ons
are
made,
1:10
is
considered
100%
– A
control
to
compare
results
to,
normal
plasma
(containing
100%
of
all
factors)
is
added
to
the
commercially
prepared
factor
deficient
plasma
in
the
same
way
– Pa<ent
sample
and
control
are
compared
to
a
standard
curve
where
the
cloZng
<mes
have
been
established
using
known
concentra<on

54. FIGURE 40-4 Factor activity curve. The factor activity curve is prepared by plotting the clotting time in seconds for
each reference plasma dilution on the y-axis and the percent factor activity for each dilution on the x-axis. (Reprinted,
with permission, from Brown BA. Hematology: Principles and Procedures, 6th ed. Philadelphia: Lea & Febiger; 1993.)

55. Coagulation Screening Test Results in Congenital Deficiencies
McKenzie