Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples.

A recently released kit (PerFix EXPOSE) was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12). The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24 h post clopidogrel dose from 19 patients hospitalized for a non-cardio-embolic ischemic stroke and treated with clopidogrel monotherapy for at least five days in an observational study. The platelet PerFix method was based on adaptation of the volume of the sample, the centrifugation speed and the incubation temperature. Poor agreement between prevention by adenosine diphosphate (ADP) of PGE1-induced cAMP-mediated VASP phosphorylation and ADP induced aggregation assessed by Light Transmittance Aggregometry was found. We found a significant correlation between the PLT VASP/P2Y12 kit and the PerFix EXPOSE kit. The PerFix EXPOSE kit may also be helpful to monitor adverse effects of second-generation tyrosine kinase inhibitors on platelets.

Clinical implications of clopidogrel non-response in cardiovascular patients: a systematic review and meta-analysis.

UNLABELLED: BSUMMARY BACKGROUND: Previous studies have shown an important risk of cardiovascular events in patients with clopidogrel biological non-response, and data have shown considerable, unexplored heterogeneity. OBJECTIVES: To evaluate the magnitude of cardiovascular risk associated with clopidogrel non-response and to explore heterogeneity. METHODS: This was a systematic review and meta-analysis of prospective studies of patients treated with clopidogrel for symptomatic atherothrombosis, evaluated by light transmission aggregometry with ADP and monitored prospectively for clinical ischemic events. RESULTS: Fifteen studies were included, totaling 3960 patients, of whom 25% were considered to be clopidogrel non-responders. The global relative risk (RR) for recurrent ischemic events in clopidogrel non-responders was 3.5 [95% confidence interval (CI) 2.4-5.2, P < 0.0001]. The results of the different studies were heterogeneous (Cochran P = 0.01 and I(2) = 52%). The most recent studies yielded lower RRs [global RR = 2.9 (95% CI 2.3-3.8) after 2007, and global RR = 6.6 (95% CI 3.7-11.9) before 2007, P = 0.01]. Heterogeneity was present in the group of studies in which more than 10% of patients took glycoprotein (GP)IIb-IIIa inhibitors [Cochran P = 0.003 and I(2) = 70%; RR = 3.8 (95% CI 2.9-5.1)] and was absent in the other studies [Cochran P = 0.88 and I(2) = 0; RR = 2.5 (95% CI 1.7-3.6)]. The RR was significantly higher in studies using higher ADP maximal aggregation cut-offs (> 65%) for clopidogrel non-response than in studies using lower cut-offs [RR = 5.8 (95% CI 3.2-10.3) and RR = 2.9 (95% CI 2.2-3.7), respectively, P = 0.03]. CONCLUSIONS: The risk of ischemic events associated with clopidogrel non-response is now more precisely defined. The risk is heterogeneous across studies, possibly because of an interaction with GPIIb-IIIa inhibitors and the use of different cut-offs to identify non-responders.

Investigation of PK-PD drug-drug interaction between acenocoumarol and amoxicillin plus clavulanic acid.

A pharmacokinetic-pharmacodynamic (PK-PD) drug-drug interaction between acenocoumarol and amoxicillin + clavulanic acid antibiotic was assessed in eight healthy volunteers, using a population PK-PD) model. Each subject received at day 1 a single dose of 8 mg of acenocoumarol. Then 1 g of amoxicillin + 250 mg of clavulanic acid was given from days 3 to 9. On day 8, each subject received a single dose of 8 mg of acenocoumarol concomitantly with the antibiotic combination. Eleven blood samples were taken during 48 h following each acenocoumarol administration. Acenocoumarol plasma concentrations and prothrombin time were measured at each sampling time. We first identified the structural PK model by pooling data from this trial with individual data from other acenocoumarol PK trials. An indirect response model was used to fit PD data. Models were built using a non-linear mixed effect modelling approach with nonmem software. Covariates were tested on PK and PD parameters, including antibiotic treatment. Acenocoumarol PK data were fitted by a two-compartment, first-order input model with log normal inter-individual variability. Weight and antibiotic treatment were found to improve significantly the fit of PK data with a 15% decrease in acenocoumarol clearance with concomitant antibiotics (P < 0.05). An indirect response model was successfully applied to the PK-PD data of acenocoumarol. No covariate, including antibiotic treatment effect, significantly affected PT. Drug-drug interaction was demonstrated at the PK level, without any PD corollary.

Basis for intracellular retention of a human mutant of the retinal rod channel alpha subunit.

A mutant of the a subunit of the retinal rod cyclic GMP-gated channel, [Arg654(1-bp del)], corresponding to a truncated alphaR654Dstop subunit, was previously described in patients with retinitis pigmentosa: when expressed in HEK-293 cells, this mutated a subunit was retained inside the cell, but had normal channel activity in one case where it reached the plasma membrane, indicating that the mechanism of targeting is altered by the mutation, but not the function of the channel. The corresponding mutants of the bovine rod channel (alphaR656D stop), and of the closely related olfactory neuron channel (alphaR632Dstop) alpha subunits were expressed in Xenopus oocytes and their activity was analyzed by patch-clamp. Like their human homologue, these two channels have no activity, and we show that their GFP fusion proteins are accumulated into intracellular compartments. The truncation alone or the R/D mutation alone do not prevent or modify channel activity, indicating that neither the R656 residue nor the C-terminal domain downstream of R656 is necessary for homomeric channel targeting and function. Several mutations of R656 and of the preceding residues in the R656Dstop mutant disclose that the motif responsible for the absence of channel activity is an endoplasmic reticulum retention signal (KXKXXstop) in which the nature of the residues in positions -1 and -4 is determinant.

Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels.

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca(2+) is increased in dystrophic muscle, leading to Ca(2+) accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca(2+) in muscle from mdx mice, an animal model of the human disease. In the present study, using plasma membrane Ca(2+)-activated K(+) channels as subsarcolemmal Ca(2+) probe, we tested the possibility of a Ca(2+) accumulation at the restricted subsarcolemmal level in mdx skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca(2+)-activated K(+) channels is significantly lower in mdx than in control muscle, suggesting a higher subsarcolemmal [Ca(2+)]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca(2+)-activated K(+) channel activation could correspond to a approximately 3-fold increase in the subsarcolemmal Ca(2+) concentration in mdx muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in mdx skeletal muscle, leading to Ca(2+) overload at the subsarcolemmal level.

Stretch-induced activation of Ca(2+)-activated K(+) channels in mouse skeletal muscle fibers.

High-conductance Ca(2+)-activated K(+) (K(Ca)) channels were studied in mouse skeletal muscle fibers using the patch-clamp technique. In inside-out patches, application of negative pressure to the patch induced a dose-dependent and reversible activation of K(Ca) channels. Stretch-induced increase in channel activity was found to be of the same magnitude in the presence and in the absence of Ca(2+) in the pipette. The dose-response relationships between K(Ca) channel activity and intracellular Ca(2+) and between K(Ca) channel activity and membrane potential revealed that voltage and Ca(2+) sensitivity were not altered by membrane stretch. In cell-attached patches, in the presence of high external Ca(2+) concentration, stretch-induced activation was also observed. We conclude that membrane stretch is a potential mode of regulation of skeletal muscle K(Ca) channel activity and could be involved in the regulation of muscle excitability during contraction-relaxation cycles.