Thrombosis

Asim K. DuttaroyERN 3110

HomeostasisState of fluid equilibrium within the blood vessels

Vessels

Platelets

Fibrinolysis/Inhibitors

Coagulation Proteins

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 cross-sectional 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.

Slide 3 of 28

Haemostasis:

• Vasoconstriction • Platelet activation• Haemostatic plug• Coagulation• Stable clot formation• Clot dissolution

BLOOD COAGULATION

ThrombinThrombin

FibrinogenFibrinogen Fibrin MonomersFibrin Monomers

Fibrin threadsFibrin threads

CaCa+2+2, factor XIII, factor XIII

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

PlateletPlateletAggregation

PlateletActivation

Blood VesselBlood Vessel Constriction

CoagulationCoagulation Cascade

Stable Hemostatic Plug

Fibrin formation

Reduced

Blood flow

Contact/ Tissue Factor

Primary hemostatic plug

Neural

formation of the platelet plug

coagulation = fibrin formation

clot retraction

fibrinolysis

RESOLUTION

NORMAL HAEMOSTASIS

platelet and coagulation activation within

blood vessel

PLATELETS AND ARTERIAL THROMBOSIS

Thrombus

“a mass of blood constituents formed within the vascular system”

“inappropriate haemostasis” ?

Overlap of Vascular Disease in Patients With Atherothrombosis

Vascular events (MI, stroke, or CV death)Vascular events (MI, stroke, or CV death)

Plaque Plaque rupturerupture

Platelet Platelet adhesion, adhesion, activation, activation,

and and aggregationaggregation

Thrombus Thrombus formationformation

MIMIUnstable anginaUnstable anginaIschemic strokeIschemic stroke PADPAD

Ness J, Aronow WS. J Am Geriatr Soc. 1999;47:1255-1256. Schafer AI. Am J Med. 1996;101:199-209.

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?

What are Platelets?

Role in Health : how do they work?

Role in Diseasebleeding disordersatherosclerosisarterial thrombosisthrombo-embolism

Platelets in cardiovascular disease

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

Platelets circulate in a resting, inactive state

Must become activated

Must stick together = Aggregation

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

Disk-shaped cellfragments producedin the megakaryocytes

Mature Platelet

Megakaryocyte

BoneMarrow

What are Platelets?

Storage and Circulation

Quantity - 200,000 - 400,000/mm3

Life Span - 10 days33%pooling

67%in the

circulation

MegakaryocyteSpleen

lipid bilayer

Glycoprotein receptors

Fig. 16-10

PlateletPlug Formation

Platelet AggregationFibrinogen binding to Glycoprotein IIb-IIIa on

activated platelets

Factors that activate platelets

ADPThrombinCollagen

5-hydroxytryptamine (serotonin)Thromboxane A2

Mechanical stimuli

Many stimuliSeveral different receptors

Multiple signalling pathways

Adhesion

GpIIb/IIIa

Platelet Activation Pathways

GpIIb/IIIaGpIIb/IIIa Aggregation

ADP

Adrenaline Platelet GpIb

Exposed Collagen

Endothelium

vWF

COLLAGEN

GpIIb/IIIaGpIIb/IIIa AggregationGpIIb/IIIaGpIIb/IIIa Aggregation

AdhesionAdhesion

ADP

Adrenaline

THROMBINTHROMBIN

                       

                 

                                                  

                                                                                                                                        

                                             Slide 23 of 79

Targets for anti-platelet therapy

AspirinNSAIDs

ADPreceptor

COX-1

TXA2

GPIIb - IIIa

Signalling

pathways

ADP receptor antagonistsClopidogrel THROMBIN

receptor

Thrombin inhibitors

II

Fibrinogen

Phosphodiesterase inhibitors

dipyridamole

Fibrinogen Receptor Antagonists

AAAA

PrimaryMetabolic

Disturbance

PrimaryMetabolic

Disturbance

Intermediate Vascular Disease

Risk Factor

Intermediate Vascular Disease

Risk Factor Intravascular

PathologyIntravascular

PathologyClinicalEvent

ClinicalEvent

Atherosclerosis

Hypercoagulability

• Coronary arteries• Carotid arteries• Cerebral arteries• Aorta• Peripheral arteries

Hypertension

Dyslipidemia

Hyperinsulinemia

Hyperglycemia

Inflammation

ImpairedFibrinolysis

Endothelial Dysfunction

Insulin Resistance

CVDOvernutrition

Hyperactivity of Platelets plays central role

                       

                 

                                                  

                                                                                                                                        

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Tissue factor,VIIa

Plaques

Effects of spices on platelet function

Spice Effects on platelets

GarlicReduced TxA2 generation, Reduced AA incorporation to membrane PL

Onion Reduced TXA2 and 12-Lipoxygenase products

Ginger Reduced Aggregation

Cloves

Cumin

TumericReduced TxA2 generation, Reduced AA incorporation to membrane PL

Antiaggregatory, Reduces cyclooxygenase and lipoxygneaseproducts

Inhibits AA-induced Aggregation

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 Event1. 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?

FUNCTIONSkin bleeding timeAggregation in vitro - response to added agonistsAssay of Products secreted by activated platelets into plasma, in vitro or in vivo

ACTIVATION STATE OF CIRCULATING PLATELETSFlow Cytometry with fluorescent markers

STRUCTURE - electron microscopy

BIOCHEMICAL PATHWAYS - signalling pathways, phosphorylation, Ca++ influx

Platelet Granule Contentsreleased on activation

Alpha-granules:Beta-thromboglobulinPlatelet Factor 4 Fibrinogenadhesive glycoproteins::P-selectin, vWF, Coagulation Factor VGrowth Factors

Dense Granules:ADP Calcium ions,

ATP Serotonin (5-HT)

Radioimmunoassay

ELISAin plasma

ASSAYS

FluorescenceHPLCradiolabelling

Platelet Function Defects

Aggregation Coagulation

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

Shape Change Release Delayed Fibrin Formation

Adhesion

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 (t-PA, 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 bufferand centrifuged at 200xg for 10 min,PRP is used to measure platelet aggregation

Washed platelets: plasma proteins are removed from PRP byColumn chromatography or centrifugation

Whole blood aggregation: blood is used as such with or without dilution

In vitro platelet aggregation is an effort to charatcerise 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 sample 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