Transcript III: Cells Utilizing Oxygen to Form Lipid Regulators and

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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)
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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) a 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 Platelets
Images From: http://www-personal.engin.umich.edu/~tkinzer/
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Mechanism of platelet aggregation to form a clot
Phospholipid
ADP
released
from platelet
granule
receptor
Ca2+
influx
in other platelets
Membrane
Phospholipase
A2 activated
Aspirin
Arachidonate
(drug)
acetylates
cyclooxygenase
2O2
Diacylglycerol (DAG)
+ inositol triphosphate
(IP3)
PLATELET
AGGREGATION
PGH2
TxA2
synthetase
drug?
Phosphatidylinositol
Membrane
Phospholipase C
Granule
release
Thromboxane A2 (TxA2)
receptor
collagen
receptor
COLLAGEN
Contraction
of microtubules
Ca2+ Influx
Fibrin clot formed by zymogen activation cascade
INTRINSIC PATHWAY
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.
Traps aggregated
platelets
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Anticoagulant drugs (stroke treatment prevents new clots) 6
.
• 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).
Anticoagulants Drugs – Vit. K antagonists
Vitamin K
O
O
CH3
O
OH
H
C
H
O
CH3
6
H
OH
O
Dicoumarol
- Spoiled sweet clover
causes fatal
hemorrhagic disease in
cattle.
O
O
Warfarin for thrombosis but high
CYP2C9 polymorphism.
Drug name Coumadin (rat poison)
2-3 days before it works 
DONT admin. again for 3d
O
H
C
OH
O
CH3
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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
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 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.
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Biochemical Structure of Typical Fatty Acids
Incorporated Into Membrane
Phospholipids

PGE1
PGE2
PGE3
Overview of Cyclooxygenase (COX) Pathways
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12
Conversion of Arachidonic Acid to PGD2, PGF2a, PGE2
2O2
O
COOH
COOH
Cyclooxygenase
CYCLOOXYGENASE
Arachidonate
O
PGG2
*
_
Aspirin
Indomethacin
Ibuprofen
Drugs, Carcinogens
Peroxidase
PEROXIDASE
*
O
Isomerase
ISOMERASE
COOH
O
PGH2
O
COOH
OH
PGE2
Oxidised (Toxic)
Toxin prev. by NSAIDs
e.g. Colon cancer
NSAIDs
Prostaglandin synthase
consists of cyclooxygenase
(COX) and peroxidase
OH
OOH
OH
Isomerase
ISOMERASE
OH
COOH
Reductase
REDUCTASE
OH
O
COOH
OH
OH
PGF2a
OH
PGD2
* NSAID now used as prophylaxis to
prevent colon carcinogenesis
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Prostaglandin and Prostacyclin and Heart Disease
OMEGA-3
H3C
Unstaurated
Fatty Acid
Formation
CH3
Essential Fatty Acids
From the diet
OMEGA-6
H3C
CH3
Linoleic Acid
(LA) 18:2
a-Linolenic acid
(LNA)18:3
Delta-6Desaturase
Stearidonic Acid
(SDA)
-Linolenic Acid
(GLA)
Elongase
Eicosatetraenoic
acid
Dihomogamma
Linolenic acid
(DGLA)
Delta-5Desaturase
Eicosapentaenoic
Acid
(EPA)
Series 3
Prostaglandins
Arachidonic
Acid
Series 1
Prostaglandins
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Series 2
Prostaglandins
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Antiplatelet drugs
Cyclooxygenase (COX) and COX inhibitors (NSAIDs)
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)
Colon cancer prevented by low daily
doses of aspirin or COX inhib.
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• 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)
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
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Role of endothelial cells in
regulating blood pressure
and
Nitric oxide formation, signaling
and toxicity
Function of Endothelial cells (line vessel walls)
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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 2.5-20 mg (10mg also for
preventing cardiovascular/stroke in diabetics.
Angiotensin converting enzyme formation and action
Angiotensinogen (411 aa) synthesised in the liver
Plasma
renin (synthesised in
kidney, a protease)
ACE inhibitors
Angiotensin II in plasma (8 aa)
angiotensin converting enzyme
(endothelial cells)
(therefore a dipeptidase)
Angiotensin I in plasma (10aa)
2aa
Vasoconstrictor
blood pressure
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Endothelial cells (line vessel walls)
Cell surface extracellular activity – (cont.)
2) ATPase and 5’-nucleotidase degrade ATP and ADP
3) Inactivate prostaglandins E and F and leukotrienes C4 and D4
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
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Synthesis and Secretion of Plasminogen Activators
(Resting State) initiate Clot Formation (reversed by clot
busters” e.g.plasmin & plasminogen activator)
Stroke Therapy (Dissolve clots)
Fibrin
Plasminogen
incorporated into
clot fibrin
Plasmin
(a serine protease)
Plasminogen Activator*
(a serine protease)
Endothelial cell
CO
CO?
Dissolve fibrin
Therefore activator
Fibrin-bound
plasmin
Fibrinolysis
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 Ca2+ with citrate or oxalate
2. Heparin* helps remove thrombin (produced by mast cells)
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Endothelial cells (con’t)
Plasma Membrane Activity
7) PROSTACYCLIN* synthesis and release (resting state) (PGI2)
prevents platelet aggregation
arachidonic
phospholipid
PGG2
PGI2
acid
phospholipase A2
prostaglandin
synthetase
(cyclooxygenase)
PGI2 synthetase
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
EDRF - Endothelial Derived Relaxing Factor (NO).
Endothelial cell regulation of blood pressure
i.e. effect on smooth muscle cells
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!
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Nitric Oxide Synthases (NOS)
O
H2N
C
H
C
O
OH
H2N
C
H
C
O
OH
H2N
C
H2
C
H2
NADPH
C
H2
NADP
+
CH C
OH
CH
2
1 NADP
H
1
NADP+
C
H2
/
CH
2
+
C
H2
O2
N
H
C
N
H2
C
H2
NOS
H2O
A
"
O2
N
H
C
NH
N
H2
CH
2
NOS
H2O
N OH
NH
C
n
P
NO
O
NH
2
Arginine
(100 uM in blood, 2 mM
cells) - NOL
synthesized -from
Nin endothelial
L
citrulline
Science 278, 425-431 (1997).
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4)
Drugs that act by Generating Nitric Oxide
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(NO.)
1) NITROGLYCERIN - antianginal, antihypertensive, vasodilator
NO
2
L
CH
NO
2O
2
r
N
O
CH2O
H
N
CH
O
NO
2
v
CH
NO
2O
2
CHO
NO
2
CH2O
NO
2
iO
C
H2O
N
2
A
C
HO
H
i2
C
H2O
N
O
2
G
g
G
t
G
G
S
u
r
S
V
a
G
cAdverse effect: oxidative stress , HEADACHE unless
e 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).
N
O
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 (Roberts)
H3
C
C
H
H
3C
m
NO
O
H
C
3
o
C
H
C
H
H
2C
2
H
3C
A
-O
N
O
N
r
m
+ GSH transferase
(GST 4-4)
G
G
G
s
r
NO
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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 methemoglobin (Poison control centre) and then Fe3+ of methemoglobin
complexes cyanide.