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Lecture 
5 
Disorders 
of 
Primary 
Hemostasis 
Qualita6ve 
Platelet 
Disorders 
Von 
Willebrand 
Disease 
2
Func6onal 
disorders 
of 
platelets 
• Congenital 
Disorders 
– Disorders 
of 
Adhesion 
• Bernard-­‐Soulier 
Syndrome 
• Platelet-­‐type 
von 
Willebrand 
Disease 
– Disorders 
of 
Aggrega6on 
• Glanzmann 
Thrombasthenia 
– Disorders 
of 
Secre6on 
• Storage 
Pool 
Disorders 
(SPD) 
– Dense 
Granule 
disorders 
– Alpha 
Granule 
disorders 
• Signal 
Transduc6on 
defects/Platelet 
Release 
defects 
– Defec6ve 
TXA2 
Pathway 
– Receptor 
Signaling 
Defects 
• Acquired 
Disorders 
3
Nature 
Reviews
Inherited 
Disorder 
of 
Platelet 
Dysfunc6on 
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
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
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
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
The 
PFA-­‐100® 
System 
Simulates 
In 
Vivo 
Condi6ons 
10 
Injured 
Blood 
Vessel 
PFA-­‐100® 
Test 
Cartridge 
Flow 
Agonist 
Collagen 
Platelet 
Plug
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)
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
Platelet Aggregation
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
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
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.
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
ADP 
18
Collagen 
and 
Arachidonic 
Acid 
19
Epinephrine 
20
Ristoce6n 
21
ADP 
and 
Arachidonic 
Acid 
22
Collagen 
(low 
dose 
and 
high 
dose) 
23
Ristoce6n 
(low 
dose 
and 
high 
dose) 
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)
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
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
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
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
Storage 
Pool 
Disease 
• Gray 
Platelet 
Syndrome 
– Congenital 
platelet 
disorder 
– Marked 
decreased 
or 
absence 
of 
platelet 
alpha-­‐granules 
– Large 
platelets 
with 
few 
granules 
àgiving 
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
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
Platelet and neutrophil images of GPS patients in comparison with controls. 
Gunay-Aygun M et al. Blood 2010;116:4990-5001 
©2010 by American Society of Hematology
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
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
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
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
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
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
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
Lab 
Tests 
in 
Disorders 
of 
Primary 
Hemostasis
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
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
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
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
vWF 
Release 
Valentijn K M et al. Blood 2011;117:5033-5043 
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
Func6on 
of 
vWF 
49 
Elsevier
50 
Elsevier
Classifica6on 
of 
vWD 
• vWD 
– 
extremely 
heterogenous, 
complex 
disorder 
with 
> 
20 
dis6nct 
subtypes 
• Types 
of 
vWD 
1. Quan6ta6ve 
Defects 
• Type 
1 
– Par6al 
quan6ta6ve 
deficiency 
– Autosomal 
dominant 
• Type 
3 
– Complete 
absence/severely 
decreased 
– Autosomal 
recessive 
2. Qualita6ve 
Defects 
• Type 
2 
– 2A 
– 2B 
– 2M 
– 2N 
– Autosomal 
recessive 
51 
Subgroups
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
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
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
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
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
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
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
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
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
Assays 
for 
vWD 
• Platelet 
Func6on 
Screen 
(BT) 
• vWF 
an6gen 
assay 
• vWF 
ac6vity 
assay 
• FVIII:C 
• Mul6mer 
Analysis 
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 
åå 
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
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
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
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

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Lecture 5, fall 2014 pdf

  • 1. Lecture 5 Disorders of Primary Hemostasis Qualita6ve Platelet Disorders Von Willebrand Disease 2
  • 2. Func6onal disorders of platelets • Congenital Disorders – Disorders of Adhesion • Bernard-­‐Soulier Syndrome • Platelet-­‐type von Willebrand Disease – Disorders of Aggrega6on • Glanzmann Thrombasthenia – Disorders of Secre6on • Storage Pool Disorders (SPD) – Dense Granule disorders – Alpha Granule disorders • Signal Transduc6on defects/Platelet Release defects – Defec6ve TXA2 Pathway – Receptor Signaling Defects • Acquired Disorders 3
  • 4. Inherited Disorder of Platelet Dysfunc6on 5
  • 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
  • 22. Collagen (low dose and high dose) 23
  • 23. Ristoce6n (low dose and high dose) 24
  • 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
  • 28. 29
  • 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 àgiving 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
  • 33. Platelet and neutrophil images of GPS patients in comparison with controls. Gunay-Aygun M et al. Blood 2010;116:4990-5001 ©2010 by American Society of Hematology
  • 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
  • 41. Lab Tests in Disorders of Primary Hemostasis
  • 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
  • 46. vWF Release Valentijn K M et al. Blood 2011;117:5033-5043 47
  • 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
  • 48. Func6on of vWF 49 Elsevier
  • 50. Classifica6on of vWD • vWD – extremely heterogenous, complex disorder with > 20 dis6nct subtypes • Types of vWD 1. Quan6ta6ve Defects • Type 1 – Par6al quan6ta6ve deficiency – Autosomal dominant • Type 3 – Complete absence/severely decreased – Autosomal recessive 2. Qualita6ve Defects • Type 2 – 2A – 2B – 2M – 2N – Autosomal recessive 51 Subgroups
  • 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
  • 60. Assays for vWD • Platelet Func6on Screen (BT) • vWF an6gen assay • vWF ac6vity assay • FVIII:C • Mul6mer Analysis 61
  • 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 åå 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