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Despite common use of clopidogrel in patients with vascular disease, monitoring of platelet inhibition is still not conventional in clinical practice. Considering substantial response variability, when some patients may experience inadequate protection, and/or increased risk of bleeding, simple and reliable methods to control adequate antiplatelet regimen is mandatory. We validated a new VerifyNow-P2Y12 assay to measure inhibition of the P2Y12 platelet receptors by clopidogrel by evaluating its receptor specificity, precision, and potential interference with platelet count, hematocrit, age, cholesterol, triglycerides, and other antiplatelet agents. Platelet aggregation induced by ADP or ADP + prostaglandin E1 (ADP + PGE1) in the presence of specific P2Y12 inhibitor 2-methylthio-AMP (2MeSAMP) for the assessment of assay specificity was performed in 10 volunteers. Seventeen medications were used for the VerifyNow-P2Y12 interference testing, and assay interplay with blood constituents was evaluated in a clinical setting in 131 patients with coronary artery disease. In the presence of 2MeSAMP, the average residual aggregation level across the 10 donors was 27% for ADP and 5% for ADP + PGE1. There also was a strong agreement between ADP + PGE1 aggregometry and VerifyNow-P2Y12 assay (93% vs. 95% average inhibition across all donors). The coefficient of variation for the test precision was less than 8%. The VerifyNow-P2Y12 readings were not influenced by age, platelet count, hematocrit, fibrinogen, cholesterol, or triglycerides level. There was an interference with abciximab before P2Y12 inhibition; however, after platelet suppression with cilostazol, the interference with all tested substances was minimal. VerifyNow-P2Y12 is a reliable, simple, and sensitive device suitable for monitoring of P2Y12 platelet receptor inhibitors in the clinical arena.

Extended release dipyridamole (DIP) is widely used in clinical practice as an Aggrenox formulation, which is proven to improve outcomes for secondary stroke prevention in patients after acute vascular events. However, presently established fluorometry techniques are not suitable for trace amount determinations, because of the variable background fluorescence. The authors sought to determine whether biological fluid pH is important for the serial measures of DIP levels in the animal experiments and in patients treated with Aggrenox after ischemic stroke. Post-stroke patient (n = 34) and mice (n = 25) samples were tested to determine DIP levels by established techniques with FluoroMax 3 spectrofluorometer. Both the absorption and emission spectra of DIP were affected by modifications in pH. Fluorescence of DIP was found to be maximal at a wavelength of 490 nm (excitation 420 nm) and the spectral pattern was independent of pH. The intensity of fluorescence, however, was drastically lower at low pH (at pH 2.6, fluorescence was 4% of intensity at pH 9.8). Background plasma fluorescence, however, was completely unaffected by changes in pH. Using these fluorometric characteristics, a regression model that facilitates the efficient and sensitive determination of DIP concentration in biological fluids was formulated. Exploiting pH-dependent characteristics of DIP versus serum fluorescence patterns permits a convenient mathematical model to determine DIP concentration. This relatively inexpensive and time-efficient procedure can quantify drug levels in human/animal plasma/serum, thereby directly determining the level of patient adherence to the prescribed drug regimen, be it in the context of clinical trials or compliance with the animal protocol.

A retrospective review of admission serum protein concentration in 18 children with hemolytic-uremic syndrome (HUS) showed significantly decreased serum total protein, albumin and globulin concentrations upon admission compared with 22 matched controls (P < 0.003). One child with atypical disease without diarrhea had normal serum protein concentrations. A strongly positive correlation (P = 0.006) was found between the age of HUS patients with diarrhea and their lowest total protein concentrations. In 10 children who eventually required hemodialysis, there was a significantly negative correlation (r = -0.8316, P = 0.01) between the admission serum albumin and the patients' highest creatinine levels, suggesting that hypoproteinemia may be a risk factor in the development of renal failure. The pathophysiological and clinical significance of hypoproteinemia in HUS needs further investigation.

Objectives. We sought to compare platelet characteristics after reteplase and alteplase therapy in the setting of the Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO)-III trial. Background. Platelet function may be impaired during thrombolysis in patients with an acute myocardial infarction. The effects of reteplase and alteplase on platelet aggregation and major surface antigen expression during the first 24 h of infarction therapy are unknown. Methods. Platelet aggregation and receptor expression by flow cytometry were determined in 23 patients before thrombolysis and thereafter at 3, 6, 12 and 24 h. Results. Aggregation was higher after reteplase at 24 h when induced by 5 μmol/liter adenosine diphosphate (ADP) (p = 0.007), 10 μmol/liter ADP (p = 0.02), collagen (p = 0.003) and thrombin (p = 0.009) than after alteplase. Reteplase therapy exhibited greater glycoprotein (GP) IIb/IIIa (p = 0.04), very late antigen-2 (p = 0.04) and platelet/endothelial cell adhesion molecule-1 (p = 0.002) expression at 24 h. Trends toward decreased receptor expression early (3 to 6 h), followed by a progressive increase at 12 h and especially at 24 h occurred after both agents. Conclusions. In this prospective clinical ex vivo platelet study, similar patterns of platelet aggregation and surface receptor expression occurred during the first 24 h of coronary thrombolysis with reteplase and alteplase. However, after reteplase, indicators of platelet activity were higher at 24 h after thrombolysis than after alteplase. These data suggest that GP IIb/IIIa inhibitors or other antiplatelet strategies may be particularly advantageous when used 12 to 24 h after thrombolysis, especially after reteplase therapy. It is at this time point during the first day of coronary thrombolysis that GP IIb/IIIa is markedly expressed and platelets are most active.

Leukocytes have been implicated in myocardial infarction (MI) development and progression. We tested the efficacy of the novel leukocyte recruitment inhibitor NPC 15669 (Scios Nova, Inc.) on myocardial stunning (MS) and preconditioned MI. NPC is a member of the leumedins, and is an inhibitor of leukocyte adhesion to endothelium via blockage of integrin binding. We also show, that NPC 15669 have strong antiplatelet effects. In an open chested swine model, NPC 15669 (10 mg NPC/kg loading dose followed by constant infusion at 6 mg/kg -1 /h -l )was administered in 6 animals. Myocardial thickening (MT) was determined by epicardial ultrasound. The left anterior descending artery was occluded for 8 min. followed by 90 min. of reperfusion, during which myocardial MT was recorded at regular intervals. We have found that treatment with NPC 15669 increases myocardial contractility and significantly decreases MS time compared to six controls (26.7 ± 4.0 min vs. 50.0 ± 4.3 min, p = 0.0026). This report also demonstrates the beneficial effects of NPC 15669 on myocardial tissue survival after a period of ischemia. In NPC 15669 treated animals 23.4 ± 6.7% of at risk tissue became necrotic compared to 53.0 ± 6.6% in controls, p = 0.0102. Our data suggests that NPC 15669 significantly reduces myocardial injury in both the stunning and infarction mod els. The precise mechanism of these effects is unknown, but may be related to the inhibition of platelet function and/or leukocyte recruitment.