Relationship between pharmacokinetics and pharmacodynamics of clopidogrel in patients undergoing percutaneous coronary intervention: comparison between vasodilator-stimulated phosphoprotein phosphorylation assay and multiple electrode aggregometry.

UNLABELLED: ESSENTIALS: The reliability of platelet tests as markers of the variable bioavailability of clopidogrel is not yet defined. Kinetics of clopidogrel active metabolite (CAM) and platelet response were studied in ischemic heart disease. CAM plasma maximum concentration (Cmax ) predicted vasodilator-stimulated phosphoprotein (VASP-P). Timely performed VASP-P, not an aggregation-based test, may be a surrogate for clopidogrel bioavailability. BACKGROUND: The high inter-individual variability in the inhibition of platelet function by clopidogrel is mostly explained by high variability in its transformation to an active metabolite (CAM). Objective We investigated the relations between pharmacokinetics and pharmacodynamics of CAM by comparing two methods of platelet function. METHODS: We enrolled 14 patients undergoing percutaneous coronary interventions for non-ST-segment elevation acute coronary syndrome or inducible myocardial ischemia. Plasma concentrations of clopidogrel and CAM, phosphorylation of vasodilator-stimulated phosphoprotein (VASP-P), expressed as a platelet reactivity index (PRI) and whole-blood platelet aggregation (multiple electrode aggregometer, MEA) were measured before and after a 600-mg clopidogrel loading dose (nine time-points) and before and after 75-mg maintenance doses on days 2, 7 and 30. RESULTS: Plasma concentrations of clopidogrel and CAM were highly variable. CAM reached maximal concentration (Cmax ) (median, 110.8 nm; range, 41.9-484.8) 0.5-2 h after the loading dose. A sigmoid dose-response curve defined the relations between CAMCmax and PRI after 3 to 24 h (IC50 , 459.6 nm; 95% confidence interval, 453.4-465.7; R(2) = 0.82). PRI was unchanged from baseline in patients with the lowest CAMCmax (< 83 nm, n = 7), indicating low sensitivity of VASP-P. PRI values were also predicted by CAMCmax at days 2, 7 and 30. Platelet aggregation measured by MEA did not show significant relations with either PRI or with CAM pharmacokinetics at any time-point. CONCLUSIONS: After 600 mg clopidogrel, VASP-P, but not whole-blood platelet aggregation measured by MEA, is almost entirely predicted by CAMCmax . VASP-P could be useful in studies aimed at investigating relations between CAM bioavailability and clinical events.

The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations.

We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long-lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Refolding of mechanically denatured HIV-1-PR monomers is also a multiple-pathway process. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, and provide information regarding possible structural features of the PF state.

Role of coronary angiography MDCT in the clinical setting: changes in diagnostic workup in the real world.

PURPOSE: The authors sought to evaluate the incremental value of introducing coronary angiography with multidetector computed tomography (MDCT-CA) compared with the conventional diagnostic workup in managing patients with suspected coronary artery disease (CAD) workup. MATERIALS AND METHODS: A total of 531 consecutive patients underwent MDCT-CA between April 2008 and August 2010. For each patient the pretest probability of CAD was obtained by using the Morise score as well as the diagnostic performance of the exercise test and of MDCT-CA, considering conventional coronary angiography (CCA) as the gold standard. Based on these results, we calculated the posttest likelihood of CAD after stress testing, comparing the incremental diagnostic value for each category of cardiovascular risk with data obtained with MDCT-CA. The conventional diagnostic workup (without MDCT-CA) was then compared with the modified workup (including MDCT-CA). RESULTS: The diagnostic performance of the exercise test for identifying patients with significant lesions had a sensitivity and specificity of 20% and 88%, respectively, with positive (PPV) and negative (NPV) predictive value of 41% and 72%, respectively. Taking CA as the gold standard, MDCT-CA had 93% sensitivity, 89% specificity, 88% PPV and 93% NPV compared with CCA in evaluating significant stenoses in the per-patient analysis. The overall diagnostic accuracy of MDCT-CA was 91%. The exercise tests provided no significant incremental diagnostic value compared with cardiovascular history in patients with a low to intermediate risk. Comparison of the diagnostic accuracy of these protocols showed improved performance results for the modified protocol. CONCLUSIONS: MDCT-CA is the reference modality for the noninvasive exclusion of critical CAD. It provides a very high incremental diagnostic value compared with exercise testing in patients with a low to intermediate risk of CAD. The use of diagnostic protocols based on MDCT-CA ensures improved diagnostic performance compared with those involving conventional exercise electrocardiograms.

Comparison between different kernel reformatting filters in 3D quantitative analysis of MDCT coronary angiography.

PURPOSE: Coronary angiography with multidetector-row computed tomography (MDCT-CA) allows quantification of coronary artery stenosis with a high level of accuracy; however, a better estimation of stenosis can be achieved by using appropriate reformatting filters, especially in stents and calcified segments. Quantitative computed tomography angiography (QCTA) is intended to overcome the limitations of the visual score. The aim of this study was to evaluate the accuracy of QCTA with different filters in comparison with quantitative coronary angiography (QCA) and visual score. MATERIALS AND METHODS: Two blinded operators visually scored 17 consecutive patients referred for MDCT-CA with a per-segment analysis. The degree of stenosis was classified as 0-20%, 20-50% (wall irregularities), 50-70% (significant disease) and 70-100% (vessel occlusion). Each segment was then analysed using the electronic callipers of the QCTA system with 15 different filters. No contour editing was performed. Data were compared with QCA and conventional coronary angiography (CCA). Comparison between QCTA, visual score and QCA were performed using Spearman's rank correlation. RESULTS: Of 25 segments analysed (mean 1.4 diseased segment per patient), 375 measurements were considered. Good correlation was found between the visual score and QCA [Pearson correlation coefficient (rho=0.852; p<0.0001)] and between QCA and CCA (rho=0.804; p<0.0001). Moderate correlation was found between QCA and QCTA only using two filters (rho=0.444; p<0.0001 for YA filter and rho=0.450; p<0.0001 for YB filter). CONCLUSIONS: Overall QCTA accuracy is low if contour editing is not applied, especially in calcified vessels. Certain filters can help to better estimate the exact percentage of stenosis.

MDCT coronary angiography vs 2D echocardiography for the assessment of left ventricle functional parameters.

PURPOSE: This study was done to compare the parameters of left ventricular (LV) function obtained by multidetector computed tomography coronary angiography (MDCT-CA) using 64-slice equipment with those obtained using twodimensional echocardiography (2D-SE) considered as reference standard. MATERIALS AND METHODS: Between April 2008 and September 2009, 116 consecutive patients were studied with both techniques. We analysed the parameters commonly sampled in echocardiography and related them with those retrieved with MDCT-CA: septal thickness, posterior wall thickness, diameter of ascending aorta, diameter and volumes in end-systolic and end-diastolic phase, ejection fraction, stroke volume, cardiac output and heart mass. RESULTS: Good correlation was found measuring septal thickness (r=0.470; p=0.001), and diameters of the ascending aorta. Correlation between systolic and diastolic diameters obtained with the two techniques was good. Poor correlation was attained measuring thickness of the posterior wall (r=0.243; p=0.104). MDCT-CA consistently overestimated the average volumes; diastolic and systolic volumes showed significant correlation (r=0.0456; p= 0.002; r=0.640; p<0.001). Ejection fraction agreement showed a significant correlation (r=0.626; p<0.001). CONCLUSIONS: MDCT-CA provides parameters of cardiac function comparable to those found in echocardiography. MDCT-CA although used primarily for coronary noninvasive imaging can provide additional information on ventricular function useful to the diagnostic workup of cardiac patients.

Anatomical variants and anomalies of the coronary tree studied with MDCT coronary angiography.

Anomalies of the coronary arteries are congenital and in most of the cases asymptomatic, although they may present with severe symptoms such as angina pectoris or cardiac arrest. Multidetector CT coronary angiography (MDCT-CA) permits, through curved multiplanar reconstructions and three-dimensional reformatting, noninvasive visualisation of the coronary tree and its variants and anomalies, providing a more accurate alternative to conventional coronary angiography (CCA). The purpose of this pictorial essay is to describe the main variants and anomalies of the coronary arteries using MDCT imaging with multiplanar and three-dimensional reconstructions.

Choice strategy of different dose-saving protocols in 64-slice MDCT coronary angiography.

PURPOSE: Multidetector-row computed tomography coronary angiography (MDCT-CA) produces high-level radiation dose because of submillimetre slice thickness and short scan time. As a result, manufacturers have produced different dose-saving protocols that may, however, reduce image quality and thus diagnostic accuracy. The aim of our study was to assess the diagnostic quality of MDCT-CA using different dose-saving protocols. MATERIALS AND METHODS: Between April and August 2008, we examined 65 patients with 64-slice MDCT-CA: 6/65 using the step-and-shoot dose-saving protocol, 45/65 the cardiac dose right protocol and 14/65 using a standard protocol. Image quality was evaluated on a per-patient and per-segment basis, and the effective dose of each protocol was recorded. RESULTS: In the per-patient analysis, image quality was excellent in 100% of the step-and-shoot protocols, in 91.1% of the cardiac dose right protocols and in 85.8% of the standard protocols. Effective dose to the patient considering the whole study (i.e. scout, calcium score, triggering and MDCT-CA) was 20.49 mSv in the standard protocol, 14.8 mSv in the cardiac dose right protocol and 6.63 mSv in the step-and-shoot protocol. CONCLUSIONS: The radiologist should apply the appropriate protocol in relation to the clinical indications, type of patient and information required in order to spare as much dose as possible while maintaining high image quality.

A monoclonal antibody specific for retinal ganglion cells of mammals.

The development of specific markers for retinal ganglion cells is an area of great interest in retinal research. In this study we report on a monoclonal antibody (AB5) which specifically labels ganglion cells in rabbit, cat and monkey, as well as a variety of other mammalian species. Labelling of ganglion cells was also observed in isolated cell preparations of rabbit retina.

The production and characterization of monoclonal antibodies against enkephalins.

The hybridoma technology of Kohler and Milstein (1975) was utilized to produce monoclonal antibodies against the enkephalins. Two hybridomas, AD4 and DB4, produced monoclonal antibodies of the IgG type 1 class against Leu(5)-enkephalin that were highly specific for Leu(5)- and Met(5)-enkephalin. AD4 exhibited almost equal reactivity with either Leu(5)- or Met(5)-enkephalin, whereas DB4 exhibited only a 20% cross-reactivity with Met(5)-enkephalin. The IC(50) of these monoclonal antibodies were approximately two orders of magnitude greater than the IC(50) a polyclonal antiserum against enkephalins (A206; Miller et al 1978) used routinely in many immunochemical and immunocytochemical studies. The monoclonal antibodies, AD4 and DB4, exhibited specific sequence and size requirements for binding enkephalin-related peptides. The amino acid sequence Gly-Gly-Phe-Leu or Gly-Gly-Phe-Met was essential for recognition by AD4 and DB4. However, Tyr-Gly-Gly-Phe which lacks Leu or Met in the fifth position did not react with our monoclonal antibodies. Moreover, enkephalin-related peptides in which the enkephalin sequence was situated at the amino terminus and which contained six or more amino acids did not react significantly with AD4 or DB4. In particular, unlike the polyclonal antiserum A206, our monoclonal antibodies do not react with dynorphins 1-6 or 1-13. However, when the monoclonal antibody (AD4) was used to localize immunohistochemically the population of enkephalinergic amacrine cells in the chicken retina, it provided a staining pattern quite comparable to that observed in previous studies (Watt et al., 1983) using the polyclonal enkephalin antiserum A206. This finding therefore demonstrates that the immunoreactive products visualized in the enkephalin-immunoreactive amacrine cells of the chicken retina with the polyclonal antiserum correspond to authentic enkephalin or peptides very closely related to the enkephalins.

N-acetylgalactosamine-6-sulfate sulfatase in man. Absence of the enzyme in Morquio disease.

Human N-acetylgalactosamine-6-sulfate sulfatase (6-sulfatase) activity is measured by using as a substrate a sulfated tetrasaccharide obtained by digesting purified chondroitin-6-sulfate (C-6-S) with testicular hyaluronidase. The amount of inorganic sulfate released is measured turbidimetrically. The enzyme from human kidney has a pH optimum of 4.8; its activity is augmented by low levels of NaCl and inhibited by phosphate and high levels of NaCl. Free glucuronate, acetylgalactosamine, inorganic sulfate, polymeric C-6-S, or tetrasaccharide obtained from chondroitin-4-sulfate do not affect the enzyme activity. The method may be used for the diagnosis of Morquio disease since extracts of Morquio fibroblasts are devoid of 6-sulfatase activity.

The effect of (+) --cyanidanol on lysosomal enzymes of I-cell fibroblasts.

(+)--Cyanidanol, a water-soluble flavonoid, when added to cultured skin fibroblasts of a patient with I-cell disease raised the intracellular concentration of beta-galactosidase but did not affect the distribution of arylsulfatase. A, alpha-mannosidase or beta-glucuronidase. The elevated accumulation of 35SO4 by I-cell, Hunter and Maroteaux-Lamy fibroblasts was decreased by the addition of (+)--cyanidanol to the culture medium, but the degradation of previously labeled, intracellular glycosaminoglycans was not. It is concluded that (+)--cyanidanol does not produce a biochemical correction of the enzymic abnormalities existing in I-cell fibroblasts.