PLATELET FUNCTION DISORDERS

Dr. Ridhi Sood

Introduction

Normal platelet structure and function

Classification of platelet function disorders
Inherited platelet function disorders
Acquired platelet function disorders
Approach to a patient with platelet function disorders
Platelet function tests
Summary

Normal Platelet Structure

Secretory Function
- Granules

Fibrinogen
Factor V
Platelet factor 4
PDGF
TGF
VEGF
vWF
P- selectin (CD 62P)

- Granules

ADP
ATP
Calcium
Serotonin

Lysosomal granules

Galactosidase
Fucosidase
Hexosaminidase
Cathepsin

Normal Platelet Function

Stages in platelet plug formation-classical model

Classification of Thrombocytopathies
Hereditary platelet dysfunction
Defects in initiation phase
Defects in extension phase
Defects in consolidation phase
Acquired platelet dysfunction

Hereditary platelet dysfunction

Initiation phase
Bernard-Soulier syndrome
Extension phase
Secretion disorders/granule deficiencies
-granule abnormalities (gray platelet syndrome)
-granule abnormalities
/ -granule deficiency
Hermansky-Pudlak syndrome
Chdiak-Higashi syndrome
Wiskott-Aldrich syndrome

Hereditary platelet dysfunction

Defects of signal transduction and secretion
Impaired liberation of arachidonic acid
Cyclooxygenase deficiency
Thromboxane synthase deficiency
Thromboxane A2 receptor abnormalities
Consolidation phase
Glanzmann thrombasthenia
Miscellaneous
Hereditary macrothrombopathy

Acquired disorders of platelet

function
Drug-induced platelet dysfunction
Analgesics
Antibiotics
Cardiovascular drugs
Psychotropic drugs
Secondary to systemic diseases
Uremia
Paraproteinemia
MDS/acute non-lymphocytic leukemia
MPDs

Defects in Initiation Phase

Bernard-Soulier syndrome (BSS)

Aka Hemorrhagiparous thrombocytic dystrophy
First described in 1948 Jean Bernard and Jean-Pierre Soulier
AR disorder; consanguinity
Etiology
Quantitative or qualitative abnormality of the membrane
GP1b complex
Type A domain of 1b binds to vWF, mediating normal
platelet adhesion
BSS platelets do not adhere to extracellular matrix

Mutations of the genes for GP Ib, GP Ib, and GP IX have been

identified

GP1b complex= 2 [GP1b (1b+1b) + GP IX] + GP V

Clinical features:
Bleeding features evident shortly after birth or in early
childhood
Degree of bleeding out of proportion to the moderate in
circulating platelets
Lab findings:
Thrombocytopenia (20,000 1,00,000/L)
Giant platelets on blood smear
Defective ristocetin-induced agglutination
Low/absent levels of GP1b complex (flow cytometry)

Bernard-Soulier syndrome (BSS)

Defects in Extension phase

Secretion defects: storage pool

diseases(SPD)
Heterogenous group of disorders that have a deficiency
of granules or their constituents resulting in defect in
ADP release
3 subtypes:
Isolated deficiency of granules
Isolated deficiency of granules
Combined granules abnormality

Syndromic Association
Storage pool deficiency can develop isolated or in
combination with complex syndromic disorders such
as:
Hermansky-Pudlak syndrome (HPS)
Chediak-Higashi syndrome (CHS)
Wiskott- Aldrich syndrome (WAS)

Chediak-Higashi syndrome
Result from mutations in lysosomal trafficking regulator
gene (LYST) located on chromosome 1q
Defective platelet granules; or absent
Clinical features Oculocutaneous albinism, photophobia, silver grey
hypopigmented hair
Severe immune deficiency, recurrent infections.
Progressive neurological dysfunction
Pancytopenia
Organomegaly

Chediak-Higashi Syndrome Cont.

Lab findingsPlatelet counts normal
Defective platelet granules; or absent
Platelet aggregation assays-secondary wave impaired
ATP:ADP ra o
platelet serotonin, calcium
Neutrophils contain abnormal large giant lysosome that can fuse
with phagosome but the ability to release their contents is
hampered.

Hermansky-Pudlak syndrome
Autosomal recessive disorder
Characterized by
Oculocutaneous albinism
Lysosomal granule defects
Platelet dense granule deficiency
Clinical features:
Lifelong h/o easy bruising, minor bleeding episodes; often require
transfusions
Pulmonary fibrosis
Infiltration by ceroid-pigmented
reticuloendothelial cells
Inflammatory bowel disease
Platelet aggregation assays- absent secondary wave in response to
ADP and epinephrine; response to collagen is abnormal

Disaggregation after primary aggregation with ADP

Dense granule deficiency

Control platelet

Br J Haematol 2007;138:671

Wiskott-Aldrich syndrome
X-linked recessive disease, Mutations in WAS gene
Clinical features Infants - bruising & purpura - risk of intracranial hemorrhage
GI bleed or prolonged bleeding on circumcision - presenting
Eczema
Immunodeficiency- infections begin in first 6 months of life
age- hemolytic anemia, vasculitis are m/c manifestations
Malignancy- usually lymphoreticular
Lab findings Platelet count- 5000 to 50,000/l, microthrombocytopenia

 Granule SPD: Gray Platelet Syndrome

First described by Raccuglia in 1971
Very rare AR disorder with <100 cases reported worldwide
The basic molecular defect involves packaging or storage of
proteins during -granule biogenesis.
Granule number during matura on (normal platelets
contain approximately 50 granules)
Mature megakaryocytes left with only small, few, abnormal
granules

Gray platelet syndrome

Defects in Consolidation phase

Glanzmann thrombasthenia
Autosomal recessive, chr 17
Qualitative or quantitative defect of either GPIIb or GPIIIa (ITG
IIb3)
GPIIb-IIIa complex is a calcium dependent heterodimer that
binds to fibrinogen or vWF
Hence, its defects leads to deficient or completely absent
platelet aggregation

Glanzmann thrombasthenia cont.

Clinical features Bleeding symptoms manifest at birth and patients are
diagnosed before the age of 5
Glanzmanns patients are stratified into three groups based
on complex expression:
Type I - <5 percent GPIIbIIIa, absent fibrinogen ( granule)
Type II - <20 percent, fibrinogen present
Type III - >50 percent; qualitative disorder

Glanzmann thrombasthenia cont.

Lab findings Normal platelet number and morphology on light
microscopy
Platelet aggregation assays- deficient platelet
aggregation with ADP, collagen, epinephrine,
thrombin; ristocetin-induced aggregation normal
Flow cytometry: decreased mAb expression of CD41
(GPIIb) and CD61 (GPIIIa)

Hereditary defects of platelet

function with thrombocytopenia
MYH-9 related thrombocytopenia syndromes
MYH-9 gene encodes for non muscle myosin IIA - a cytoskeletal
contractile protein
Defective megakaryopoiesis & thrombocytopenia
Clinical features- Bleeding + SNHL + Glomerulonephritis
Lab findings Platelet count- 20,000 to 130,000/l
mean platelet volume- population of very large platelets
Dhle-like bodies within neutrophils on stained blood smears
Definitive based on identification of MYH9 mutation

Giant platelet syndromes associated

with MYH9 mutations
Syndrome

Neutrophil
inclusions

Hereditary
nephritis

Deafness

May-Hegglin

Yes

No

No

Fechtner

Yes

Yes

Yes

Sebastian

Yes

No

No

Epstein

No

Yes

Yes

Acquired disorders of platelet function

Common Causes:
Drugs & food substances
Uremia
Cardiopulmonary bypass
Myeloproliferative disorders
Paraproteinemia
Platelet antibodies

Acquired disorders (cont.)

Drug-induced platelet dysfunction Analgesics- aspirin, NSAIDs, acetaminophen
P2Y12 antagonists- clopidogrel, ticlopidine
Antibiotics- -lactams: penicillins, cephalosporins
Integrin GPIIb-IIIA antagonists- abciximab, tirofiban, eptifibatide
Uremia Uremic pa ents have defect in COX ac vity TXA2) and nitric oxide
(limits platelet adhesion)
stores of platelet ADP and serotonin
ability of integrin IIb3 to bind vWF and fibrinogen
Typical bleeding sites- GIT, intracranial

Acquired Disorders cont.

Paraproteinemias
Multiple myeloma, Waldenstrom macroglobulinemia, monoclonal
gammopathies, polyclonal hypergammaglobulinemia
Non-specific binding of Ig to platelet surface or specific receptor
Clonal hematopoietic disorders
MDS, acute nonlymphocytic leukemia
Peripheral blood- platelet granules in number or larger in size
Bone marrow- typical- micromegakaryocytes with hypolobulation
and hypogranularity
Ultrastructural abnormalities

Approach to patient
1.

History- THE SINGLE BEST MEANS FOR ASSESING PLATELET

FUNCTION
2. Examination
Bleeding manifestations typical of platelet dysfunction:
Unexplained or extensive bruising
Epistaxis, particularly lasting > 30 minutes
Menorrhagia, particularly if present since menarche
Oral cavity bleeding
Bleeding during childbirth
Bleeding following invasive procedures
Bleeding following dental extraction

Approach to patient cont.

4. Laboratory assessment

Platelet count
Coagulation tests
Bleeding time
Platelet function tests

Platelet function tests

Platelet function tests

Bleeding time
Adhesion tests
PFA-100
Aggregation tests
Turbidometric techniques
Investigations of granular content and release
Electron microscopy
ADP and ATP content (bioluminiscence)
Serotonin release
Flow cytometry

Bleeding Time
Platelet function testing began with the application of
the in vivo bleeding time by Duke in 1910.
The bleeding time was further refined by the Ivy
technique
It is the time taken by a standardized skin wound to
stop bleeding
Previously recommended as a clinically useful
screening test of platelet function
A prolonged BT with a normal platelet count Indicates a qualitative platelet disorder

Bleeding Time

43

Platelet Function Analyser (PFA-100)

The PFA-100 device is a test system in which
citrated whole blood is aspirated at high shear rates
through an aperture
Widely used as a screening tool to measure global
platelet haemostatic function

Principle
PFA-100
principle
Simulates primary hemostasis
Utilizes a cartridge with a biologically active
membrane - These agonists trigger platelet adhesion,
activation and aggregation leading to rapid occlusion
of the aperture and cessation of blood flow
The end-points for test is time to occlusion of blood
flow (closure time [CT]) or non-closure if the CT
exceeds 300 seconds.

45

46

48

49

50

51

52

z
Reference Ranges:
C/EPI cartridge= 78 - 199 s
C/ADP cartridge= 55 - 137 s

53

Advantages of the PFA-100 include:

Only small volumes of citrated venous blood (800L) are needed and so the test is useful for
investigating platelet function in children.

Can be used by non-skilled personnel and is both rapid and automated

The PFA-100 was designed as a screen to detect problems with primary haemostasis and in
part to replace the bleeding time and in this respect it is better standardised.

Measurement of platelet function at high shear [physiological] rates

Relatively insensitive to clotting factor deficiencies

High negative predictive value i.e. if the PFA-100 gives a normal result then with some
exceptions primary haemostasis is intact [Exceptions: SPD, Primary Secretion Defects, mild
Type 1 VWD]

Light Transmission Aggregometry (LTA)

Invented in the 1960s
Most widely used platelet function assay
Specific assays of platelet function
The gold standard for platelet function testing

55

LTA- Sample
Platelet rich plasma
Light spin 200g x 10 min

56

LTA-Principle
Addition of an aggregating agent to a
suspension of PRP
Change in platelet shape & aggregation
Monitored as increasing transmittance
Changes recorded in form of graph

LTA-AGONIST
Aggregating agents

ADP
Epinephrine
Epinephrine
Collagen
Ristocetin
Arachidonic acid
59

Patients citratecitrate-anticoagulated blood is centrifuged at low speed.

Platelet--rich plasma is separated and aliquoted into different cuvettes
Platelet
cuvettes..
Standard concentrations of agonists; standardized temp
temp;; constant agitation.
Aggregometer detects aggregation as changes in light transmission through cuvettes
cuvettes..

0%
0%
Epinephrine
100%

0%
Collagen
100%

0%
ADP
100%

Ristocetin
100%
60

Platelet Aggregation In BSS Patients

BSS patients showed no aggregation in

response to Ristocetin (A); (B) ADP;
(C) collagen - normal

There is lack of agglutination with ristocetin.

Possible diagnosis are - Von Willebrand Disease or
Bernard Soulier Syndrome

Patient Plasma + Control

Platelets

Patient Platelets + Control

Plasma

Platelet Aggregation In Glanzmann

Thrombasthenia

Reduced aggregation in response to (A)

Ristocetin and no aggregation with (B) ADP
and (C) Collagen

Suggest a failure of granule release and is

consistent with either platelet storage pool
disorder or a defect in nucleotide release.

Disorder

Characteristic Findings on LTA

Glanzmann's Thrombasthenia
OR afibrinogenaemia

Absent or markedly impaired aggregation to

all agonists except ristocetin.

Bernard Soulier Syndrome OR Von

Willebrand Disease

Absent or markedly reduced platelet

agglutination with ristocetin.

Storage Pool Disorder OR Platelet Release

Defect

Primary aggregation only with ADP,

adrenaline and collagen and only partial
agglutination with ristocetin suggesting a
failure of granule release or a deficiency of
platelet granules.

Aspirin [or defects in the COX pathway]

Absent aggregation to arachadonic acid.

Primary wave aggregation only with ADP.
Decreased or absent aggregation with
collagen.

Clopidogrel

Absent aggregation with ADP

Tests for platelet secretion

Dense bodies
Electron microscopy
ADP & ATP content
Serotonin release
granules
thromboglobulin
PF- 4
vWF
80

Flow cytometry
Identification & quantitation of membrane receptors
eg. (GP IIb- IIIa) density
Measure granule content

81

Measurement of total and released

nucleotides
Diagnostic tool usually in conjunction with
aggregometry
For determining any specific deficiency in dense
granule numbers or their content (e.g. storage pool
disease) or defects in degranulation (e.g. release
defects).
These defects can be misdiagnosed if relying on
platelet aggregometry alone

Other tests
Electron microscopy has also proven very useful for
defining ultrastructural abnormalities associated
with dense granule defects
Platelet alpha granule proteins measured by ELISA,
RIA or western blotting may be helpful for the
diagnosis of Quebec platelet disorder
Molecular genetic diagnosis of heritable platelet
disorders offer valuable confirmation of diagnosis in
affected individuals, in family members and for antenatal diagnosis

Summary

Differential diagnosis

Thank you