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.
5. Platelet Activation Pathways
Phospholipase A2 Phospholipase C
PL PIP2
Arachidonic Acid DAG IP3
PGG2 PGH2
TXA2
Protein Kinase C
CA2+
CA2+
Protein Phosphorylation
Procoagulant
Activity
Shape
Change
GpIIb/IIIa
Receptor
a-granules
Dense
Bodies
ATP
ADP
5HT
Ca2+
PF4
B-TG
PAI-1
vWF
Fibrinogen
Factor V
PDGF
TXA2
Fibrinogen
Adrenaline
ADP
Thrombin
TxA2
*
Arachidonic Acid
DAG
Lipase
COX-1
Thromboxane
Synthetase
TXA2
*
TXB2
Low Collagen High Collagen
Aspirin
Inhibition
Created by Krystal McGarvey, Applications Specialist, Chrono-log Corp.
Reference: Platelets in Thrombotic and Non-thrombotic Disorders. 2002. Pages: 119, 127-129, 222-223, 238-239, 339, 361, 371, 471-472
6. Laboratory
Inves6ga6on
of
Primary
Hemostasis
• Includes
tests
for
platelet
number
and
func6on
– Platelet
Count
– Peripheral
Blood
Smear
Evalua6on
– Platelet
Func6on
Analyzer
(PFA)
• Bleeding
Time
– VerifyNow
(Accumetrics)
– Platelet
Aggrega6on
7. Bleeding
Time
• Bleeding
6me—overall
test
of
hemosta6c
func6on
• Measures
1. Vessel
integrity
2. Platelet
integrity
3. Protein/Platelet
interac6on
– Methods
– Duke
(1912)—ear
lobe
– Ivy
(1941)–
volar
surface
of
forearm
with
blood
pressure
cuff
inflated
to
40
mm
of
mercury
– Meckel
(1969)—standardized
template
device
– Reference
range
=
2-‐9
minutes
– Prolonged
in:
• Thrombocytopenia
• Platelet
disorders
• vWD
• Low
or
abnormal
fibrinogen
• Vascular
disorders
– Disadvantages
1. Lack
of
consistency
with
the
results
2. No
correla6on
with
pre-‐surgical
bleeding
3. No
evidence
to
suggest
that
it
will
predict
a
post-‐surgical
bleed
– Procedure
– Standardized
cut
made
in
forearm
– 1
mm
deep
and
5
mm
long
– 40
mm
Hg
pressure
(blood
pressure
cuff)
used
to
provide
constant
hemosta6c
stress
– Reference
range:
generally
2
-‐
9
minutes
8
8. PFA-‐100:
Platelet
Func6on
Screen
• Test
cartridges
containing:
1. Collagen/Epinephrine
2. Collagen/ADP
• Monitors
platelet
adhesion
and
aggrega6on
• Results
reported
as
a
“Closure
6me”
in
seconds
(CT)
• Correla6on
to
Bleeding
Time
9
9. The
PFA-‐100®
System
Simulates
In
Vivo
Condi6ons
10
Injured
Blood
Vessel
PFA-‐100®
Test
Cartridge
Flow
Agonist
Collagen
Platelet
Plug
10. VerifyNow
• Cartridge-‐based
system
that
uses
fibrinogen-‐coated
beads
• Method
1. Citrated
blood
is
drawn
into
each
of
two
sample
channels
in
a
disposable
cartridge
2. Mixed
with
platelet
agonist
FLLRN
and
Fibrinogen
coated
polystyrene
beads
by
a
steel
ball
3. Light
is
transmiied
through
the
sample
4. Agglu6na6on
occurs
between
ac6vated
platelets
and
the
fibrinogen-‐
coated
beads
such
that
they
fall
out
of
suspension
-‐à
increased
light
transmission
5. Reported
as
PAU
(platelet
aggrega6on
units)
11. OPTICAL PLATELET AGGREGOMETRY: BORN PRINCIPLE
MAGNET PRP with
PPP BLANK,
NO MAGNET PRP with
MAGNET PRP with
MAGNET PRP with
MAGNET
AGONIST ADDED TIME MINUTES
0%
100%
L I GHT IN LIGHT
OUT
L
I
G
H
T
T
R
A
N
S
M
I
S
S
I
O
N
Monophasic
Curve
13. Platelet
Aggrega6on
§ Primary
wave
§ Reversible
§ Measures
ability
of
platelets
to
respond
to
an
external
agonist
and
to
start
to
aggregate
§ Without
enough
s6mulus
or
without
an
intact
prostaglandin
pathway
à
TXA2
–
platelets
disaggregate
§ Secondary
wave
§ Irreversible
§ Results
in
complete
release
of
dense
granules
contents,
most
importantly
ADP
Graphic
accessed
URL
hip://evolvels.elsevier.com/sec6on/default.asp?id=1138_ccalvo7_0001,
2008.
Biphasic
Curve
14. ANATOMY
of
a
BIPHASIC
AGGREGATION
CURVE
ATP
ATP
Resting
disk-shaped
cells
2
3
DIS-AGGREGATION
Activation:
shape change Irreversible Aggregation
4
ADP
time
aggregation (%)
PRIMARY WAVE
REVERSIBLE
AGGREGATION
1
SECONDARY WAVE
MAXIMUM
AAGREGATION
15. PLT
Aggrega6on:
WB
• Parallel
electrodes
(DC)
immersed
in
saline-‐diluted
whole
blood
• Add
agonist
• PLTs
aggregate
on
electrodes,
reducing
current
• Change
is
current
directly
propor6onal
to
level
of
PLT
aggrega6on
The
aggrega6ng
platelets
form
a
layer
on
the
electrodes,
and
current
is
impeded
by
the
platelet
layer.
Resistance
(Ω)
is
propor6onal
to
aggrega6on,
providing
a
tracing
that
resembles
op6cal
aggregometry.
Graphic
accessed
URLhip://evolvels.elsevier.com/sec6on/default.asp?id=1138_ccalvo7_0001,
2008.
16. Agonists
• Collagen
– Membrane
defects
– General
ability
of
platelets
to
aggregate
– SPD,
RD,
NSAID
• Epinephrine
– Membrane
defects
– COX
– NSAID
• Arachidonic
Acid
– Most
useful
in
detec6ng
aspirin-‐like
deficiencies
– Aspirin,
NSAID
• ADP
– Membrane
defects
– COX,
SPD
– NSAID
• Ristoce6n
– Membrane
defects
– Measures
agglu7na7on
– Differen6ate
Aggrega6on
between
BS
vs.
vWD,
vWD
2B
vs.
Platelet-‐type
vWD
Agglu6na6on
24. Bernard-‐Soulier
Syndrome
(BSS)
• Laboratory
findings
– Thrombocytopenia
– Giant
platelets
• 5-‐8
um
vs
20-‐30
um
diameter
– Prolonged
PFA/BT
– Abnormal
aggrega6on
with
ristoce6n
– Decreased
to
absent
expression
of
GPIb
and
or
GPIX
(CD42b,
CD42a)
– CD61
=
GPIIIa
25
Green = control, Red = patient
FS = size
Absence
of:
• CD42a,
42b,
and
42c
• (components
of
the
Ib/IX
complex)
25. Bernard-‐Soulier
Syndrome
• Laboratory
Findings
– Mild
to
moderate
thrombocytopenia
common
• 30
–
200
x109/L
– Giant
platelets
on
peripheral
blood
smear
– Platelet
aggrega6on
studies
• Absent
aggrega6on
with
ristoce6n
• Normal
aggrega6on
with
all
other
agonists
Why
is
platelet
agglu6na6on
with
ristoce6n
s6ll
abnormal
when
vWF
is
added?
Missing
GPIb/IX
receptor
Why
is
platelet
aggrega6on
normal
with
other
agonists?
In
vitro
aggrega6on
does
not
first
require
adhesion
26. Glanzmann
Thrombasthenia
(GT)
• First
described
in
1918
–
Switzerland
–Dr.
Glanzmann
– Described
in
children
from
a
6ny
village
–
Le
Valais
in
Swiss
Alps
–
where
intermarriage
was
common
• Autosomal
recessive
disorder
involving
one
of
two
genes
coding
for
either
GPIIb
or
GPIIIa
–
both
found
in
chromosome
17q
• GPIIb/IIIa
func6on
in
platelet
aggrega6on
–
binding
to
fibrinogen
• Bleeding
appears
during
the
1st
year
of
life
– Epistaxis,
gingival
bleeding,
purpura,
heavy
menorrhagia
• Three
clinical
presenta6ons
– Type
I
–
severe
deficiency
with
less
than
5%
IIb/IIIa
receptors
present
– Type
II
–
mild-‐moderated
deficiency
with
5-‐20%
IIb/IIIa
receptors
present
– Type
III
–
normal
to
almost
normal
amount
of
IIb/IIIa
receptors
present
but
defec6ve
func6on
27
27. Glanzmann
Thrombasthenia
(GT)
• Laboratory
findings
– Prolonged
PFA/BT
– Normal
platelet
count
– Normal
platelet
retrac6on
– Abnormal
platelet
aggrega6on
response
to
ADP,
Arachidonic
Acid,
Collagen,
Epinephrine
– Normal
platelet
aggrega6on
response
to
ristoce6n
– Decrease
expression
by
flow
cytometry
–
confirmatory
diagnosis
• GPIIb
(CD41)
or
• GPIIIa
(CD61)
• Why
is
aggrega6on
with
ristoce6n
normal?
28
29. Storage
Pool
Disease
• Affect
the
secre6on
phase
of
platelet
func6on
• Autosomal
dominant
or
autosomal
recessive
mode
of
inheritance
– Dense
Granules
Deficiency
• Decrease
or
absence
of
dense
granules
on
EM
• Morphologically
normal
appearing
platelets
on
peripheral
blood
smear
• Prolonged
PFA/BT
• Abnormal
aggregaAon
due
to
lack
of
ADP
in
Dense
Granules
• Abnormal
aggrega6on
with
ADP,
Epinephrine
à
normal
primary
wave
BUT
blunted
secondary
wave
• Low
levels
of
collagen
–
collagen
requires
endogenous
ADP
and
this
is
lacking
30
30. Storage
Pool
Disease
• Alpha
Granules
Deficiency
– Absence
of
the
alpha
granules
causes
the
platelets
to
appear
agranular
on
peripheral
blood
smear
(EM)
– Mgk
synthesis
of
the
alpha
granules
is
normal
BUT
there
are
defects
involving
targe6ng
endogenously
synthesized
proteins
to
developing
alpha-‐granules
– Platelet
aggrega6ons
studies
are
normal
/decreaased
in
alpha
granule
deficiency
– AKA
gray
platelet
syndrome
31
31. Storage
Pool
Disease
• Gray
Platelet
Syndrome
– Congenital
platelet
disorder
– Marked
decreased
or
absence
of
platelet
alpha-‐granules
– Large
platelets
with
few
granules
àgiving
the
“gray”
appearance
– Bleeding
is
usually
mild
to
moderate
but
can
be
exacerbated
by
aspirin
– Clinical:
easy
bruising,
menorrhagia,
and
excessive
postpartum
or
postopera6ve
bleeding
– Typical
Lab
Findings
• Usually
normal
platelet
count
with
variable
morphology
• Platelet
aggrega6on
shows
normal
primary
wave
but
absence
of
secondary
wave
when
s6mulated
with
ADP,
epinephrine,
arachidonic
acid
–
• Ristoce6n
agglu6na6on
is
normal
32
32. Gray
Platelet
Syndrome
• Quebec
Platelet
Defect
– Deficiency
of
α-‐granule
mul6merin
–
a
protein
that
binds
FV
within
the
α-‐granule
à
decreased
content
of
platelet
FV
– Abnormal
proteolysis
of
alpha-‐
granule
proteins
due
to
increased
levels
of
platelet
urinary-‐type
plasminogen
ac6vator
– Platelets
are
morphologically
normal
by
light
microscopy
– Slight
thrombocytopenia
33
34. Gray
Platelet
Syndrome
• ScoD
Syndrome
– Due
to
a
defect
in
a
platelet
mechanism
required
for
blood
coagula6on
– Defec6ve
procoagulant
ac6vity
of
platelets
– During
normal
platelet
ac6va6on
–
PS
on
the
inner
leaflet
is
transported
to
the
outer
membrane
surface
–
provides
a
binding
site
form
the
tenase
and
prothrombinase
complexes
– In
Scoi
Syndrome
the
mechanism
for
transloca6ng
PS
is
defec6ve
à
impaired
thrombin
genera6on
Nature.com
35
35. Gray
Platelet
Syndrome
• SPD
versus
RD
–
need
EM
to
differen6ate
between
the
two
– SPD
may
have
decreased
number
of
dense
bodies
– RD
will
have
normal
number
of
dense
bodies
36
36. Hermansky
-‐
Pudlak
Syndrome
• Due
to
a
decreased
number
of
dense
granules
• Described
in
1959
by
Hermansky
and
Pudlak
– Described
a
55-‐year
old
man
with
oculocutaneous
albinism
and
history
of
frequent
bruising
following
minimal
trauma
• Autosomal
recessive
disorder
à
muta6on
in
the
HPS1
gene
on
chromosome
10q23
– HSP1
responsible
for
produc6on
and
control
of
melanosomes,
dense
granules,
and
lysosomes
• Most
commonly
found
in
Swiss
Alps
and
Puerto
Rico
• Triad
phenotype
1. Albinism—blond
hair
pale
skin
2. Prolonged
bleeding
due
to
storage
pool
granular
deficiency
• Platelet
func6on
requires
dense
granules
filled
with
proaggrega6on
chemical
reagent
3. Accumula6on
of
ceroid
pigment
in
lysosomal
organelles
• Ceroid
à
wax-‐like
substance
made
by
certain
cells
• Ceroid
accumula6on
may
cause
organ
dysfunc6on
[intes6nes,
lungs,
kindeys]
• Lab
findings
– Normal
PT/PTT,
BT
variably
normal
to
prolonged
– Platelet
aggrega6on
shows
blunted
response
in
biphasic
curves
– Diagnosis
made
by
EM
à
absence
of
dense
granules
37
37. Chediak
-‐
Highashi
Syndrome
• Autosomal
recessive
disorder
resul6ng
in
recurrent
infec6ons
with
ocular,
neurological,
and
skin
manifesta6ons
• Described
in
1943
by
a
Cuban
pediatrician
à
Chediak
and
Higashi
gavedetailed,
published
descrip6on
in
1954
• Caused
by
a
muta7on
in
the
LYST
gene
– Lysosomal
trafficking
regulator
gene
on
chromosome
1
– Abnormal
membrane
fluidity
with
uncontrolled
granule
membrane
fusion
– Giant
cytoplasmic
granules
in
all
granule-‐containing
cells
(leukocytes,
melanocytes
and
platelets)
– Platelets
have
deficient
or
reduced
storage
pools
of
ADP,
ATP,
and
serotonin
à
loose
platelet
aggrega6on
forma6on
• Clinical
manifesta7on
– Decreased
pigmenta6on
of
the
hair
and
eyes
– Photophobia,
Nystagmus
– Large
eosinophilic,
peroxidase-‐posi6ve
inclusion
– Pa6ents
are
suscep6ble
to
bacterial
infec6ons
• Laboratory
findings
– Normal
platelet
counts,
prolonged
bleeding
6me
– Normal
PT/aPTT
– Leukocytes
with
darkly
stained
giant
granula6on
– Platelet
aggrega6on
decreased
with
collagen
and
ADP
38
ASH
Image
Bank
38. Acquired
Platelet
Defects
• Cardiopulmonary
Bypass
• Chronic
Renal
Failure
– Seen
in
uremic
pa6ents
related
to
the
accumula6on
of
waste
products
in
the
blood
– Prolonged
PFA/BT
– Decreased
aggrega6on
with
collagen
– Secondary
aggrega6on
with
ADP
and
epinephrine
is
decreased
à
abnormal
secretory
response
– Platelet
procoagulant
ac6vity
is
defec6ve
• Myeloprolifera6ve
Disease
and
Acute
Leukemia
• Drugs
– Aspirin
– Alcohol
– An6bio6cs
– Cardiopulmonary
Bypass
Surgery
39
39. Cardiopulmonary
Bypass
• Causes
a
deple6on
of
α-‐granules
• Func6onal
defect
results
from
increased
platelet
ac6va6on
and
fragmenta6on
in
the
bypass
mechanical
process
• Causes
of
defects
and
granule
deple6on
a. Aggrega6on
of
platelets
by
fibrinogen
absorbed
onto
the
surfaces
of
the
bypass
circuit
material
b. Hypothermia
c. Complement
ac6va6on
d. Mechanical
trauma
and
shear
stresses
e. Bypass
pump-‐priming
solu6ons
• Lab
findings
– Increases
the
BT
by
>30
minutes
– Platelet
fragments
–
Ø Typically
platelet
func6on
returns
to
normal
~
1-‐3
hours
awer
surgery
Ø Platelet
count
returns
to
normal
several
days
later
– Thrombocytopenia
can
be
amplified
by
hemodilu6on
as
blood
passes
through
the
bypass
mechanism
– Significant
post-‐surgical
bleeding
is
seen
in
3%
of
pa6ents
40
40. Uremia
• Related
to
accumula6on
of
waste
products
in
the
blood
including
inhibitory
and
dialyzable
molecules
1. BT
correlates
with
severity
of
disease
2. Procoagulant
ac6vity
may
be
impaired
3. Nitric
Oxide
may
inhibit
platelet
func6on
4. Thought
to
be
due
to
impaired
platelet-‐vessel
interac6on
5. Hemosta6c
abnormality
partly
corrected
by
RBC
transfusion
or
EPO
• Failue
of
HGB
to
quench
excess
NO
synthesis
may
be
partly
responsible
for
platelet
dysfunc6on
42. vWD—Disorder
of
Primary
Hemostasis
} Most
common
of
the
congenital
bleeding
disorders
} 1-‐2
%
of
the
general
popula6on
} Symptoma6c
in
only
about
1/10,000
} 1926
–
Erik
von
Willebrand
à
5
y-‐o-‐f
and
her
family
who
lived
on
the
Åland
Islands
–
Hereditär
pseudohemofili,
1926
} Ini6ally
described
as
“pseudohemophilia”
43
43. vWD—Disorder
of
Primary
Hemostasis
} Clinical
manifesta6ons
} Mucocutaneous
bleeding
of
varying
severity
in
males
and
females
1. Ecchymoses
2. Epistaxis
3. Gastrointes6nal
bleeding
4. Menorrhagia
} Defec6ve
platelet
adhesion
} Reduced
FVIII
levels
44
44. vWF
• Large
mul6meric
protein
–
ranges
from
600
kD
to
>20
million
kD
– Synthesized
by
endothelial
cells
and
megakaryocytes
• Endothelial
cells
source
of
plasma
vWF
• Gene
for
vWF
is
located
on
chromosome
12p
• 178
kB,
52
exons
Hoffman:
Adapted
from
Ginsburg
D,
Bowie
EJW:
Molecular
geneAcs
of
von
Willebrand
disease.
Blood
79:2507,
1992.)
45
45. Synthesis
of
vWF
} vWF
synthesized
in
endothelial
cells
and
megakaryocytes
1. Stored
in
Weibel-‐Palade
bodies
of
endothelial
cells
2. Stored
in
α-‐granules
of
platelets
Steps
in
synthesis
of
vWF
1. First
synthesized
as
a
pre-‐
pro-‐vWF
monomer
2. DimerizaAon
occurs
in
ER
3. Pre-‐pro-‐vWF
monomers
linked
together
at
the
carboxyl
terminal
end
4. Dimeric
molecules
pass
to
the
Golgi
apparatus
5. Dimers
mulAmerize
6. Propep6de
is
cleaved
off
à
mature
subunit
46
N-Terminal
Multimerization
C-Terminal
Dimerization
High
Molecular
Weight
Multimer
ER
Golgi
47. Func6on
of
vWF
} vWF
serves
two
important
biologic
func6ons
1. Serves
as
a
carrier
protein
for
plasma
FVIII
a. VWF
protects
Factor
VIII
in
circula6on
b. VWF
co-‐localizes
FVIII
at
sites
of
vascular
injury
2. Serves
as
a
ligand
that
binds
to
the
gpIb
receptor
on
platelets
to
ini6ate
platelet
adhesion
to
the
damaged
endothelium
a. VWF
binds
to
extravascular
collagen
b. Platelets
adhere
to
the
bound
vWF
c. Adherent
platelets
become
ac6vated
48
Platelets
VWF
Clotting factors Vessel wall
51. Type
I
vWD
} Most
common
type
of
vWD
} 80%
of
pa6ents
with
vWD
fall
into
this
category
} Caused
by
heterozygous
muta6on
leading
to
a
par6al
quan6ta6ve
deficiency
of
vWF
} Gene6c
abnormality
in
ONE
of
the
vWF
alleles
} Accounts
for
a
50%
reduc6on
in
vWF
} Mild
secondary
deficiency
in
FVIII
} Endothelial
cells
and
platelets
contain
normal,
but
reduced
levels
of
vWF
} DDAVP
can
induce
the
release
the
stored
vWF
} Bleeding
symptoms
range
from
asymptoma6c
to
mild
52
52. Type
I
vWD
} Lab
findings
} Normal
to
decreased
1. FVIII
(aPTT)
2. vWF:Ac6vity
(Ristoce6n
Cofactor)
3. vWF:An6gen
4. Prolonged
BT
• (PFA-‐100—Col/EPI,
Col/ADP)
5. Propor6onal
decrease
of
ALL
vWF
mul6mers
53
53. Type
3
vWD
} Most
severe
form
of
the
disease
} Results
from
the
homozygous
muta6on
leading
to
a
deficiency
of
vWF
with
absent
or
profound
deficiency
in
levels
of
plasma
vWF
} Autosomal
recessive
} vWF
levels
are
<5%
} FVIII
is
markedly
cleared
from
the
plasma
with
levels
below
5-‐10%
} FVIII
is
not
as
severely
depressed
as
in
severe
Hemophilia
A
} Spontaneous
bleeding
} Severe
mucocutaneous
bleeding
} Sow
6ssue/musculoskeletal
bleeding
} 1-‐5%
of
case
} Prevalence
increases
in
regions
of
consanguineous
marriages
54
54. Type
2A
vWD
• Muta6ons
commonly
occur
in
the
A2
region
• Presence
of
only
the
smaller
vWF
mul6mers
in
plasma
à
reduced
binding
to
platelets
• LOSS
platelet-‐dependent
funcAon
• Two
proposed
mechanisms:
▫ Abnormal
assembly
and
secre6on
of
large
vWF
mul6mers
▫ Increased
suscep6bility
of
vWF
to
proteolysis
in
circula6on
• Pa6ents
exhibit
moderate
to
severe
mucocutaneous
bleeding
55
55. Type
2B
• Muta6on
in
the
A1
domain
of
the
vWF
gene
• Absence
of
the
high-‐molecular-‐weight
mul6mers
– Caused
by
“gain
of
func7on”
muta6on
in
vWF
à
increased
affinity
to
bind
to
the
gpIb
platelet
receptor
– Spontaneous
binding
of
vWF
to
platelets
– Large
mul6mers
are
synthesized
but
rapidly
cleared
due
to
increased
binding
to
platelets
– Thrombocytopenia
• DDAVP
contraindicated
à
would
cause
increased
thrombocytopenia
as
platelets
would
be
hyper-‐reacAve
to
the
released
vWF
56
56. Type
2M
vWD
• Muta6ons
in
Exon
28
in
A1
domain
N D1 D2 D‘D3 A1 A2 A3 D4 B1B2 B3 C1 C2 C
• Defect
leads
to
decreased
or
absent
binding
of
vWF
to
platelet
gpIb
receptor
• Decreased
platelet
dependent
func6on
• Normal
mul6mer
profile
• Plasma
binding
to
FVIII
is
normal
57
FVIII GPIb collagen RGDS
collagen GPIIb/IIIa
57. Type
2N
vWD
• Also
referred
to
as
“autosomal
hemophilia”
or
the
Normandy
variant
• Caused
by
muta6ons
in
the
FVIII
binding
region
of
vWF
N D1 D2 D‘D3 A1 A2 A3 D4 B1B2 B3 C1 C2 C
• Markedly
decreased
affinity
for
binding
to
FVIII
– Rapid
turnover
of
the
unbound
FVIII
à
reduced
levels
• Lab
findings
1. Decreased
FVIII
2. Normal
vWF
an6gen
and
ac6vity
3. Normal
bleeding
Ames
(PFA-‐100)
4. Platelet
binding
to
vWF
is
normal
5. Similar
to
“mild”
hemophilia
• GeneAc
counseling
and
treatment
is
different
from
hemophilia
58
FVIII GPIb collagen RGDS
collagen GPIIb/IIIa
58. Pseudo-‐von
Willebrand
Disease
–
(Platelet
type
vWD)
} NO
gene6c
defect
of
the
vWF
molecule
–
vWF
molecule
is
NORMAL
} “Gain
in
func6on”
muta6on
in
the
platelet
gpIb
receptor
} Increased
affinity
of
platelets
for
vWF
} Enhanced
clearance
of
vWF
and
platelets
from
circula6on
} Defect
is
in
the
platelet
à
standard
approaches
to
trea6ng
vWD
are
not
helpful
} Lab
findings
1. Loss
of
high
molecular
weight
mul6mers
2. Platelet
count
is
low**
3. Platelet
aggrega6on
with
low
dose
ristoce6n
(RIPA)
59
59. Acquired
vWD
• Qualita6ve,
structural,
or
func6onal
disorder
of
vWF
not
inherited
and
is
associated
with
an
increased
risk
of
bleeding
• Associated
with
– Autoimmune
clearance
–
lymphoprolifera6ve,
MGUS,
SLE,
hypothyroidism
• Autoan6bodies
à
increased
clearance
of
vWF
from
plasma
– Fluid
shear
stress-‐induced
proteolysis
–
aor6c
stenosis,
LVAD
60
Aspect
Acquired
vWD
Congenital
vWD
Personal
History
Late
onset
bleeding
Early
onset
bleeding
Family
History
Nega6ve
Posi6ve
AVWS
associated
Posi6ve
Nega6ve
disorder
Laboratory
associated
disorder
Inhibitor
to
vWF
Gene6c
muta6on
Treatment
• Remission
awer
IVIg
• Short
lived
response
awer
vWF-‐containing
product
vWF-‐containing
product
61. Assays
for
vWD
• vWF:An6gen
– Immunoassay
that
measures
the
concentra6on
of
vWF
protein
in
plasma
• Actual
protein
responsible
for
binding
to
FVIII
and
gp
Ib/IX/V
complex
– Detects
all
forms
of
vWF
(func6onal
and
nonfunc6onal
forms)
– Cannot
discriminate
between
mul6mer
size
62
Patient
vWF å
åå
å
Testing
well
LIA
based
tes7ng
Reagent beads
coated with anti-vWF
å
å
å
å
å
å
å
å
Incub
ate
Instrument reading—changes in optical density secondary to aggregates
62. Assays
for
vWD
• vWF:Ac6vity
– Ristoce6n
cofactor
assay
(gold
standard)
• Measures
the
ability
of
vWF
(pa6ent)
to
induce
agglu6na6on
of
normal
fixed
platelets
in
the
presence
of
Ristoce6n
• Mix
paAent’s
plasma
+
normal
donor
platelets
+
ristoceAn
à
platelet
aggluAnaAon
reacAon
occurs
on
platelet
aggregometer
63
63. Assays
for
vWD
• vWF:Ac6vity
– Latex
par6cle
enhanced
immunoturbidimetric
assay
• Specific
anA-‐vWF
monoclonal
anAbody
adsorbed
onto
latex
reagent
directed
against
the
platelet
binding
site
of
vWF
(gp
Ib
receptor)
• Reacts
with
vWF
in
the
pa6ent’s
plasma
• Degree
of
agglu6na6on
is
directly
propor6onal
to
ac6vity
of
vWF
in
pa6ent's
plasma
–
Mix
paAent’s
plasma
+
latex
beads
coated
with
an
anA-‐vWF
monoclonal
anAbody
è
aggluAnaAon
of
parAcles
64
64. Assays
for
vWD
• FVIII
▫ Circula6ng
level
of
FVIII
▫ Clot-‐based
assay
that
measures
the
ability
of
plasma
FVIII
to
shorten
the
clo}ng
6me
in
FVIII-‐deficient
plasma
• Mul6mer
Analysis
▫ QualitaAve
assay
(electrophoresis)
to
depict
the
variable
concentraAons
of
different-‐sized
vWF
mulAmer
65