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UNIT 6A

Platelets use Oxygen to form Lipid Regulators while Endothelial cells form NO

A) Platelet function:i. Blood clotting and stroke (anticoagulant drugs) ii. Prostaglandin and thromboxane formation from

polyunsaturated fatty acidsiii. Nutritional approach to prevent heart diseaseiv. COX Inhibitors (NSAIDs) (antiplatelet drugs)

B) Endothelial cell functions: Prostacyclin and plasminogen

activator formationRole of endothelial cells in regulating blood pressure:

- Angiotensin and EDRFNitric oxide formation, signaling and toxicity:

- Use of Nitric Oxide generators: (vasodilator / antihypertensive drugs)

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Plateletst1/2 = 4 days Fragments of Megakaryocytes of bone marrow Contain: glycogen granules Mitochondria Lysosomes No nucleus, DNA, protein synthesis.1) dense granules – ADP, ATP, serotonin (5HT)2) granules contain clotting factors and PDGF (platelet derived growth factor) 80% ATP from glycolysis and 20% from mitochondria

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Platelet Function is to plug blood leaks (blood clotting) Activated by collagen (damaged vascular surface) or thrombin which binds to receptors. Platelet disc shape changes to sphere i.e. swell, form pseudopods, become sticky and attach to collagen

Resting platelets Activated PlateletsImages From: http://www-personal.engin.umich.edu/~tkinzer/

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Mechanism of platelet aggregation to form a clot

ADPreleasedfrom plateletgranule

receptor

Ca2+ influxin other platelets

MembranePhospholipaseA2 activated

Phospholipid

Arachidonate

cyclooxygenaseacetylates

Aspirin(drug)

PGH2

Thromboxane A2 (TxA2)

Diacylglycerol (DAG)+ inositol triphosphate (IP3)

PhosphatidylinositolMembrane

Phospholipase C

collagenreceptor

COLLAGEN

TxA2 synthetasedrug?

receptor

Ca2+ InfluxContraction

of microtubules

Granulerelease

PLATELETAGGREGATION

2O2

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Modified from Fig. 10.37, Stryer (5th ed).A fibrin clot (blood clot) is formed by the interplay of the intrinsic, extrinsic, and final common pathways.Intrinsic pathway:• initiated when factor XII is activated by contact with abnormal surfaces due to injury.Extrinsic pathway:• triggered by trauma, which activates factor VII which releases tissue factor.

Fibrin clot formed by zymogen activation cascadeINTRINSIC PATHWAY

Traps aggregatedplatelets

6Anticoagulant drugs (stroke treatment prevents new clots).• Vitamin K is essential for prothrombin synthesis (and other clotting

factors).

• Abnormal prothrombin is formed (does not bind Ca2+) in the absence of Vit. K or in the presence of Vit. K antagonists (see below).

O

O

CH3

CH3

H

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Vitamin K

OO

OH

C

O O

OH

H

H

O

OO

OH

CH

CH3

Dicoumarol- Spoiled sweet clover causes fatal hemorrhagic disease in cattle.

Warfarin for thrombosis but high CYP2C9 polymorphism.Drug name Coumadin (rat poison)

Anticoagulants Drugs – Vit. K antagonists

2-3 days before it works DONT admin. again for 3d

Drug induced thrombocytopenia

• Life threatening platelet depletion caused by drug or heparin induced antibodies which bind to platelet factor 4 complex. This is not associated with immune memory.

• Hemorrhage, bruising, brain, nose bleeds, stools

• Drugs heparin, quinine, antimicrobials, NSAIDs,

penicillin, ranitidine, furosemide

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Platelets Repair Broken Blood Vessel

a. Platelets secrete platelet derived growth factor (PDGF): a growth factor migration and division of vascular endothelial cells, smooth muscle cells, fibroblasts REPAIR OF DAMAGED VASCULAR WALLS b. MEMBRANE ACTIN AND MYOSIN CONTRACT platelet attached to fibrin are retracted CLOT RETRACTS edges of broken blood vessel are pulled together

c. Platelets collected and stored for use in surgeries , transplants and cancer therapy to stop bleeding.

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Prostaglandin and thromboxane synthesis stages1. Membrane Phospholipid Containing a Glycerol Backbone and a Fatty Acid at the First Position, b Arachidonic Acid at the Second Position c Polar Phospholipid Moiety at the Third Position.

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Overview of Cyclooxygenase (COX) PathwaysCellular Phospholipid

Free Arachidonic Acid

Prostaglandin H

Prostaglandins

Phospholipase A2

PG H Synthase

ProstacyclinSynthase

ENDOTHELIAL CELL

Prostacyclins

TX Synthase

Thromboxanes

PLATELET

PG Synthases

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Conversion of Arachidonic Acid to PGD2, PGF2, PGE2

Prostaglandin synthase consists of cyclooxygenase (COX) and peroxidase

COOHO

O

COOH

OOH

O

O

COOH

OH

OH

O

COOH

OH

OH

OH

COOH

OH

OH

O

COOH

OH

Arachidonate

CYCLOOXYGENASE

2O2

AspirinIndomethacin

Ibuprofen

PGG2

PEROXIDASE

Drugs, Carcinogens

Oxidised (Toxic)*

*

PGH2

PGE2

PGF2

PGD2

ISOMERASE

ISOMERASE

REDUCTASE

Cyclooxygenase

_

Peroxidase

Isomerase

Reductase

Isomerase

* NSAID now used as prophylaxis to prevent colon carcinogenesis

Toxin prev. by NSAIDs e.g. Colon cancer

NSAIDs

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Prostaglandin and Prostacyclin and Heart Disease

O

O

COOH

OHPGH2 Prostaglandin H2

Endothelial Cells

COOH

OH

O

PGI2 ProstacyclinCOOH

OH

O

O

Platelets

Prevents Platelet Aggregation

TXA2 Thromboxane A2 COOH

OH

O

Causes Platelet Aggregation

TXB2 Thromboxane B2

H2O

OH

HO

OH

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COX-1 (ALL CELLS) COX-2 (INFLAMMATORY CELLS) Physiological Function – all cells - PG’s cytoprotective (stomach, kidney) - TXA platelet (clotting function)

Inflammatory cells (also brain) - mediate inflammation and pain - inducible by cytokines, mitogens,

endotoxins (contributes to rheumatoid arthritis)

- promote colon cancer by preventing apoptosis

- causes premature labour Inhibitors - aspirin, NSAIDS, antithrombosis drugs but cause gastric/renal lesions

Inhibitors - selective NSAIDS e.g. nimesulide - chemoprevention/colon cancer - antipyretic (knockout mice – infertile

females, no intestinal polyps)

Antiplatelet drugs Cyclooxygenase and inhibitors (NSAIDs)

14 NSAIDSCOX-1 inhibitors bind COX enzyme (prevents PGE formation) GI toxicity as PGE protects intestinal mucosa

1) Aspirin - irreversible - acetylates Ser 530, prevents arachidonic acid binding2) Mefenamate, Ibuprofen - reversible, competitive with fatty acid binding .3) Flurbiprofen - slow binding (salt bridge) competitive inhibition by

binding in the hydrophobic channel4) Indomethacin - non-selective - binds deepest in hydrophobic channel(incr. risk of hypertension, congestive heart failure unlike Celecoxib)

COX-2 inhibitors on the market – much less GI toxicity 4) Celecoxib (celebrex). Vioxx withdrawn

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Role of endothelial cells in regulating blood pressure

andNitric oxide formation, signaling

and toxicity

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1) Angiotensin converting enzyme (inhibited by specific dipeptides) used to lower blood pressure. Captopril 25-50mg (but agranulocytosis risk, cough); Enalapril 1-20 mg; Ramipril 2-20 mg (10mg also for preventing cardiovascular/stroke in diabetics.

Function of Endothelial cells (line vessel walls)

Angiotensin converting enzyme formation and action

Angiotensinogen (411 aa) synthesised in the liver Plasma

renin (synthesised in kidney, a protease)

Angiotensin I in plasma (10aa)

2aa

angiotensin converting enzyme(endothelial cells) (therefore a dipeptidase)

ACE inhibitors

Angiotensin II in plasma (8 aa)

Vasoconstrictor

blood pressure

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Intracellular endothelial cell activity

4) Monoamine oxidase plasma norepinephrine or serotonin

5) Thromboplastin synthesis and secretion (activated state) initiate blood clotting (extrinsic pathway)

6) Synthesis and secrete plasminogen activators (resting state) initiates clot fibrinolysis

Endothelial cells (line vessel walls)

2) ATPase and 5’-nucleotidase degrade ATP and ADP

3) Inactivate prostaglandins E and F and leukotrienes C4 and D4

Cell surface extracellular activity – (cont.)

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Synthesis and Secretion of Plasminogen Activators (Resting State) - initiate clot formation and reversed by

clot busters e.g. plasmin & plasminogen activator)Stroke Therapy (Dissolve clots)

Plasminogen incorporated into

clot fibrin

Plasmin(a serine protease)

Fibrin

Plasminogen Activator*(a serine protease)Endothelial cell

CO

Dissolve fibrinTherefore activator

Fibrin-boundplasmin

Fibrinolysis

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Stroke therapy (con’t)

Drug companies are using recombinant DNA technology to makePLASMINOGEN ACTIVATORS to reduce myocardial damagefollowing acute coronary thrombosis (but x 10 expensive and nobetter/safer.

Cigarette smoke CO damages endothelial cellCO Prevents clotting Smoking and CO induce a procoagulant and anti-fibrinolytic state, thepathological substrate of thrombophilia.

One likely mechanism that plays a major role1. Complex Ca2+ with citrate or oxalate2. Heparin* helps remove thrombin (produced by mast cells)

20Endothelial cells (con’t)

7) PROSTACYCLIN* synthesis and release (resting state) (PGI2) prevents platelet aggregation

phospholipidarachidonic

acid PGG2 PGI2

phospholipase A2PGI2 synthetaseprostaglandin

synthetase(cyclooxygenase)

Therefore endothelial cells function to prevent thrombosisProstacyclin synthase inhib. by COX2 inhibitor e.g. rofecoxib.

8) EDRF (i.e. nitric oxide, a vasodilator) formed from arginine

Plasma Membrane Activity

21Nitric Oxide (NO) = Endothelial Derived Relaxing Factor

NO The endothelium (inner lining) of blood vessels uses nitric oxide to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow.

Nitric oxide is a chemical radical thus highly reactive and has a lifetime of a few seconds - yet diffuses freely across membranes.

Endothelial cell regulation of blood pressurei.e. effect on smooth muscle cells

22Nitric Oxide Synthases (NOS)

Arginine (100 uM in blood, 2 mM in endothelial cells) - NO synthesized from citrulline

Science 278, 425-431 (1997).

H2N CH C

CH2

OH

O

CH2

CH2

NH

C

NH2

NH

H2N CH C

CH2

OH

O

CH2

CH2

NH

C

NH2

N

H2N CH C

CH2

OH

O

CH2

CH2

NH

C

NH2

O

NOS NOS

OH

NADPH NADP+

O2 H2O

NADPH NADP+1/21/2

O2 H2O

Analogous to"P450 + reductase"

L-Arginine N-OH Arginine L-Citrulline

+ NO

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CH2ONO2

CHONO2

CH2ONO2

LiverNADPH microsomes

via reduced P450

NO2reduced by mitochondria NO

CH2OH

CHONO2

CH2ONO2

CH2ONO2

CH2OH

CH2ONO2

Glycerol

glucuronidatedurine

CO2

GSH GSHtransferase GSNO GSH

GSSG

NO

Vascular smooth musclecells and endothelial cells

Drugs that act by Generating Nitric Oxide (NO.)1) NITROGLYCERIN – anti-anginal, anti-hypertensive, vasodilator

Adverse effect: oxidative stress , HEADACHE unless tolerant Tolerance because vascular ALDH in mitochondria reduces NG but is inactivated.by peroxynitrite formed when NO reacts with O2

(formed when NO inhibits respiratory chain) (J.Clin.Invest.113,482-9(2004);J.Am.Coll.Cardiol.57,93-8(2011). Tolerance reversed antioxidants atorvastatin,carvediol,thiols,hydralazine,,HMG-CoA red.inhib.

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“AMYL” NITRITE (i.e. isopentyl nitrite) - potent vasodilators which are inhaled

+ GSH transferase (GST 4-4)

CHCH2CH2H3C

H3C

ON O mitochondria

or P450 reductase-ONO

CHCH2CH2OHH3C

H3C

NOAmyl nitrite

GSHGSNO

GSH or reducingsystem

Nitrite anionradical

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Amyl nitrite (con’t)

A) Prescription drug In glass ampules enclosed in mesh - crushed in the fingers = popping sound. “Poppers” - inhaled to relieve angina.

B) Recreational inhalant (“locker room”) - Butyl nitrite aphrodisiac – used for enhancing sexual pleasure as nitrite induces vasodilation of the rectal mucosal muscles. but causes acute hemolysis if G6PDH deficient J.of Toxicol-Clin Toxicol.42,313-6(2004);J. Neuroimmunol.83, 157-61 (98)

C) Cyanide antidote - nitrite oxidises oxyhemoglobin to met- hemoglobin (Poison control centre) and then Fe3+ of methemoglobin complexes cyanide.