Dual Antiplatelet Therapy in ACS: Time-Dependent Variability in Platelet
Aggregation During the First Week
Santanu Guha.*, Partha Sardar. (Post-Graduate Trainee)**, Pradipta Guha. (Post-Graduate Trainee)**,
Suryyani Deb. SRF ****, Rathindranath Karmakar. Senior Resident*, Prantar Chakraborti. Asst
Professor ***, Soura Mookerjee, Asst Professor *, P. K.Deb *****, Rajib De DM Resident***, Arnab
Dutta ,RMO*, Utpal Chaudhuri . ***
* Department of Cardiology, Medical College, Kolkata, India
**Department of Medicine, Medical College, Kolkata, India
*** Institute of Hematology & Transfusion Medicine, Medical College, Kolkata, India
****Department of Biochemistry, Calcutta University
*****ESI Hospital, Kolkata

recruited from consecutive patients presenting within 12 hours of onset of symptoms, admitted to the Cardiology ward and ICCU of Medical College, Kolkata. The study was reviewed and approved by the Institutional Ethical Committee. All patients were >21 years old and received both aspirin and
clopidogrel (325 mg loading dose and 150 mg of maintenance dose of aspirin and 300 mg loading dose and 75 mg maintenance dose of clopidogrel). Exclusion criteria were use of nonsteroidal anti-inflammatory drugs, family or personal history of bleeding disorders, platelet count
<150X103/mL or >450 x103/mL. Forty-one ST elevated acute coronary syndrome (STEACS) patients and twentythree Non ST elevated acute coronary syndrome (NSTEACS) patients made up a total of sixty four ACS patients. Blood samples Blood samples for platelet function assays were collected from an antecubital vein using a 21-gauge needle 2 to 4 hours
after antiplatelet therapy intake. The first 2 to 4 mL of blood was discarded to avoid spontaneous platelet activation. Blood samples were collected in 3.2% citrated plasma. Platelet Function Analysis Platelet aggregometric study was done at IHTM, Medical College, Kolkata. Platelet aggregation with 10mM epinephrine, 2mg/ml collagen and 10mM ADP was performed
with light transmittance aggregometry in all patients according to a standard protocol.3 Briefly, platelet rich plasma (PRP) was obtained after centrifuging blood at 200g for 15 min. Platelet poor plasma (PPP) served as an appropriate blank and it was obtained by centrifugation of blood at 1500g for
15 min. Platelet aggregation induced by Collagen and Epinephrine was measured by standard aggregometric technique based on optical density in an aggregometer (Chrono-Log, USA, Model 530BS). Platelet aggregation
was defined as the difference in light transmission measured in PPP and PRP. Agonist (epinephrine, collagen, ADP) induced aggregation was studied initially at 48 hours and also at 7th day after initiation of anti-platelet therapy2. Statistical Analysis Normally distributed continuous variables are presented as mean+/-SD. Variables have been analyzed for a normal
distribution with the Kolmogorov-Smirnov test. Categorical variables are expressed as frequencies and percentages. Differences between groups were assessed with the Fisher exact test for categorical variables. Unpaired t tests were

used for comparison of normally distributed continuous variables between the 2 groups. p<0.05 was considered statistically significant. Statistical analysis was performed with SPSS v10.0 software (SPSS Inc. Chicago). RESULTS Platelet function after dual antiplatelet therapy with aspirin and clopidogrel was analyzed in all 64 patients by conventional aggregometry. Demographic parameters, risk factors, medications used and left ventricular function of the patients are depicted in table 1. Majority of patients had one or more risk factors. Diabetes Mellitus was present in 59.4% of cases. Most of the patients were hypertensive (56.2%). 43.7% of the patients were smokers (including 16 females) and elevated LDL (LDL>100mg/dL) was found in 46.9% cases. Platelet aggregation study with 10μM of epinephrine 48 hours and 7 days after initiation of dual antiplatelet therapy with aspirin and clopidogrel showed a mean aggregation of 52.3±14.2% and 30.4±17.2% respectively (p value < 0.0001) (fig 1). Mean platelet aggregation with ADP(10μM) and collagen (2μg/ml) also showed a higher mean aggregation initially and a significant decrease after 7 days of antiplatelet treatment (71.2±8.3% to 47.3±12.4% and 48.2±16.2% to 22.7±11.3% respectively). Considering the risk factors, the diabetics showed an increased initial aggregation in response to epinephrine when compared to non-diabetics. There was no difference in the initial aggregation pattern among those with hypertension and elevated LDL. The initial aggregation pattern was higher in smokers than in non smokers although this difference did not reach a statistically significant level.
DISCUSSION
Platelet activation results from the combined action of several agonists that bind to their respective membrane receptors on adherent platelets, mobilize intracellular calcium, and transmit platelet-activating intracellular signals5. These platelet stimuli include humoral mediators in plasma (e.g., epinephrine, thrombin), mediators released from activated cells (e.g., ADP, serotonin), and vessel wall extracellular matrix constituents that come in contact with adherent platelets (e.g., collagen, vWF). Several of these stimuli can synergistically activate platelets and may also act in concert with shear forces that platelets encounter simultaneously2. Activated platelets synthesize de novo and release the potent platelet activator and vasoconstrictor TxA2, the major

cyclooxygenase product of arachidonic acid metabolism in platelets. The clinical effects of blocking TxA2 and ADP induced activation are well known. The present study was designed to gain an insight into the pathophysiological aspects occurring during this initial period of ACS in patients who are under the therapeutic regime of dual antplatelet therapy. Catecholamine-induced changes of platelet response have been reported to be involved in the mechanism linking stress, sympathoadrenal hyperactivation and cardiovascular disease6. Stress commonly increases the adrenaline levels to 1-2nmol/ L, but in other conditions of sympathetic activation, such as shock, myocardial infarction, intense physical activity, catecholamine levels can even reach upto 5-10 nmol/L 7-10. However, a sudden surge in the level of catecholamines can potentiate the aggregation mediated by other agonists like ADP and arachidonic acid by increasing the sensitivity of the platelets to such agonists. The presence of even a small concentration of ADP and/or arachidonic acid may activate platelets in such situations. The documented decrease in number of adrenoceptors immediately after myocardial infarction, as a consequence of the raised levels of catecholamines11 perhaps leads more credence to the fact that there indeed is a catecholamine surge during such episodes. In addition to circulating catecholamines, recent studies have underlined the importance of intraplatelet catecholamines in the control of platelet responses. Intracellular noradrenaline content is directly correlated with platelet sensitivity to collagen and negatively correlated with the sensitivity to the inhibitory effects of prostacycline12,13 Epinephrine induced increase in platelet aggregation has been found to be concentration dependent14. It has also been proven that there is a direct correlation between the plasma concentration of adrenaline early in the course of acute myocardial infarction
and the size of the infarct determined by enzyme release15. There are certain difficulties in interpreting results of the studies of platelet aggregation in patients with Acute Coronary Syndrome. In vitro tests of platelet function may not truly reflect the situation in vivo, because of artefacts implicit in the formulation of platelet rich plasma (PRP)16, and because metabolic alterations in other blood components may also modulate platelet aggregation in these situations12. Some studies of platelet aggregation after myocardial infarction have demonstrated platelet hypoaggregability13,17, which may result either from dissociation of circulating platelet aggregates during preparation of platelet rich plasma, or
from in vivo consumption of the more active platelets.