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Introduction:
Platelets play a key role in the pathophysiology of thrombosis after plaque
rupture
(1)
(3-5)
.In addition, it
potentiates the effect of aspirin in reducing ischemic and vascular events in the
setting of cardiovascular diseases (6-8). However, a substantial percentage of patients
with Cardiovascular Disease (CAD) showed low or no response to clopidogrel
therapy (9-11) .
The term Clopidogrel Resistance has been used to denote nonresponsiveness of Adenosine Di-Phophatase ADP induced platelet aggregation
following standard clopidogrel therapy(12). Several factors were suggested to
explain low response to clopidogrel; including genetic polymorphisms, cellular
factors e.g. accelerated platelet turnover and drug-drug interaction (13) .
Platelet Aggregation:
1
(14,15)
adhere to the vessel wall through interactions with the subendothelium constituents
(collagen, von Willebrand factor, and other adhesive proteins such as fibronectin,
laminin, and vitronectin)
(15,16)
can
no
longer
phosphorylate
the
vasodilator
stimulated
(20)
(20)
(21)
surface of the platelets (GPIa/IIa, GPIb/V/IX, GPVI, etc.) bind the former
molecules, causing the platelet to adhere to the site of the endothelial injury (23,24).
Platelet adhesion leads to activation of the cell through intracellular
metabolic cascades. As a result,platelets aggregate through fibrinogen bridges,
which bind to the activated platelet receptor-integrin IIb3 (GPIIb/IIIa). Activated
platelets release biologically active substances, stored inside the cell or newly
synthesised,among them adenosine diphosphate (ADP), arachidonic acid, plateletactivating factor (PAF) and serotonin, which induce and preserve platelet
activation and aggregation through positive feedback mechanisms(24,25).
The secretion of pre-coagulant factors from the platelets (e.g. Factor V) and
the interaction with the negatively charged phospholipids of the platelet membrane
maximise the reaction of thrombin synthesis,which has been initiated by the
intravascular exposure of tissue factor and is one of the most powerful platelet
activators. These mechanisms may partly explain the recurrence of thrombotic
episodes in patients already on antiplatelet medication, and justify the need for this
type of drug in cases of acute ischaemic events(26,27) .
Apart from the established importance of platelet actions in the thrombotic
procedure, they play a significant role in the formation of the atheromatous plaque.
According to recent reports, they adhere to the endothelium under mild
inflammatory conditions and attract monocytes, which penetrate the subendothelium and are transformed into macrophages and foam cells.
Several adhesion molecules (P-selectin, ICAM-1) and chemokines (MCP-1,
SDF-1, IL1, IL- 8, CD40L, RANTES, ENA-78, etc.) participate in these
intercellular interactions and enhance the inflammation in the arterial wall(28).
5
is
hydrolyzed
by
carboxylase
to
an
inactive
carboxylic
acid
In the first step, the thiophene ring of clopidogrel is oxidized to 2-oxoclopidogrel, which is then hydrolyzed to a highly labile active metabolite, R130964, which has both carboxylic acid and thiol groups (30-32).
Recent studies indicate that CYP2C19, CYP1A2, and CYP2B6 participate in
the first metabolic step, whereas CYP2C19, CYP2C9, CYP2B6, and CYP3A are
responsible for the second step (30,31) (Fig. 3).
The highly unstable active metabolite, R-130964, covalently binds to
platelet P2Y12 receptor specifically and irreversibly during passage through the
hepatic circulation resulting in inhibition of ADP-induced platelet activationaggregation for the life span of the platelet (33) (Fig 1).
This metabolic activation scheme is consistent with the time-dependent
cumulative inhibition of ADP-induced platelet aggregation as observed with
repeated daily dosing of clopidogrel and is further highlighted by slow recovery of
platelet function following drug withdrawal (34-36).
Multiple lines of evidence strongly suggest that variable and insufficient
active metabolite generation are the primary explanations for clopidogrel response
variability and nonresponsiveness, respectively (37).
Variable levels of active metabolite generation following clopidogrel
administration could be explained by:
1. Variable or limited intestinal absorption, which may be affected by an ABCB1
gene polymorphism (38-40).
2. Functional variability in P450 isoenzyme activity influenced by drug-drug
interactions as well as other factors.
3. Single nucleotide polymorphisms of specific genes encoding CYP450
isoenzymes (41,42).
(43)
. It is a product, which is
absorbed in the gut with the aid of the ABCB1/MDR1 protein transporter.
Subsequently, it is converted to the active metabolite by several isoforms of
cytochrome P450 in the liver, mainly CYP2C19. CYP3A4, CYP3A5,CYP1A2,
CYP2B6 and CYP2C9 also participate in the procedure.
The maximum concentration of the active metabolite in blood is reached
within 1 hour after administration of 600 mg clopidogrel
8
(44)
. interestingly, 85% of
the absorbed drug is hydrolysed by plasma and intestinal mucosa esterases to form
inactive product. The half-life of the active metabolite after a single or multiple
doses is about 8 hours
(45)
the P2Y12ADP receptor, which exerts its action by forming disulphuric bonds with
2 serine residues (ser-17, ser-270) of the receptor molecule.
The maximum inhibitory activity of clopidogrel is reached in 24 hours after
administration of 75 mg, in six hours after 300 mg, and in two hours after 600 mg.
as the P2Y12 receptor blockade is irreversible, and 10% of platelets are renewed
daily, at 5 days after treatment cessation 50% of the circulating platelets will be
completely functional and capable of producing adequate hemostasis(46).
Procedure:
Whole blood impedance aggregometry will be carried out using the novel
multiplate analyzer (Dynabyte medical, Munich, Germany)
(50,51)
. The instrument
with each test. Aggregation is recorded for 6 min. Results are expressed in arbitrary
units. Parameters are the aggregation (maximal aggregation), velocity (steepness of
the curve), and the area under curve (AUC). The application of different activators
facilitates to study, e.g., the effect of several platelet active drugs.
In the present study, thrombin inhibitor(TI)-blood samples (3 ml withdrawn
in a hirudin anticoagulant containing test tube) will be used for the Multiplate
analyses which should be carried out within 30-180 min after blood sampling in
each study participant. The TI-blood diluted with NaCl 0.9% in a 1:1 ratio, and
platelet aggregation determined thereafter in response to stimulation with thrombin
receptor activator peptide 6 with a final concentration of 32 M (TRAP test),
adenosine- 5-diphosphate (ADP) with a final concentration of 6.4 M (ADP
test),and arachidonic acid (ASPI test).Obtained results will be expressed as areas
under the curve (AUC).
The ADP test is used to detect copidogrel response, the ASPI test is for
detection of function of aspirin.
Multiplate- sensitivity to antiplatelet drugs:
Tests
ADP
Sensitivity
Clopidegrel
Aspirin
Positive +
Negative
12
ASPI
Negative
Positive ++
TRAP
Positive/negative
Negative
Laboratory data of complete blood count CBC , fasting blood sugar ,blood
urea ,serum creatinine ,lipid profile (s.cholestrol,s.triglyceride , s.HDL
,s.VLDL,s.LDL) Viral screen (HBs Ag,HCV Ab ,HIV Ab) ,body mass index
(height and weight) ,and clinical history will be taken from patients medical file
according to questionnaire designed for this purpose.
Questionnaire:
Date . //.20
procedure:
..
Mobile number: .
Name:
..
Age: years
..
Months
Sex:
13
Residence:
City Center:
Village:
Ethnicity:
Kurdish
Arabic Turkish ..
others.
Waist circumference ..cm Ht..cm Wt..kg
Lab tests
CBC:
Hb:. gm/dl WBC .
Platelet count
Lipid profile :
s.cholestrol mg/dl
s.triglyceridemg/dl
s.HDL.mg/dl
s.VLDLmg/dl
s.LDL..mg/dl
B.sugar
.mg/dl
Bl.urea
S.creatinine..mg/dl
Viral screen :
14
..mg/dl
HBs ..
HCV..
HIV
Clinical history:
Diabetes mellitus ..
hypertention .
cigarette/day
Smoking.
duration years.
Drug history :
Lipid lower agent.
Proton pump inhibitor
NSAID
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23