Thrombosis
Asim K. Duttaroy
ERN 3110
Homeostasis
State of fluid equilibrium within the blood vessels
Vessels
Coagulation
Proteins
Platelets
Fibrinolysis/Inhibitors
Hemostasis
• Arrest of bleeding
• Events preventing excessive blood loss
– Vascular spasm: Vasoconstriction of
damaged blood vessels
– Platelet plug formation
– Coagulation or blood clotting
Functions of Blood
• Transport of:
– Gases, nutrients, waste products
– Processed molecules
– Regulatory molecules
•
•
•
•
Regulation of pH and osmosis
Maintenance of body temperature
Protection against foreign substances
Clot formation
Composition of Blood
HEMOSTASIS
 VASCULAR SPASM
 PLATELET PLUG
 BLOOD COAGULATION (will talk later)
 GROWTH OF FIBROUS TISSUE IN CLOT
 Fibrinolysis (will talk later)
What is thrombosis?
• Thrombosis is the formation of a blood clot inside a
blood vessel, obstructing the flow of blood through the
circulatory system
• When a thrombus occupies more than 75% of crosssectional area of the lumen of an artery, blood flow to the
tissue supplied is reduced enough to cause of symptoms
because of decreased oxygen supply and accumulation
of lactic acid.
• More than 90% obstruction can result in anoxi, complete
deprivation of oxygen, and the infarction , a mode of cell
death
Thrombosis
• Arterial Thrombosis :
– Adherence of platelets to arterial walls - White in
color - Often associated with MI, stroke and
ischemia
• Venous Thrombosis :
– Develops in areas of stagnated blood flow (deep
vein thrombosis), Red in color- Associated with
Congestive Heart Failure, Cancer, Surgery.
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Haemostasis:
•
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Vasoconstriction
Platelet activation
Haemostatic plug
Coagulation
Stable clot formation
Clot dissolution
BLOOD COAGULATION
Thrombin
Fibrinogen
Fibrin Monomers
Ca+2, factor XIII
Fibrin threads
Coagulation:
•
•
•
•
•
•
Fibrinogen to Fibrin – Coag. Cascade
Several factors – proenzymes-activation.
Enzyme amplication –
Plasma, Endothelium & Platelets
Stable hemostatic plug.
Clot lysis – starts soon after clot formation.
Haemostasis overview:
BV Injury
Contact/
Tissue
Factor
Neural
Blood Vessel
Constriction
Platelet
Aggregation
Coagulation
Cascade
Primary hemostatic plug
Reduced
Platelet
Activation
Blood flow
Stable Hemostatic Plug
Fibrin
formation
NORMAL HAEMOSTASIS
 formation of the platelet plug
 coagulation = fibrin formation
 clot retraction
 fibrinolysis
 RESOLUTION
PLATELETS AND ARTERIAL THROMBOSIS
platelet and coagulation activation within
blood vessel
Thrombus
“a mass of blood constituents formed within the
vascular system”
“inappropriate haemostasis” ?
Overlap of Vascular Disease in Patients With
Atherothrombosis
Ischemic stroke
Unstable angina
Plaque
rupture
Platelet
adhesion,
activation,
and
aggregation
MI
Thrombus
formation
Vascular events (MI, stroke, or CV death)
Ness J, Aronow WS. J Am Geriatr Soc. 1999;47:12551256. Schafer AI. Am J Med. 1996;101:199-209.
PAD
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
What are platelets?
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
Normal Function of Platelets
Haemostasis
•
Preventing bleeding from wounds
•
Integrity and repair of the vessel wall
Haemostatic role of platelets in health:
how do they work?
 Platelets circulate in a resting, inactive state
 Must become activated
 Must stick together =
Aggregation
What are Platelets?
Disk-shaped cell
Mature
fragments produced
Platelet
in the
megakaryocytes
Megakaryocyt
e
Bone
Marrow
Storage and Circulation
Quantity - 200,000 - 400,000/mm3
Life Span - 10 days
33%
pooling
67%
in the
circulation
Megakaryocyte
Spleen
lipid bilayer
Glycoprotein receptors
Fig. 16-10
Platelet
Plug Formation
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
Platelet Activation Pathways
COLLAGEN
THROMBIN
ADP
Aggregation
GpIIb/IIIa
GpIIb/IIIa
GpIIb/IIIa
Adrenaline
Platelet
Adhesion
vWF
Endothelium
Exposed Collagen
Slide 23 of 79
Targets for anti-platelet therapy
ADP receptor
antagonists
Clopidogrel
Phosphodiesterase
inhibitors
dipyridamole
ADP
receptor
Signalling
Fibrinogen Receptor
Antagonists
II
THROMBIN
receptor
COX-1
TXA2
- IIIa
Aspirin
AA
pathways
GPIIb
Thrombin
inhibitors
NSAIDs
Primary
Metabolic
Disturbance
Intermediate
Vascular Disease
Risk Factor
Intravascular
Pathology
Insulin
Resistance
Clinical
Event
Hyperactivity of
Platelets plays central
role
Hypertension
Dyslipidemia
Hyperglycemia
Overnutrition
Hyperinsulinemia
Inflammation
Atherosclerosis
•
•
•
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Coronary arteries
Carotid arteries
Cerebral arteries
Aorta
Peripheral arteries
Hypercoagulability
Impaired
Fibrinolysis
Endothelial
Dysfunction
CVD
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Plaques
Tissue factor,
VIIa
Effects of spices on platelet function
Spice
Effects on platelets
Garlic
Reduced TxA2 generation, Reduced
AA incorporation to membrane PL
Onion
Reduced TXA2 and 12-Lipoxygenase products
Ginger
Reduced Aggregation
Cloves
Antiaggregatory, Reduces cyclooxygenase and
lipoxygnease
products
Cumin
Inhibits AA-induced Aggregation
Tumeric
Reduced TxA2 generation, Reduced
AA incorporation to membrane PL
In vitro anti-aggregatory properties of fruit extracts
Fruits
% of inhibition
Tomato
92
Kiwifruit
85
Strawberries
51-65
Melon
30
Plum
25
Banana
21
Avocado
21
Mango
19
Cranberry
18
Orange
18
Nectarine
15
Pineapple
12
Pear
5
Apple
2
Disorders of platelets
• Decreased Number: Thrombocytopenia
– Decreased Production
– Decreased Survival – Immune (ITP)
– Increased utilization - DIC
• Defective Platelet function:
– Acquired – Drugs – Aspirin, MPS, MDS
– Congenital – Eg. Thrombasthenia.
Disorders of Hemostasis
• Vascular disorders
– Scurvy, easy bruising,
• Platelet disorders
– Low Number or abnormal function
• Coagulation disorders
– Factor deficiency.
• Mixed/Consumption: DIC
Platelet
Petechiae, Purpura
Role of Platelets in Acute Ischaemic Event
1. Growth of atherosclerotic plaque
2. Plaque rupture
3. Thrombus formation
4. Occlusion
NEXT
 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
Platelet Granule Contents
ASSAYS
released on activation
Alpha-granules:
Beta-thromboglobulin
Platelet Factor 4
Fibrinogen
adhesive glycoproteins::P-selectin, vWF,
Coagulation Factor V
Growth Factors
Dense Granules:
ADP
Calcium ions,
ATP
Serotonin (5-HT)
Radioimmunoassay
ELISA
in plasma
Fluorescence
HPLC
radiolabelling
Platelet Function Defects
Adhesion
Shape Change Release
Aggregatio
n
Coagulatio
n
Delayed Fibrin
Formation
1. Failure of platelets to adhere
2. Failure to release ADP
3. Failure to release TxA2
4. Failure to aggregate
5. Failure of surface binding of coagulation
factors
Diagnostic Procedures
• Platelet count
• Peripheral smear
• Platelet
aggregation
• Bleeding time
• Platelet
adhesiveness
• Clot retraction
Drugs That Affect Platelets
• Analgesics (aspirin, NSAIDs) affecting prostanoid synthesis
or action
• Caffeine, theophylline, dipyridamole and drugs which
increase platelet cyclic AMP
• Antimicrobials (penicillins, cephalosporins, nitrofurantoin)
• Cardiovascular agents (quinidine, diurectics, vasodilators
• Anticoagulants (coumadin, heparin) and Thrombolytics (tPA, streptokinase)
• Psychotropics (tricyclics/phenothiazines) and anesthetics
• Chemotherapeutic agents
• Miscellaneous agents (dextrans, clofibrate, ETOH, Vitamin E,
onions, garlic, ginger, fish oil)
Plaetelet aggregation can be measured in
Platelet rich plasma (PRP): Blood collected in citrate buffer
and centrifuged at 200xg for 10 min,
PRP is used to measure platelet aggregation
Washed platelets: plasma proteins are removed from PRP by
Column chromatography or centrifugation
Whole blood aggregation: blood is used as such
with or without dilution
In vitro platelet aggregation is an effort to charatcer
the in vivo ability if the platelets to form the primary
hemostatic plug
Whole blood aggregation: by which platelets are tested in
anti-coagulated blood, without the need to islolate them from
other components of blood.
The chrono-log whole blood aggregometer consists of samp
Compartments heated to 37C with stiring facility using
magnetic bars
Principle of whole blood aggregation:
Impedence method (electrical resistance)
Two electrodes are inserted in blood sample (0.5 ml) at 37C.
Platelet monolayer is developed on the electrode surface and
basal resistance is developed.
As platelet aggregation proceeds with the addition of agonist the more
platelets aggregates
are deposited on the electrodes, and the resistance (ohm) is increased
Disadvantages of some 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)
Dutta-Roy, AK, Dietary components and human platelet activity.
Platelets. 2002 Mar;13(2):67-75.
.
Dutta-Roy, AK Effects of tomato extract on human platelet aggregation
in vitro Platelets. 2001
12:218-27
Andrews and Berndt Platelet physiology and thrombosis. Thromb Res.
2004;114(5-6):447-53
Kroll MH, Schafer AI. Biochemical mechanism of platelet activation
Blood 1989; 74: 1181-95.
Colquhoun DM, Nutraceuticals: vitamins and other nutrients in coronary
heart disease, Curr Opin Lipidol , 2001, 12, :639-46