1. 1 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 acids iii.Nutritional approach to prevent heart disease iv.COX Inhibitors (NSAIDs) (antiplatelet drugs) B)Endothelial cell functions: Prostacyclin and plasminogen activator formation Role of endothelial cells in regulating blood pressure: - Angiotensin and EDRF Nitric oxide formation, signaling and toxicity: - Use of Nitric Oxide generators: (vasodilator / antihypertensive drugs)

2. 2 Platelets t1/2 = 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

3. 3 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 plateletsActivated Platelets Images From: http://www-personal.engin.umich.edu/~tkinzer/

4. 4 Mechanism of platelet aggregation to form a clot ADP released from platelet granule receptor Ca 2 + influx in other platelets Membrane Phospholipase A 2 activated Phospholipid Arachidonate cyclooxygenase acetylates Aspirin (drug) PGH 2 Thromboxane A2 (TxA 2 ) Diacylglycerol (DAG) + inositol triphosphate (IP 3 ) Phosphatidylinositol Membrane Phospholipase C collagen receptor COLLAGEN TxA 2 synthetasedrug? receptor Ca 2 + InfluxContraction of microtubules Granule release PLATELET AGGREGATION 2O 2

5. 5 Modified from Fig. 10.37, Stryer (5 th 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 cascade INTRINSIC PATHWAY Traps aggregated platelets

6. 6 Anticoagulant drugs (stroke treatment prevents new clots). Vitamin K is essential for prothrombin synthesis (and other clotting factors). Abnormal prothrombin is formed (does not bind Ca 2+ ) in the absence of Vit. K or in the presence of Vit. K antagonists (see below). Vitamin K 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

7. 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 7

8. 8 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.

9. 9 Prostaglandin and thromboxane synthesis stages 1. 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.

10. 10 Biochemical Structure of Typical Fatty Acids Incorporated Into Membrane Phospholipids PGE 1 PGE 2 PGE 3 

11. 11 Overview of Cyclooxygenase (COX) Pathways

12. 12 Conversion of Arachidonic Acid to PGD 2, PGF 2 , PGE 2 Prostaglandin synthase consists of cyclooxygenase (COX) and peroxidase * NSAID now used as prophylaxis to prevent colon carcinogenesis Toxin prev. by NSAIDs e.g. Colon cancer NSAIDs

13. 13 Prostaglandin and Prostacyclin and Heart Disease

14. 14 Unstaurated Fatty Acid Formation OMEGA-3OMEGA-6 18:3 18:2

15. 15 Antiplatelet drugs Cyclooxygenase (COX) and COX inhibitors (NSAIDs)

16. Colon cancer prevented by low daily doses of aspirin or COX inhib. Aspirin inhibits COXI which catalyse the peroxidase activation of environmental arylamines to human carcinogens e.g. benzidine,naphthylamine. Azoxymethane rodent colon carcinogen. COX2 activity & extracellular matrix metalloproteinases is induced by inflammation or colorectal cancer (CRC). Inhibitors of these enzymes decrease CRC metastasis Diseases Colon Rectum 51,342-347(2008) 16

17. 17 NSAIDS COX-1 inhibitors bind COX enzyme (prevents PGE formation) GI toxicity as PGE protects intestinal mucosa 1) Aspirin - irreversible - acetylates Ser 530, prevents arachidonic acid binding 2) Mefenamate, Ibuprofen - reversible, competitive with fatty acid binding. 3) Flurbiprofen - slow binding (salt bridge) competitive inhibition by binding in the hydrophobic channel 4) 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

18. 18 Role of endothelial cells in regulating blood pressure and Nitric oxide formation, signaling and toxicity

19. 19 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 2.5-20 mg (10mg also for preventing cardiovascular/stroke in diabetics. Function of Endothelial cells (line vessel walls)

20. 20 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.)

21. 21 Synthesis and Secretion of Plasminogen Activators (Resting State) initiate Clot Formation (reversed by clot busters” e.g.plasmin & plasminogen activator) CO?

22. 22 Stroke therapy (con’t) Drug companies are using recombinant DNA technology to make PLASMINOGEN ACTIVATORS to reduce myocardial damage following acute coronary thrombosis (but x 10 expensive and no better/safer. Cigarette smoke  CO  damages endothelial cell Prevent clotting e.g., stored blood 1. Complex Ca 2+ with citrate or oxalate 2. Heparin* helps remove thrombin (produced by mast cells)

23. 23 Endothelial cells (con’t) 7) PROSTACYCLIN* synthesis and release (resting state) (PGI2) prevents platelet aggregation Therefore endothelial cells function to prevent thrombosis Prostacyclin synthase inhib. by COX2 inhibitor e.g. rofecoxib. 8) EDRF (i.e. nitric oxide, a vasodilator) formed from arginine Plasma Membrane Activity

24. 24 EDRF - Endothelial Derived Relaxing Factor (NO). i.e. acetylcholine relaxes vascular smooth muscle only if endothelial cells are present. ENDOTHELIAL derived RELAXING FACTOR: - unstable and inactivated by oxygen and hemoglobin  Local hormone - indentified as nitric oxide! Endothelial cell regulation of blood pressure i.e. effect on smooth muscle cells

25. 25 Nitric Oxide Synthases (NOS) Arginine (100 uM in blood, 2 mM in endothelial cells) - NO synthesized from citrulline Science 278, 425-431 (1997).

26. 26 4)

27. 27 Drugs that act by Generating Nitric Oxide (NO. ) 1) NITROGLYCERIN - antianginal, antihypertensive, 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 O 2  (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.

28. 28 “AMYL” NITRITE (i.e. isopentyl nitrite) - potent vasodilators which are inhaled (Roberts) + GSH transferase (GST 4-4)

29. 29 Amyl nitrite (con’t) A) Prescription drug (except 1960-1969) 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 Fe 3+ of methemoglobin complexes cyanide.