The Role of platelets aggregation tests in the diagnosis of clopidogril

resistance rate in patients undergoing pecutaneous coronary

intervension

Introduction:

Platelets play a key role in the pathophysiology of thrombosis after plaque

rupture (1) .Plaque rupture occurs spontaneously in patients with acute coronary

syndromes (ACS), or may be iatrogenically induced in patients undergoing

percutaneous coronary interventions (PCI).significant reduction in such vascular

events in high risk patients with coronary artery diseases or strokes has been

achieved using antiplatelet therapy (2) .

Clopidogrel is a potent antiplatelet drug that is effective in reducing the risk

of vascular events in patients with established vascular disease (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 non-

responsiveness 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

Adenosine diphosphate plays an important role in platelet activation and

aggregation (14,15). Upon damaged or disrupted endothelium, circulating platelets

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). After adhesion, these anchored platelets undergo

conformational changes through the action of several extrinsic activators such as

collagen, thrombin, and epinephrine.Once activated, platelets release the contents

of their dense (platelet agonists such as ADP and serotonin) and alpha-granules

(fibrinogen, von Willebrand factor, other adhesive proteins, proinflammatory

factors, and prothrombotic factors), which trigger platelet-activating intracellular

signals in surrounding platelets. Activated platelets also synthesize and release

thromboxane A2 in circulation. Activated and degranulated platelets expose

glycoprotein (GP) IIb/IIIa receptors at their surface allowing fibrinogen binding,

which forms bridges between adjacent activated platelets causing platelet

aggregation. In addition, the release of granule contents amplifies the coagulation

and inflammatory processes.

ADP and its receptor. Adenosine diphosphate binds to neighboring

platelets through two G-protein coupled receptors (P2Y1 and P2Y12) and the

cation channel-coupled P2X1 receptor (17) (Fig. 1).

The activation of P2X1 receptor mediates a rapid transient calcium ion

influx in platelets but does not play a major role in platelet aggregation (17).

Stimulation of the Gq-coupled P2Y1 receptor activates phospholipase C and

induces a transient rise in cytosolic calcium resulting in a platelet conformational

change and in weak, transient platelet aggregation (18).

2

Activation of the Gi-coupled P2Y12 receptor by ADP liberates the Gi

protein subunits αGi and βγ, which couple to independent signaling events and

lead to a sustained platelet aggregation (19).

The subunit αGi decreases the platelet cyclic adenosine monophosphate

(cAMP) level through the inhibition of adenylyl cyclase. This decrease in cAMP

production leads, in turn, to a reduction in the activation of specific protein

kinases,which can no longer phosphorylate the vasodilator stimulated

phosphoprotein (VASP); VASP phosphorylation is crucial for GP IIb/IIIa receptor

inhibition (19).

The subunit βγ activates the phosphatidylinositol 3-kinase, which is an

important signaling molecule for P2Y12-mediated platelet-dense granule secretion

and GP IIb/IIIa receptor activation (20). Signaling events downstream of the P2Y12

receptor mediate thromboxane A2 production, α-granule release, and subsequent

expression of P-selectin on activated platelets (21). The P2Y12 receptor is also

involved in thrombus growth and stability (20). Stimulation of both P2Y1 and

P2Y12 receptors is required to cause ADP-induced platelet aggregation (17).

3

Figure 1: Mechanism of action of clopidogrel.

Platelets in the pathogenesis of atherothrombosis:

The complicated pathophysiological procedure underlying acute thrombotic

episodes includes interactions between platelets, plaque components and

coagulation factors (22,23) Figure 2.

The rupture of the intima of acoronary artery, following a plaque rupture or

a iatrogenic injury during PCI, exposes the sub-endothelial elements, such as von

Willebrand factor and collagen,to the bloodstream. Specific receptors on the

4

cAMP= Cyclic adenosine monophosphateVASP=Vasodilator stimulated phosphoprotienDotted arrows indicate inhibitionSolid arrows represent activation

CYP450= Cytochrome P450PGE1 =Prostaglandin E1PKA =Protien Kinase activationPLC= Phospholipase CPI3K= Phosphatidylinositol 3-KinaseAc= adenylyl cyclase

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 αIIbβ3 (GPIIb/IIIa). Activated

platelets release biologically active substances, stored inside the cell or newly

synthesised,among them adenosine diphosphate (ADP), arachidonic acid, platelet-

activating 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 sub-

endothelium and are transformed into macrophages and foam cells.

Several adhesion molecules (P-selectin, ICAM-1) and chemokines (MCP-1,

SDF-1, IL1β, IL- 8, CD40L, RANTES, ENA-78, etc.) participate in these

intercellular interactions and enhance the inflammation in the arterial wall(28).

5

Apart from monocytes,endothelial progenitor cells (EPCs) are also recruited

by the activated platelets and have the potential to transform into either foam cells,

promoting atherogenesis, or into endothelial cells, leading to endothelial

regeneration (29).

Figure2: Central role of ADP-P2Y12 Receptor Interaction In Platelet Activation And

Aggregation.

Mechanism of action and metabolism of Clopidogrel:

Clopidogrel is a prodrug that requires hepatic conversion into an active

metabolite to exert its antiplatelet response .Most of absorbed clopidogrel (85% to

90%) is hydrolyzed by carboxylase to an inactive carboxylic acid

metabolite,SR26334, whereas the remaining 10% to 15% is rapidly metabolized by

hepatic cytochrome (CYP) P450 isoenzymes in a 2-step process ( Fig. 3).

6

In the first step, the thiophene ring of clopidogrel is oxidized to 2-oxo-

clopidogrel, which is then hydrolyzed to a highly labile active metabolite, R-

130964, 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 activation-

aggregation 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).

7

Figure 3:Clopidogrel response variability is a pharmacokinetic problem primarily influenced by the activity of cytochrome P450 isoenzymes in the generation of the active metabolite.Absorption may be affected by polymorphism of the ABCB1 gene.The activity of hepatic cytochrome isoenzymes are influenced by drug-drug interactions, single nucleotide polymorphisms, and environmental influences (smoking).

Pharmacokinetic and Pharmacodynamic properties of Clopidegrel:

Clopidogrel is a member of the thienopyridine family, along with

ticlopidyne and prasugrel, and is a powerful antiplatelet agent (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.

8

The maximum concentration of the active metabolite in blood is reached

within 1 hour after administration of 600 mg clopidogrel (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). This active metabolite is a potent selective inhibitor of

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).

Clopidogrel resistance — definition

No single receptor signaling pathway mediating platelet activation is

responsible for all thrombotic complications. Therefore, a single treatment strategy

directed against a specific receptor cannot overcome all thrombotic complications.

With this in mind, it is our opinion that the optimal definition of resistance or non

responsiveness to an antiplatelet agent is the failure of the antiplatelet agent to

inhibit the target of its action. The identification of resistance would therefore

9

utilize a laboratory technique that detects residual activity of the target. Therefore,

clopidogrel resistance is best demonstrated by evidence of residual post-treatment

P2Y12 activity by measuring ADP-induced platelet aggregation before and after

treatment. Since thrombosis involves multiple signaling pathways, treatment

failure is not synonymous with drug resistance (47).

Aim of the study:

To estimate the incidence of clopidogrel resistance among Kurdish

population and to find the predictors of its resistance.

Materials and methods:

This is a prospective study, 100 patients planned to undergo

elective therapeutic percutaneous coronary intervention (PCI) admitted

in the coronary care unit (CCU) will be included in this study. All

patient will receive a loading dose of 300mg of clopidogrel "Plavix®" 12

hours before the PCI and after that will receive a maintenance dose of

75mg of clopidogrel. The patients are already on maintaining dose of

100mg of aspirin. Inform consent will be taken from indicated patients.

The first blood sample will be withdrawn for analysis of platelet

function before clopidogrel intake and the second sample will be

withdrawn 24 hours after PCI (i.e the patient received 300mg loading

dose and 75mg maintaining dose of clopidogrel) (48,49).

10

Platelet aggregation will be tested using the Multiplate analyzer®

(Verum Diagnostica GmbH, Munich, Germany) in the hematology

department in the central laboratory.

The following instruments will be required:

1. Hirudin containing test tubes.

2. Prewarmed dilute tubes.

3. Agonists reagent (adenosine diphosphate (ADP), arachidonic acid

(ASPI), thrombin receptor activating peptide (TRAP).

4. Multiplate test cell

5. Multiplate analyzer

Estimated cost: 12,500,000 ID

Procedure:

11

Whole blood impedance aggregometry will be carried out using the novel

multiplate® analyzer (Dynabyte medical, Munich, Germany) (50,51). The instrument

analyzes platelet function in whole blood at 37 °C by the attachment of platelets

onto metal electrodes, leading to a change of the electrical conductivity (or

impedance), which is continuously recorded. The Multiplate® instrument is an

improvement of impedance aggregometry using a computer-controlled 5-channel

device and disposable test cells with a dual sensor unit allowing duplicate analyses

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 μM (TRAP test),

adenosine- 5-diphosphate (ADP) with a final concentration of 6.4 μM (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.

12

Multiplate- sensitivity to antiplatelet drugs:

Tests Sensitivity

Clopidegrel Aspirin

ADP Positive + Negative

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:

13

Date …. /…/….20 procedure:

…………………..

Mobile number: ………………….

Name: ……………… ………..…… …………..

Age: ………… years ……… Months

Sex: …………………………

Residence:

City Center: ……………

District: ………… / Town: ….…. /Sub district: …..…….. /

Village: ……

Ethnicity: Kurdish………… Arabic ………… Turkish ………..

others………….

Waist circumference …………..cm Ht……..cm Wt………..kg

Lab tests

CBC:

Hb:……. gm/dl WBC ………. Platelet count ………

Lipid profile : 14

s.cholestrol …………mg/dl

s.triglyceride…………mg/dl s.HDL…….mg/dl s.VLDL…………mg/dl

s.LDL…………..mg/dl

B.sugar ………….mg/dl Bl.urea ……..mg/dl

S.creatinine………..mg/dl

Viral screen :

HBs ……..

HCV……..

HIV………

Clinical history:

Diabetes mellitus ……….. hypertention …………. Smoking……….

cigarette/day ……… duration ………years.

Drug history :

Lipid lower agent…….

Proton pump inhibitor………

NSAID………

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