Platelets in cardiovascular disease

Dr Isobel Ford
Dept of Medicine & Therapeutics

i.ford@abdn.ac.uk

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 Platelets in cardiovascular disease

 What are Platelets?

 Role in Health : how do they work?

 Role in Disease
bleeding disorders
atherosclerosis
arterial thrombosis
thrombo-embolism
 Techniques for the study of platelets

 Anti-platelet Therapy in Cardiovascular Disease

 WHAT ARE PLATELETS ?

 white, discoid
 smallest element of flowing blood
1 - 2 microns diameter
 lipid bilayer membrane
 normal range 150 - 400 x 10 9 / Litre blood
 Formed from cytoplasm of megakaryocytes
 No nucleus
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Pla
te l et

Peripheral Blood Film

Magnification x100
 lipid bilayer
Glycoprotein receptors
 Normal Function of Platelets

Haemostasis
• Preventing bleeding from wounds

• Integrity and repair of the vessel wall

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 Haemostatic role of platelets in health:
how do they work?

 Platelets circulate in a resting, inactive state

 Must become activated

 Must stick together = Aggregation

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 Blood Vessel
Endothelium
Subendothelium
INJURY
Collagen VWF Tissue
Vaso-
Factor
constriction
Platelet Adhesion Coagulation Cascade
& Secretion
Thrombin

Platelet aggregation Fibrin

Haemostatic plug
Platelet Adhesion
adhesion, shape change, spreading and secretion
 Platelet Aggregation
Fibrinogen binding to Glycoprotein IIb-IIIa on
activated platelets
Factors that activate platelets

ADP
Thrombin
Collagen
5-hydroxytryptamine (serotonin)
Thromboxane A2
Mechanical stimuli

Many stimuli
Several different receptors
Multiple signalling pathways
SUMMARY
Stimulus e.g. damaged vessel wall

• Adhesion to exposed vWF via glycoprotein (GP Ib)

receptor, or collagen (GPIa, GPVI receptors)
• Signalling events, Shape Change
• Exposure of Fibrinogen binding site on GPIIb/IIIa
• Secretion from granules
• Exposure of anionic phospholipids

Platelet Aggregation
• Promotion of clotting on activated platelet surface
 NORMAL HAEMOSTASIS

 formation of the platelet plug

 coagulation = fibrin formation

 clot retraction
 fibrinolysis
 RESOLUTION

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 PLATELETS IN DISEASE:
ARTERIAL THROMBOSIS

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 PLATELETS AND ARTERIAL THROMBOSIS

platelet and coagulation activation within

blood vessel

Thrombus

“a mass of blood constituents formed within the

vascular system”

“inappropriate haemostasis” ?
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 THROMBOSIS

Predisposing factors: “Virchow’s Triad”

abnormalities of
vessel wall

blood flow blood constituents

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 How do we know platelets are important in CVD?

 Platelets are present in atherosclerosis, thrombosis,

embolism i.e. at early and late stages of cardiovascular
disease

 Activated platelets are present in circulation of patients

with cardiovascular disease

 Modification of platelet activity affects the development

and progression of cardiovascular disease

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 Activating Factors for Platelets in Cardiovascular
Disease
 Damage to endothelium :

Atherosclerosi  Loss of inhibitory

s function of
endothelium

 Exposure of von
Willebrand Factor and
collagen
Activating Factors for Platelets in Cardiovascular Disease

 Disturbed blood
flow -
 shear stresses,
narrowing of artery,
turbulence

Others
Low density lipoprotein or oxidised LDL
 Bacteria - myocardial infarction?
 Immune Complexes - present in MI
 Role of Platelets in Acute Ischaemic Event
1. Growth of atherosclerotic plaque 3. Thrombus formation

2. Plaque rupture 4. Occlusion

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 What techniques can we use to study platelets ?

 Modification of platelets in the prevention and
treatment of cardiovascular disease
 Drugs in current use and some newer ones
 How can we study platelets in the laboratory?

 STRUCTURE - electron microscopy

 BIOCHEMICAL PATHWAYS - signalling pathways,
phosphorylation, Ca++ influx
 FUNCTION
Skin bleeding time
Aggregation in vitro - response to added agonists
Assay of Products secreted by activated platelets
into plasma, in vitro or in vivo
 ACTIVATION STATE OF CIRCULATING PLATELETS
Flow Cytometry with fluorescent markers

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 Platelet Granule Contents ASSAYS
released on activation

Alpha-granules:
Beta-thromboglobulin Radioimmunoassay
Platelet Factor 4 ELISA
Fibrinogen in plasma
adhesive glycoproteins::P-selectin, vWF,
Coagulation Factor V
Growth Factors

Dense Granules:
ADP Calcium ions, Fluorescence
HPLC
radiolabelling
ATP Serotonin (5-HT)

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Platelet Aggregometry
 Disadvantages of some older techniques

 Require large sample of fresh blood

 Susceptible to ex vivo activation during
venepuncture and sample preparation, particularly
centrifugation or washing
 Take a long time to perform
 Better at detecting hypo-functional platelets
(bleeding disorders) than
hyper-active platelets (pro-thrombotic)

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 Point-of-Care (“bedside”)
Platelet Testing

● Cartridge-based, whole blood

● Ultegra rapid platelet function analyzer
Aggregation of fibrinogen-coated beads: COX or Thrombin pathways
● Platelet Function Analyser PFA100
Aperture closure time: collagen/ADP or collagen/adrenaline

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 Flow Cytometer

Detector &
Computer System
Mechanical Fluidics
 Flow Cytometry
Labelling platelets with fluorescently-conjugated
antibodies

USES:
 Detection of activated platelets in
circulation
 Effects of treatment on platelet activation
CAN MEASURE:
 Changes in platelet surface receptors
 Expression of markers of activation:
Fibrinogen binding, fibrinogen receptor, P-selectin
 Calcium ion flux
 Procoagulant changes in membrane phospholipid - by
binding of Annexin V
 Adhesion to other blood cells

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 GPIIb-IIIa

Alpha granules

lysosomes

ADP/adrenaline Phorbyl ester

Thrombin

Budding of
microvesicles
GPIIb/IIIa Exposure of negatively
charged phospholipid

Fusion of granule membrane proteins

Fibrinogen Receptor P-selectin (CD62P)
Fibrinogen exposure CD63
Binding
Flow cytometry- Platelets
Anti-GPIIb/IIIa/FITC
Platelet activation
 Potential Problems in Detection Of Platelet
Activation Markers

 Platelets very susceptible to in vitro handling

artefacts
 Platelets are very small
 Fixation and chemical inhibition of platelets may
cause changes in the receptors you are trying to
measure
To overcome:
Careful venepuncture and sample handling
Analyse whole blood so no centrifugation or
separation required
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 FLOW CYTOMETRY IN WHOLE BLOOD

 Very small sample volume - < 1mL

 Rapid analysis
 Single platelets can be analysed
 No need to isolate platelets from blood - electronic
gating used to exclude other cells
 Low levels of activation markers can be accurately
measured, reflecting in vivo situation

Disadvantages:
Expensive instrument
Needs trained operator
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 We have talked about:

 Key role of Platelets in normal haemostasis

 Structure and function
 Mechanisms of Activation
in health and in disease
 Role of platelets in cardiovascular disease
atherosclerosis thrombosis embolism