Faculty Bibliography

OBJECTIVES/OBJECTIVE:The aim of this study was to evaluate the relationship between reticulated platelets (RPs), platelet size, and platelet function in patients with stable coronary artery disease (CAD) taking aspirin and clopidogrel. BACKGROUND:Reticulated platelets are young platelets that are larger and possibly more active than non-RPs. METHODS:Flow cytometry was used to measure RPs after staining with thiazole orange and to define the upper 20% and lower 20% of platelets by size. Platelet aggregation was measured with light transmission aggregometry (LTA); platelet activation was assessed by measuring activated platelet surface expression of P-selectin and glycoprotein (GP) IIb/IIIa. RESULTS:Ninety patients were recruited and stratified into tertiles of %RPs. Patients in the upper tertile displayed greater platelet aggregation to 5-mumol/l adenosine diphosphate (ADP) (50.7 +/- 16.4% vs. 34.2 +/- 17.3%, p < 0.001), 1.5-mmol/l arachidonic acid (AA) (27.3 +/- 16.9% vs. 11.7 +/- 9.3%, p < 0.001), and 1-mug/ml collagen (18 +/- 11.6% vs. 12.1 +/- 8.7%, p < 0.05) and greater expression of GP IIb/IIIa (4.7 +/- 1.8% vs. 3.1 +/- 2.2%, p < 0.001). Frequency of low response to aspirin (AA LTA >20%) was higher in the upper tertile (53% vs. 17%, p < 0.001) compared with the lower tertile; low response to clopidogrel (ADP LTA >50%) was also elevated in the upper tertile (50% vs. 13%, p = 0.003). The larger platelet gate had a higher % of RPs compared with the smaller gate (15.4 +/- 16.7% vs. 1.7 +/- 2.3%, p < 0.001) and greater GP IIb/IIIa (5.7 +/- 3.1 vs. 2.1 +/- 1.2, p < 0.001) and P-selectin expression (7.8 +/- 4.9 vs. 4.6 +/- 2.7, p < 0.001). CONCLUSIONS:The proportion of circulating RPs strongly correlates with response to antiplatelet therapy in patients with stable CAD. Large platelets exhibit increased reactivity despite dual antiplatelet therapy, compared with smaller platelets.

BACKGROUND:Clopidogrel inhibits the platelet P2Y12 receptor, leading to increased intracellular cyclic AMP (cAMP) levels. Caffeine also causes a rise in platelet cAMP. We aimed to test the effect of acute caffeine administration on platelet inhibition by clopidogrel, in healthy volunteers and patients with coronary artery disease. METHODS:Cohort 1: 12 healthy subjects were enrolled in a 2-week crossover study. Blood samples were drawn at baseline, 2, 4, and 24 hours after 300 mg clopidogrel intake. At the first week, 6 subjects received caffeine (300 mg pill, equivalent to a medium sized coffee drink) 30 minutes after clopidogrel. At week 2, the other 6 subjects received caffeine. One month later the effect of caffeine alone was tested. Platelet function was evaluated by aggregation in response to 5, 10, and 20 micromol/L adenosine diphosphate, 1 microg/mL collagen, and flow cytometric determination of P-selectin expression, PAC-1 binding, and vasodilator-stimulated phosphoprotein phosphorylation. Cohort 2: 40 patients with coronary artery disease receiving aspirin and clopidogrel (75 mg daily) for > or = 1 week were tested at baseline and 2.5 hours after caffeine (300 mg). RESULTS:In cohort 1 (crossover study), caffeine was associated with lower adenosine diphosphate-induced aggregation at 4 hours, lower activation markers at 2 hours, and lower vasodilator-stimulated phosphoprotein phosphorylation at 4 hours after clopidogrel. Caffeine alone had no effect on the assessed platelet surface biomarkers. In cohort 2, caffeine administration was associated with lower platelet activation markers (P-selectin, PAC-1 binding), without significant effect on aggregation. CONCLUSIONS:Acute caffeine administration after clopidogrel loading appears to be associated with enhanced platelet inhibition 2 to 4 hours after clopidogrel intake. The mechanism probably involves synergistic increase in cAMP levels.