Thrombosis Asim K Duttaroy ERN 3110 Homeostasis State

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 p. H and osmosis Maintenance of body temperature Protection against foreign substances Clot formation

Composition of Blood

HEMOSTASIS Ø VASCULAR SPASM Ø PLATELET PLUG Ø BLOOD COAGULATION (will talk later) Ø GROWTH OF FIBROUS TISSUE IN CLOT Ø 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: • • • 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 Blood flow Platelet Activation 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 Ì What are Platelets? Ì Role in Health : how do they work? Ì Role in Disease bleeding disorders atherosclerosis arterial thrombosis thrombo-embolism è Techniques for the study of platelets è 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? l Platelets circulate in a resting, inactive state l Must become activated l 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/mm 3 Life Span - 10 days 33% pooling 67% in the circulation Megakaryocyte Spleen

lipid bilayer Glycoprotein receptors

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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 A 2 Mechanical stimuli Many stimuli Several different receptors Multiple signalling pathways

Platelet Activation Pathways COLLAGEN THROMBIN ADP Aggregation Gp. IIb/IIIa Platelet G p. I Adrenaline b Adhesion v. WF Endothelium Exposed Collagen

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Targets for anti-platelet therapy ADP receptor antagonists Clopidogrel Phosphodiesterase inhibitors dipyridamole ADP receptor Fibrinogen Receptor Antagonists THROMBIN receptor COX-1 Signalling Aspirin AA pathways TXA 2 GPIIb Thrombin inhibitors II - IIIa Fi b rin og en 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 • • • 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 Tx. A 2 generation, Reduced AA incorporation to membrane PL Onion Reduced TXA 2 and 12 -Lipoxygenase products Ginger Reduced Aggregation Cloves Antiaggregatory, Reduces cyclooxygenase and lipoxygnease products Cumin Inhibits AA-induced Aggregation Tumeric Reduced Tx. A 2 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 Ì What techniques can we use to study platelets ? Ì Modification of platelets in the prevention and treatment of cardiovascular disease Ì 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, v. WF, 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 Tx. A 2 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 (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 buffer and centrifuged at 200 xg 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 37 C 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 37 C. 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