Eli I. Lev

Role of Reticulated Platelets and Platelet Size Heterogeneity on Platelet Activity After Dual Antiplatelet Therapy With Aspirin and Clopidogrel in Patients With Stable Coronary Artery Disease

OBJECTIVES 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.

Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin.

Background: The mechanisms for the variability in antiplatelet effects of aspirin are unclear. Immature (reticulated) platelets may modulate the antiplatelet effects of aspirin through uninhibited cyclooxygenase (COX)‐1 and COX‐2. Objectives: To evaluate the role of reticulated platelets in the antiplatelet effects of aspirin. Methods: Sixty healthy volunteers had platelet studies performed before and 24 h after a single 325‐mg dose of aspirin. Platelet studies included light transmission aggregometry; P‐selectin and integrin αIIbβ3 expression, and serum thromboxane B2 (TxB2) levels. Reticulated platelets and platelet COX‐2 expression were measured using flow cytometry. Results: Subjects were divided into tertiles based on the percentage of reticulated platelets in whole blood. Baseline platelet aggregation to 1 μg mL−1 collagen, and postaspirin aggregations to 5 μm and 20 μm ADP and collagen, were greater in the upper than in the lower tertile of reticulated platelets. Stimulated P‐selectin and integrin αIIbβ3 expression were also higher in the upper tertile both before and after aspirin. Platelet COX‐2 expression was detected in 12 ± 7% (n = 10) of platelets in the upper tertile, and in 7 ± 3% (n = 12) of platelets in the lower two tertiles (P = 0.03). Postaspirin serum TxB2 levels were higher in the upper (5.5 ± 4 ng mL−1) than in the lower tertile (3.2 ± 2.5 ng mL−1, P = 0.03), and decreased even further with ex vivo additional COX‐1 and COX‐2 inhibition. The incidence of aspirin resistance (≥ 70% platelet aggregation to 5 μm ADP) was significantly higher in the upper tertile (45%) than in the lower tertile (5%, P < 0.0001). Conclusions: Reticulated platelets are associated with diminished antiplatelet effects of aspirin and increased aspirin resistance, possibly because of increased reactivity, and uninhibited COX‐1 and COX‐2 activity.

Blood thrombogenicity in type 2 diabetes mellitus patients is associated with glycemic control

OBJECTIVES This study was designed to determine whether blood thrombogenicity is related to chronic glycemic control in type 2 diabetes mellitus (T2DM). BACKGROUND Type 2 diabetes mellitus is associated with accelerated atherosclerosis and a high rate of arterial thrombotic complications. Whether increased blood thrombogenicity is associated with glycemic control has not been properly tested. METHODS Forty patients with T2DM with hemoglobin A1c (HbA1c) > or =7.5% were selected. Maintaining their current hypoglycemic therapies, patients were randomized into a conservative (diet modification plus placebo) or intensive (diet modification plus troglitazone) hypoglycemic regimen for three months. Blood thrombogenicity was measured at baseline and after three months with the Badimon ex vivo perfusion chamber and assessed as platelet-thrombus formation. The repeated measurements allowed every patient to be his/her own control. RESULTS Patients in both groups (48% and 74% of the conservative and intensive groups, respectively) improved glucose control (HbA1c reduction > or =0.5%), showing a significant decrease in blood thrombogenicity. A significant positive correlation was observed between the reduction in thrombus formation and the reduction in HbA1c (r = 0.47, p < 0.01). The reduction in HbA1c achieved by both treatments was comparable. Patients without glycemic improvement showed no change in blood thrombogenicity. Improved glycemic control was the only significant predictor of a decrease in blood thrombogenicity. CONCLUSIONS In T2DM, there is an association between improved glycemic control and blood thrombogenicity reduction. The effect of glycemic control on the thrombotic complications of T2DM patients deserves further investigation.

In Vivo Plaque Characterization Using Intravascular Ultrasound–Virtual Histology in a Porcine Model of Complex Coronary Lesions

Objective—To determine the accuracy of detection of different tissue types of intravascular ultrasound–virtual histology (IVUS-VH) in a porcine model of complex coronary lesions. Methods and Results—Coronary lesions were induced by injecting liposomes containing human oxidized low-density lipoprotein into the adventitia of the arteries. IVUS-VH imaging was performed in vivo at 8.2±1.6 weeks after injection. A total of 60 vascular lesions were analyzed and compared with their correspondent IVUS-VH images. Correlation analysis was performed using linear regression models. Compared with histology, IVUS-VH correctly identified the presence of fibrous, fibro-fatty, and necrotic tissue in 58.33%, 38.33%, and 38.33% of lesions, respectively. The sensitivity of IVUS-VH for the detection of fibrous, fibro-fatty, and necrotic core tissue was 76.1%, 46%, and 41.1% respectively. A linear regression analysis performed for each individual plaque component did not show strong correlation that would allow significant prediction of individual values. Conclusions—In a porcine model of complex coronary lesions, IVUS-VH was not accurate in detecting the relative amount of specific plaque components within each individual corresponding histological specimen.

Acute Antithrombotic Effect of a Front-Loaded Regimen of Clopidogrel in Patients With Atherosclerosis on Aspirin

There is a need for a rapid antithrombotic effect after the administration of antiplatelet drugs in the setting of acute coronary syndromes and percutaneous interventions. Clopidogrel, a new thienopyridine derivative, is an efficient antiplatelet agent. However, the standard regimen of clopidogrel (75 mg/d) requires 2 to 3 days before significant antithrombotic effects. Patients with stable arterial disease on chronic aspirin therapy (n=20) were treated with clopidogrel either with a front-loaded regimen, 300 mg the first day and 75 mg/d the next 7 days, or with a standard regimen, 75 mg/d for 8 days. Blood thrombogenicity was assessed by quantification of platelet-thrombus formation in an ex vivo perfusion chamber, by ADP-induced platelet aggregation, and by ADP-induced fibrinogen binding. At 2 hours, mean total thrombus area with the standard regimen was not significantly reduced. In contrast, at 2 hours, the mean total thrombus area with the front-loaded regimen was significantly decreased by 23.1±8.5% versus baseline (P <0.05). ADP-induced platelet aggregation (with 5 and 10 &mgr;mol/L) was also significantly (P <0.05) reduced with the front-loaded regimen at 2 hours, with the mean platelet aggregation being 82.2±4.4% and 81.8±4.5%, respectively, versus baseline. Similarly, flow cytometry demonstrated a significant decrease (P <0.05) in the ADP-induced fibrinogen binding (with 0.12 and 0.6 &mgr;mol/L) at 2 hours in this front-loaded regimen group (36.1±2.0% and 53.2±9.3%). With the standard regimen, platelet activity was not significantly reduced at 2 hours. Our data suggest that a front-loaded regimen of clopidogrel added to aspirin achieves a significant antithrombotic effect at 2 hours in patients with known atherosclerotic disease on chronic aspirin therapy. This provides a rationale for using front-loaded clopidogrel in combination with aspirin in percutaneous coronary interventions.

Comparison of the Predictive Value of Four Different Risk Scores for Outcomes of Patients With ST-Elevation Acute Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention

Accurate risk stratification has an important role in the management of patients with acute coronary syndromes. Even in patients with ST-elevation acute myocardial infarction (STEMI), for whom early therapeutic options are well defined, risk stratification has an impact on early and late therapeutic decision making. We aimed to compare the prognostic value of 4 risk scores used to evaluate patients with STEMI. We conducted a prospective registry of all patients treated with primary percutaneous coronary intervention for STEMI from January 2001 to June 2006. Excluded were patients with cardiogenic shock. A total of 855 consecutive patients were included in the analysis (age 60.5 +/- 13 years, 19% women, 28% with diabetes, and 48% with anterior wall myocardial infarction). For each patient, the Thrombolysis In Myocardial Infarction (TIMI), Controlled Abciximab and Device Investigation to Lower Late Angioplasty complications (CADILLAC), Primary Angioplasty in Myocardial Infarction (PAMI), and Global Registry for Acute Coronary Events (GRACE) risk scores were calculated using specific clinical variables and angiographic characteristics. Thirty-day and 1-year clinical outcomes were assessed. The predictive accuracy of the 4 risk scores was evaluated using the area under the curve or C statistic method. The CADILLAC, TIMI, and PAMI risk scores all had relatively high predictive accuracy for 30-day and 1-year mortality (C statistic range 0.72 to 0.82), with slight superiority of the CADILLAC score. These 3 risk scores also performed well for prediction of reinfarction at 30 days (C statistic range 0.6 to 0.7). The GRACE score did not perform as well and had low predictive accuracy for mortality (C statistic 0.47). In conclusion, risk stratification of patients with STEMI undergoing primary percutaneous coronary intervention using the CADILLAC, TIMI, or PAMI risk scores provide important prognostic information and enables accurate identification of high-risk patients.

Treatment of Aspirin-Resistant Patients With Omega-3 Fatty Acids Versus Aspirin Dose Escalation

OBJECTIVES The aim of this study was to evaluate whether addition of omega-3 fatty acids or increase in aspirin dose improves response to low-dose aspirin among patients who are aspirin resistant. BACKGROUND Low response to aspirin has been associated with adverse cardiovascular events. However, there is no established therapeutic approach to overcome aspirin resistance. Omega-3 fatty acids decrease the availability of platelet arachidonic acid (AA) and indirectly thromboxane A2 formation. METHODS Patients (n = 485) with stable coronary artery disease taking low-dose aspirin (75 to 162 mg) for at least 1 week were screened for aspirin response with the VerifyNow Aspirin assay (Accumetrics, San Diego, California). Further testing was performed by platelet aggregation. Aspirin resistance was defined by > or =2 of 3 criteria: VerifyNow score > or =550, 0.5-mg/ml AA-induced aggregation > or =20%, and 10-micromol/l adenosine diphosphate (ADP)-induced aggregation > or =70%. Thirty patients (6.2%) were found to be aspirin resistant and randomized to receive either low-dose aspirin + omega-3 fatty acids (4 capsules daily) or aspirin 325 mg daily. After 30 days of treatment patients were re-tested. RESULTS Both groups (n = 15 each) had similar clinical characteristics. After treatment significant reductions in AA- and ADP-induced aggregation and the VerifyNow score were observed in both groups. Plasma levels of thromboxane B2 were also reduced in both groups (56.8% reduction in the omega-3 fatty acids group, and 39.6% decrease in the aspirin group). Twelve patients (80%) who received omega-3 fatty acids and 11 patients (73%) who received aspirin 325 mg were no longer aspirin resistant after treatment. CONCLUSIONS Treatment of aspirin-resistant patients by adding omega-3 fatty acids or increasing the aspirin dose seems to improve response to aspirin and effectively reduces platelet reactivity.

Testing Platelet Activation With a Shear-Dependent Platelet Function Test Versus Aggregation-Based Tests Relevance for Monitoring Long-Term Glycoprotein IIb/IIIa Inhibition

BackgroundTests developed to monitor glycoprotein (GP) IIb/IIIa blockade do not properly reflect platelet function in vivo and need a baseline (pretreatment) value. Because GP IIb/IIIa is essential in platelet aggregation and thrombosis under shear conditions, a flow-dependent approach to monitor its inhibition can be used. Methods and ResultsWe compared a test based on flow-dependent platelet deposition, the Cone and Platelet Analyzer (CPA), with in vitro platelet aggregometry and the Rapid Platelet Function Assay (RPFA) on platelet function after GP IIb/IIIa inhibition. In vitro, increasing concentrations of abciximab (0% to 100% receptor occupancy) were tested. Ex vivo, platelet function was monitored with the CPA and with aggregometry for up to 1 week after abciximab administration. The CPA was better correlated with the percentage of free GP IIb/IIIa receptors than was aggregometry or the RPFA. Only the RPFA, when expressed as a ratio over baseline (pretreatment), was comparable to the CPA. Ex vivo, the CPA, but not aggregometry, showed prolonged platelet inhibition with gradual recovery from GP IIb/IIIa receptor blockade in the first week after abciximab administration. ConclusionsPlatelet function assessment by shear-induced deposition is a reliable test to monitor a wide range of GP IIb/IIIa inhibition. Its accuracy does not require a baseline reference. The effects of GP IIb/IIIa blockade on platelet function should be examined under high shear conditions.

Major bleeding complicating contemporary primary percutaneous coronary interventions-incidence, predictors, and prognostic implications.

BACKGROUND Major bleeding is one of the most frequent procedural-related complications of primary percutaneous coronary intervention (PCI) for ST-elevation myocardial infraction (STEMI). We investigated the incidence, predictors, and prognostic impact of peri-procedural bleeding in a cohort of unselected patients undergoing contemporary primary PCI. METHODS A total of 831 consecutive patients who underwent primary PCI between 1/2001 and 6/2005 were studied. Major bleeding was defined as hemorrhagic stroke, hemoglobin (Hb) drop of >5 g%, or 3-5 g% with a need for blood transfusion. Clinical outcomes were evaluated at 30 days and 6 months. RESULTS Major bleeding occurred in 27 patients (3.5%). Those who experienced major bleeding were older (66+/-15 vs. 61+/-13, P=.02), more frequently female gender (48% vs. 27%, P=.0001), presented more often with cardiogenic shock (37% vs. 8%, P=.0001), and had higher CADILLAC score (7.8+/-4.5 vs. 5.1+/-4.0, P=.002) and activated clotting time (ACT) levels (284+/-63 vs. 248+/-57 s, P=.007). In multivariate analysis, significant predictors of major bleeding were female gender (OR 5.1, 95% CI 1.7-15.2, P=.004), ACT levels >250 s (OR 3.6, 95% CI 1.1-12.1, P=.04), and use of intra-aortic balloon pump (IABP) (OR 3.5, 95% CI 1.0-12.1, P=.047). Major bleeding was associated with increased 6-month mortality rates (37% vs. 10%, P=.0001), which remained significant after adjustment for baseline CADILLAC score (37% vs. 19.4%, P=.05). CONCLUSIONS Major bleeding complicating primary PCI is associated with increased 6-month mortality. Women and those who need IABP support are at particularly high risk. Tight monitoring of anticoagulation may reduce the risk of bleeding.

Circulating Endothelial Progenitor Cells and Coronary Collaterals in Patients with Non-ST Segment Elevation Myocardial Infarction

Background: Endothelial progenitor cells (EPCs) are bone marrow-derived cells that are augmented in response to ischemia and incorporated into neovascularization sites. We sought to determine whether circulating EPCs are related to collateral formation following non-ST segment elevation myocardial infarction (NSTEMI). Methods: Twenty patients who underwent percutaneous coronary intervention (PCI) within a week of NSTEMI were divided into two groups: patients without collaterals (coll–, n = 10) and patients with Rentrop grade 3–4 collaterals (coll+, n = 10). Blood samples were drawn before PCI and 24 ± 2 h after PCI. EPC colonies were grown from peripheral blood mononuclear cells, characterized, and counted. Using flow cytometry the percentage of cells coexpressing vascular endothelial growth factor receptor-2 and CD133 was determined. Results: The coll+ group had higher degree of culprit vessel stenosis and lower initial thrombolysis in myocardial infarction flow grade. The relative number of EPCs before PCI was significantly higher in the coll+ group than in the coll– group (1.49 ± 0.9% vs. 0.77 ± 0.4%, p = 0.045). There were no significant intergroup differences in the number of EPC colony-forming cells. The number of EPC colonies increased in the coll– group after PCI (9.5 ± 4.8 to 14.0 ± 5.9/106 cells, p = 0.01). Conclusions: This study supports an association between circulating EPC levels and collateral formation in patients with an NSTEMI.