Crowd-Sourced Verification of Computational Methods and Data in Systems Toxicology: A Case Study with a Heat-Not-Burn Candidate Modified Risk Tobacco Product.

Systems toxicology intends to quantify the effect of toxic molecules in biological systems and unravel their mechanisms of toxicity. The development of advanced computational methods is required for analyzing and integrating high throughput data generated for this purpose as well as for extrapolating predictive toxicological outcomes and risk estimates. To ensure the performance and reliability of the methods and verify conclusions from systems toxicology data analysis, it is important to conduct unbiased evaluations by independent third parties. As a case study, we report here the results of an independent verification of methods and data in systems toxicology by crowdsourcing. The sbv IMPROVER systems toxicology computational challenge aimed to evaluate computational methods for the development of blood-based gene expression signature classification models with the ability to predict smoking exposure status. Participants created/trained models on blood gene expression data sets including smokers/mice exposed to 3R4F (a reference cigarette) or noncurrent smokers/Sham (mice exposed to air). Participants applied their models on unseen data to predict whether subjects classify closer to smoke-exposed or nonsmoke exposed groups. The data sets also included data from subjects that had been exposed to potential modified risk tobacco products (MRTPs) or that had switched to a MRTP after exposure to conventional cigarette smoke. The scoring of anonymized participants' predictions was done using predefined metrics. The top 3 performers' methods predicted class labels with area under the precision recall scores above 0.9. Furthermore, although various computational approaches were used, the crowd's results confirmed our own data analysis outcomes with regards to the classification of MRTP-related samples. Mice exposed directly to a MRTP were classified closer to the Sham group. After switching to a MRTP, the confidence that subjects belonged to the smoke-exposed group decreased significantly. Smoking exposure gene signatures that contributed to the group separation included a core set of genes highly consistent across teams such as AHRR, LRRN3, SASH1, and P2RY6. In conclusion, crowdsourcing constitutes a pertinent approach, in complement to the classical peer review process, to independently and unbiasedly verify computational methods and data for risk assessment using systems toxicology.

Comparison of new adenosine diphosphate receptor antagonists with clopidogrel in patients with coronary artery disease: a meta-analysis.

Recent data suggest the superiority of new adenosine diphosphate (ADP) receptor antagonists compared with clopidogrel in acute coronary syndrome patients. We aimed to assess the risks and benefits of new ADP receptor antagonists in patients with coronary artery disease (CAD). Relevant studies published through February 28, 2014 were searched and identified in the MEDLINE, EMBASE, and Cochrane databases. Summary estimates were obtained using a random-effects model. All nine published randomized controlled studies comparing new ADP receptor antagonists with clopidogrel in CAD were included. The database consisted of 66,900 patients; 33,782 on novel agents, and 33,118 on clopidogrel. New ADP receptor antagonists reduced the composite incidence of all-cause mortality, myocardial infarction or stroke (odds ratio [OR] 0.89, 95 % confidence interval [CI] 0.81-0.97, p = 0.01) but increased the incidence of non-coronary artery bypass grafting-related major bleeding (OR 1.24, 95 % CI 1.08-1.42, p = 0.003). The composite end point of the net rate of adverse clinical events, which was the combination of the primary efficacy end point and the primary safety end point, was significantly lower in the new agent group compared to the clopidogrel group (9.7 versus 10.6 %, OR 0.92, 95 % CI 0.85-1.00). Use of recently introduced new ADP receptor antagonists results in a reduction in adverse clinical outcomes but a substantial increase in bleeding. New agents revealed an improved combined efficacy and safety outcome compared to that of clopidogrel in patients with CAD.

SAR216471, an alternative to the use of currently available P2Y12 receptor inhibitors?

P2Y12 antagonism is a key therapeutic strategy in the management and prevention of arterial thrombosis. The objective of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of SAR216471, a novel P2Y12 receptor antagonist. SAR216471 blocks the binding of 2MeSADP to P2Y12 receptors in vitro (IC50=17 nM). This inhibition was shown to be reversible. It potently antagonized ADP-induced platelet aggregation in human and rat platelet-rich plasma (IC50=108 and 62 nM, respectively). It also inhibited platelet aggregation when blood was exposed to collagen or thromboxane A2. Its high selectivity was demonstrated against a large panel of receptors, enzymes, and ion channels. Despite its moderate bioavailability in rats, oral administration of SAR216471 resulted in a fast, potent, and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition were directly proportional to its circulating plasma levels. Pre-clinical study of SAR216471 in a rat shunt thrombosis model demonstrated a dose-dependent antithrombotic activity after oral administration (ED50=6.7 mg/kg). By comparison, ED50 values for clopidogrel, prasugrel and ticagrelor were 6.3, 0.35 and 2.6 mg/kg, respectively. Finally, the anti-hemostatic effect of SAR216471 and its competitors was investigated in a rat tail bleeding model, revealing a favorable safety profile of SAR216471. Together, these findings have established a reliable antiplatelet profile of SAR216471, and support its potential use in clinical practice as an alternative to currently available P2Y12 receptor antagonists.

Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors.

BACKGROUND: Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. METHODS: We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. RESULTS: The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the light scattering method could detect platelet shape change and aggregation in response to nanomolar concentrations of extracellular nucleotides. Furthermore, our results demonstrate that the advantages of the light scattering method make it a choice method for platelet receptor monitoring and for investigation of both murine and human platelets in disease models. CONCLUSIONS: Our data demonstrate the suitability and superiority of this new low angle light scattering method for comprehensive analyses of platelet receptors and functions. This highly sensitive, quantitative, and online detection of essential physiological, pathophysiological and pharmacological-response properties of human and mouse platelets is a significant improvement over conventional techniques.

The new INNOVANCE® PFA P2Y cartridge is sensitive to the detection of the P2Y12 receptor inhibition.

Insufficient response on antiplatelet medication has become an intensively discussed issue because of the risk factor of recurrent adverse cardiovascular events. However, the monitoring of antiplatelet therapy requires appropriate, robust and reliable test methods. For the measurement of thienopyridine effects, the manufacturer of the PFA-100® System provides the INNOVANCE® PFA P2Y * cartridge. We tested this cartridge for its capacity to detect the inhibition of the P2Y12 receptor, which is the target for thienopyridine medication (e.g. clopidogrel). We compared the INNOVANCE® PFA P2Y * results with those obtained by the receptor specific flow cytometric vasodilator stimulated phosphoprotein (VASP) assay that expresses the status of the P2Y12 receptor as "platelet reactivity index" (PRI). The in vitro addition of the P2Y12 receptor antagonist cangrelor (AR-C69931MX) to citrated human whole blood resulted in a dose-dependent prolongation of closure times (CTs) of the INNOVANCE® PFA P2Y * cartridge correlating with decreased PRI levels. In volunteers, the intake of a 600 mg clopidogrel loading dose caused an increase of the CTs in all volunteers, although some of these volunteers were identified as "poor responders" by the VASP assay (no significant reduction of PRI levels). In 50 patients with stable coronary artery disease undergoing percutaneous coronary intervention (PCI) and under dual antiplatelet therapy, the new cartridge had a detection rate of 84% (CT 106 s as cut-off) for clopidogrel medication. After dividing the 50 patients into two groups according to their response to clopidogrel INNOVANCE® PFA P2Y * recognized all "responders" (defined by a PRI > 50%) using >106 s as cut-off but the specificity for a "good response" was only 42% because several "poor responders" (defined by a PRI > 50%) also showed CTs above the cut-off. The best correlation (substantial agreement) between the results of INNOVANCE® PFA P2Y * and of the VASP phosphorylation assay was achieved using CT > 200 s and PRI < 55% as cut-offs. Then, the sensitivity of INNOVANCE® PFA P2Y * was 97% and the specificity for a "good response" 65%. In summary, INNOVANCE® PFA P2Y * showed a high sensitivity for the detection of P2Y12 receptor blockade, but had only a limited specificity for a "good response" to clopidogrel. Therefore, this new cartridge is a useful tool to rule out P2Y12 receptor inhibition, if normal or only slightly prolonged CTs are obtained. Its predictive value for risk assessment of thromboembolic events, e.g. after coronary stent implantation, needs to be evaluated in clinical trials.

Resolvin E1 regulates adenosine diphosphate activation of human platelets.

OBJECTIVE: To investigate the ability of resolvin E1 (RvE1) to regulate adenosine diphosphate (ADP) activation of platelets via specific receptors because RvE1 reduces platelet aggregation with certain agonists, including ADP. METHODS AND RESULTS: RvE1 is an eicosapentaenoic acid-derived specialized proresolving mediator generated during the resolution of acute inflammation. RvE1 exhibits potent organ-protective actions in vivo and acts on specific cell types, including platelets. RvE1, 0.1 to 100 nmol/L, incubated with platelets gave reduced ADP-stimulated P-selectin mobilization (IC(50), approximately 1.6×10(-12) mol/L) and polymerized actin content compared with control platelets. RvE1, 1 to 100 nmol/L, did not stimulate or block intracellular Ca(2+) mobilization. By using a new P2Y(12)-ß-arrestin-coupled cell system, ADP-activated P2Y(12) with an EC(50) of 5×10(-6) mol/L and RvE1 did not directly stimulate P2Y(12) or block the ADP-P2Y(12) signals. In this system, another eicosanoid, leukotriene E(4) (LTE(4)) (EC(50), 1.3×10(-11) mol/L), dose dependently activated P2Y(12). When recombinant P2Y(12)-expressing cells were transiently transfected with an RvE1 receptor, human ChemR23 (present on human platelets), with the addition of RvE1 (0.1-10.0 nmol/L), blocked ADP signals (IC(50), approximately 1.6×10(-11) mol/L) in P2Y(12)-ChemR23-expressing cells compared with mock transfections. CONCLUSIONS: RvE1's regulatory actions (ie, reducing ADP-stimulated P-selectin mobilization and actin polymerization) are human (h)ChemR23-dependent. Moreover, specific platelet actions of RvE1 selectively engaged with ADP-activated platelets that illuminate a new cellular mechanism and affect ω-3 eicosapentaenoic acid, which may contribute to both resolution of vascular inflammation and ADP-dependent platelet activation relevant in pathological cardiovascular events.

Impact of P2Y12 inhibition by clopidogrel on cardiovascular mortality in unselected patients treated by percutaneous coronary angioplasty: a prospective registry.

OBJECTIVES: The aim of this study was to determine whether low platelet response to the P2Y(12) receptor antagonist clopidogrel as assessed by Vasodilator-stimulated phosphoprotein flow cytometry test (VASP- FCT) predicts cardiovascular events in a high-risk population undergoing percutaneous coronary intervention (PCI). BACKGROUND: Impaired platelet responsiveness to clopidogrel is thought to be a determinant of cardiovascular events after PCI. The platelet VASP-FCT is a new assay specific to the P2Y(12) adenosine diphosphate receptor-pathway. In this test, platelet activation is expressed as platelet reactivity index (PRI). METHODS: Four-hundred sixty-one unselected patients undergoing urgent (n = 346) or planned (n = 115) PCI were prospectively enrolled. Patients were classified as low-response (LR) and response (R) to clopidogrel, depending on their PRI. Optimal PRI cutoff was determined by receiver-operator characteristic curve analysis to 61% (LR: PRI > or =61% and R: PRI <61%). Follow-up was obtained at a mean of 9 +/- 2 months in 453 patients (98.3%). RESULTS: At follow-up, total cardiac mortality rates and possible and total stent thrombosis were higher in LR patients. Multivariate analysis identified creatinine clearance (hazard ratio [HR]: 0.95; 95% confidence interval [CI]: 0.93 to 0.98, p < 0.001), drug-eluting stent (HR: 5.73; 95% CI: 1.40 to 23.43, p = 0.015), C-reactive protein (HR: 1.01; 95% CI: 1.001 to 1.019, p = 0.024), and LR to clopidogrel (HR: 4.00; 95% CI: 1.08 to 14.80, p = 0.037) as independent predictors of cardiac death. The deleterious impact of LR to clopidogrel on cardiovascular death was significantly higher in patients implanted with drug-eluting stent. CONCLUSIONS: In patients undergoing PCI, LR to clopidogrel assessed by VASP-FCT is an independent predictor of cardiovascular death at the PRI cutoff value of > or =61%. The LR clinical impact seems to be dependent on the type of stent implanted.

Novel antiplatelet agents in the prevention of cardiovascular complications--focus on ticagrelor.

Atherothrombosis, thrombus formation as a result of atherosclerotic plaque rupture, is a major modern health problem, often underlying coronary artery disease, stroke, and peripheral arterial disease. After the treatment of an acute thrombotic episode, long-term therapy is warranted as a secondary prophylaxis of such events and their complications. Because of the importance of platelets' involvement in the initiation and propagation of thrombosis, antiplatelet drugs have come to the forefront of atherothrombotic disease treatment. Dual antiplatelet therapy of aspirin plus clopidogrel--the current standard--has its benefits, but it also has its limitations with regard to its pharmacologic properties and adverse effects. For these reasons, within the last decade or so, the investigation of novel antiplatelet agents has prospered. Here, we review the main pathways through which platelets participate in acute thrombosis and the interruption of these pathways by using novel antiplatelet agents, including P2Y12 receptor antagonists (the recently approved prasugrel, the probable next-in-line ticagrelor, and others). The need for a more individualized patient therapy is evident; although most of the aforementioned pharmaceuticals have the potential to contribute to this, their clinical utility remains to be seen.

Clopidogrel response: head-to-head comparison of different platelet assays to identify clopidogrel non responder patients after coronary stenting.

OBJECTIVES: We investigated the agreement between different platelet tests to identify clopidogrel non response. BACKGROUND: Biological definition of clopidogrel non response remains controversial. Different platelet tests have been linked with recurrent ischemic events and proposed for daily practice. METHODS: We prospectively investigated the agreement of platelet tests to isolate clopidogrel non response in patients receiving high 150 mg clopidogrel maintenance dose after coronary stenting. Clopidogrel response was assessed with ADP-induced aggregation (ADP-Ag) (non response if >70%), Platelet reactivity index VASP (PRI VASP) (non response if >50%) and Verify Now Point-of-care assay (VN) (non response if PRU > 240 AU). RESULTS: Seventy consecutive patients were included. The rates of non-responders were respectively: 13% (n = 9) with the ADP-Ag, 39% (n = 27) with the PRI VASP and 33% (n = 23) with the VN. We observed significant correlation between different platelet tests assessing clopidogrel response: r = 0.55 (p < 0.0001) for ADP-Ag and PRI VASP, r = 0.64 (p < 0.0001) for ADP-Ag and VN and r = 0.59 (p < 0.0001) for PRI VASP and VN. However, using the most common thresholds, the agreement between the difference tests was poor: 0.35 for ADP-Ag and PRI VASP, 0.36 for ADP-Ag and VN and 0.46 for PRI VASP and VN. CONCLUSION: This study showed that assessment of platelet function inhibition by clopidogrel is highly test-specific. Indeed, our results demonstrated a poor agreement between different platelet assays and suggested that identification of clopidogrel non responders is test-dependent.

A new role for the A2b adenosine receptor in regulating platelet function.

BACKGROUND: Activation of platelets is a critical component of atherothrombosis and plays a central role in the progression of unstable cardiovascular syndromes. Adenosine, acting through adenosine receptors, increases intracellular cAMP levels and inhibits platelet aggregation. The A2a adenosine receptor has already been recognized as a mediator of adenosine-dependent effects on platelet aggregation, and here we present a new role for the A2b adenosine receptor (A2bAR) in this process. METHODS AND RESULTS: As compared with platelets from wild-type controls, platelets derived from A2bAR knockout mice have significantly greater ADP receptor activation-induced aggregation. Although mouse megakaryocytes and platelets express low levels of the A2bAR transcript, this gene is highly upregulated following injury and systemic inflammation in vivo. Under these conditions, A2bAR-mediated inhibition of platelet aggregation significantly increases. Our studies also identify a novel mechanism by which the A2bAR could regulate platelet aggregation; namely, ablation of the A2bAR leads to upregulated expression of the P2Y1 ADP receptor, whereas A2bAR-mediated or direct elevation of cAMP has the opposite effect. Thus, the A2bAR regulates platelet function beyond mediating the immediate effect of adenosine on aggregation. CONCLUSIONS: Taken together, these investigations show for the first time that the platelet A2bAR is upregulated under stress in vivo, plays a significant role in regulating ADP receptor expression, and inhibits agonist-induced platelet aggregation.

Which platelet function test is suitable to monitor clopidogrel responsiveness? A pharmacokinetic analysis on the active metabolite of clopidogrel.

BACKGROUND: Multiple platelet function tests claim to be P2Y12-pathway specific and capable of capturing the biological activity of clopidogrel. OBJECTIVES: The aim of the present study was to determine which platelet function test provides the best reflection of the in vivo plasma levels of the active metabolite of clopidogrel (AMC). PATIENTS/METHODS: Clopidogrel-naive patients scheduled for elective percutaneous coronary intervention (PCI) received a 600 mg loading dose of clopidogrel and 100 mg of aspirin. For pharmacokinetic analysis, blood was drawn at 0, 20, 40, 60, 90, 120, 180, 240 and 360 min after clopidogrel loading and peak plasma concentrations (C(max)) of the AMC were quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Platelet function testing was performed at baseline and 360 min after the clopidogrel loading. RESULTS: The VASP-assay, the VerifyNow P2Y12-assay and 20 micromol L(-1) adenosine diphosphate (ADP)-induced light transmittance aggregometry (LTA) showed strong correlations with C(max) of the AMC (VASP: R(2) = 0.56, P < 0.001; VerifyNow platelet reactivity units (PRU): R(2) = 0.48, P < 0.001; VerifyNow %inhibition: R(2) = 0.59, P < 0.001; 20 micromol L(-1) ADP-induced LTA: R(2) = 0.47, P < 0.001). Agreement with C(max) of the AMC was less evident for 5 micromol L(-1) ADP-induced LTA or whole blood aggregometry (WBA), whereas the IMPACT-R ADP test did not show any correlation with plasma levels of the AMC. CONCLUSION: The flow cytometric VASP-assay, the VerifyNow P2Y12 assay and, although to a lesser extent, 20 micromol L(-1) ADP-induced LTA correlate best with the maximal plasma level of the AMC, suggesting these may be the preferred platelet function tests for monitoring the responsiveness to clopidogrel.

Adenosine diphosphate-inducible platelet reactivity shows a pronounced age dependency in the initial phase of antiplatelet therapy with clopidogrel.

BACKGROUND: Until recently, there were hardly any data on the antiplatelet effect of clopidogrel in advanced age. Like other metabolic processes, the conversion of clopidogrel to its active metabolite may be impaired in older patients, leading to high on-treatment residual ADP-inducible platelet reactivity. OBJECTIVE: To investigate the age dependency of clopidogrel-mediated platelet inhibition. PATIENTS AND METHODS: This was a prospective observational study. We determined adenosine 5'-diphosphate (ADP)-inducible platelet reactivity using light transmission aggregometry (LTA) and the VerifyNow P2Y12 assay in 191 patients on dual antiplatelet therapy after angioplasty and stenting for cardiovascular disease. RESULTS: ADP-inducible platelet reactivity increased linearly with age after adjustment for cardiovascular risk factors, type of intervention, medication, C-reactive protein (CRP) and renal function [using LTA 0.36% of maximal aggregation per year, 95% CI 0.08-0.64%, P = 0.013; using the VerifyNow P2Y12 assay 3.2 P2Y12 reaction units (PRU) per year, 95% CI 1.98-4.41 PRU, P < 0.001]. ADP-inducible platelet reactivity was significantly higher in patients aged 75 years or older compared with younger patients (P = 0.003 for LTA and P < 0.001 for the VerifyNow P2Y12 assay). Further, high on-treatment residual ADP-inducible platelet reactivity was significantly more common among patients aged 75 years or older (P = 0.02 for LTA and P < 0.001 for the VerifyNow P2Y12 assay). CONCLUSION: ADP-inducible platelet reactivity shows a pronounced age dependency in the initial phase of antiplatelet therapy with clopidogrel. The clinical implications of these findings need to be addressed in future trials.

Ticagrelor--a new platelet aggregation inhibitor in patients with acute coronary syndromes. An improvement of other inhibitors?

Antiplatelet agents play an essential role in the treatment of acute coronary syndrome (ACS). Thienopyridines are a class of drugs that function via inhibition of the adenosine diphosphate (ADP) P2Y12 platelet receptors. Currently, clopidogrel, a second generation thienopyridine, is the main drug of choice and the combination of aspirin and clopidogrel is administered orally for the treatment of ACS. Clopidogrel, is a pro-drug that needs to be metabolized in the liver and intestines to form active metabolites. Prasugrel, a third generation thienopyridine, was approved for use in Europe in February 2009, and is currently available in the United Kingdom. All thienopyridines however, have pharmacological limitations that lead to a search for more effective non-thienopyridine P2Y12 inhibitors. Promising results have been reported with ticagrelor, an oral first reversible, direct-acting inhibitor of the P2Y12 receptor. Ticagrelor is the first oral P2Y12 receptor binding antagonist that does not require metabolic activation. Furthermore, ticagrelor has at last 1 active metabolite, which has very similar pharmacokinetics to the parent compound. Therefore, ticagrelor has more rapid onset and more pronounced platelet inhibition than other antiplatelet agents. The safety and efficacy of ticagrelor compared with clopidogrel in ACS patient has been recently evaluated by the PLATelet inhibition and patient Outcomes (PLATO) trial. Ticagrelor compared with clopidogrel had a significantly greater reduction in the death rate from vascular causes, myocardial infarction, or stroke without major bleeding. There was however, an increase in non-procedure related bleeding, dyspnoea and ventricular pauses in the first week of treatment. Further studies on new antiplatelet agents are needed to establish a new "gold standard" antiplatelet therapy.

The dual nature of extracellular ATP as a concentration-dependent platelet P2X1 agonist and antagonist.

Patient groups subject to higher occurrence of stroke (e.g., people with diabetes, cystic fibrosis, pulmonary hypertension) have reduced release of ATP from their erythrocytes (ERYs) when subjected to flow-induced deformation or pharmacological stimuli. These same groups also have platelets that are more adhesive in comparison to controls. Here we show platelet aggregation, and inhibition of that aggregation, is affected by free Ca(2+) entering the platelet through the ATP-gated P2X1 receptor. The addition of ATP (10 microM) increased the platelet NO by 26.7 +/- 7.7%. This value was decreased significantly to below basal levels in the presence of NF 449 (p < 0.001), an inhibitor of the P2X1 receptor on the platelet. Aggregation profiles measured in the presence of ATP revealed that when the P2X1 receptor was blocked, or when the measurements were performed in Ca(2+) free buffer, platelet aggregation was nearly eliminated. Our findings employing standard aggregation measurements suggest that ATP behaves as a platelet inhibitor below 1.6 x 10(-19) moles ATP per platelet; however, above this value, ATP behaves as a platelet activator. These findings suggesting a dual nature of ATP with regard to platelet behavior were confirmed by passing platelets over endothelial cells that were coated in the channels of a microfluidic device. Importantly, it was determined that ERY-derived ATP release was a major determinant of platelet adhesion to the endothelium. These findings may have implications in anti-platelet drug design as most current therapies focus on the inhibition of P2Y-type receptors. Moreover, through the use of microfluidic technologies, we have provided in vitro evidence for a possible relationship between ERY properties and platelet behavior in vivo.

Platelet function testing and implications for clinical practice.

Platelets are key mediators in the pathophysiology of atherothrombotic disease. Inappropriate platelet activation can lead to thrombosis and ischemic events. Platelet function testing has been used to monitor patient response to antiplatelet therapy. Variability in response to antiplatelet therapy may be due in part to incomplete inhibition of targeted pathways (thromboxane A(2) and adenosine diphosphate [ADP]) by currently available agents (aspirin and P2Y(12) ADP receptor antagonists). Low responsiveness to antiplatelet therapy (as measured in various platelet function assays) correlates with a high rate of ischemic events. However, tailoring treatment based on platelet response remains to be definitively proven in clinical trials, correlations between assays are modest, and concordance in defining suboptimal response is poor. Additional studies are needed to determine whether changes in therapy based on results of platelet function testing improve clinical outcomes, and thus will determine whether broader use of platelet function testing in clinical practice is warranted.

Dysfunctional inflammasome in Schnitzler's syndrome.

OBJECTIVE: IL-1beta plays a key role in the pathogenesis of Schnitzler's syndrome (SS). We have investigated inflammasome activity in peripheral blood mononuclear cells (PBMCs) from a patient affected by a variant type of SS. METHODS: PBMCs were purified by Ficoll and examined for ability to secrete IL-1beta and -18, expression and function of the P2X(7) receptor and expression of apoptosis-associated speck-like protein containing a caspase recruitment domaine (ASC) and NOD-like receptor protein 3 (NLRP3) before and after the therapy with steroid. Furthermore, extracellular adenosine 5'-triphosphate (ATP) blood levels were determined by luciferase assay. Expression of inflammasome components was measured by real time PCR and western blotting. RESULTS: PBMCs of patient with SS showed a high, spontaneous and lipopolysaccharide-stimulated, IL-1beta release but low response to stimulation with the P2X(7) agonist benzoyl ATP. P2X(7) expression was several fold increased, whereas ASC expression was dramatically decreased compared with PBMCs from healthy controls. NLRP3 expression was unchanged. Prednisone treatment induced remission of clinical symptoms and normalized IL-1beta secretion and P2X(7) and ASC expression. CONCLUSION: These findings reveal the presence of an overall derangement of the inflammasome and IL-1beta processing and release in SS.

Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates.

Platelet adhesion is a complex and important event for prevention of blood loss after vessel injury. This study investigated fundamental adhesive mechanisms occurring in an in-vitro assay developed for the measurement of static adhesion of human platelets in plasma. The aim was to gain methodological knowledge that could be used for interpretations of results from other studies using this specific assay. Involvement of adhesive receptors was investigated by the use of various antibodies as well as therapeutic drugs (abciximab, eptifibatide and tirofiban). Inhibitors of adenosine 5'-diphosphate receptors (cangrelor, MRS2179) and of thromboxane A(2) signalling (BM-531) were used to estimate the role of autocrine activation. Adhesion to collagen was found to be mainly mediated by alpha(2)beta(1) and to some extent by alpha(IIb)beta(3). Adhesion to fibrinogen was mediated by alpha(IIb)beta(3). In addition, adenosine 5'-diphosphate-induced adhesion to albumin was dependent on alpha(IIb)beta(3). Furthermore, experiments with cangrelor and BM-531 showed that the majority of the adhesive interactions tested were dependent on adenosine 5'-diphosphate or thromboxane A(2). We conclude that the mechanisms of adhesion measured by the static platelet adhesion assay are in accordance with the current knowledge regarding platelet activation and adhesion. Despite its simplicity, we suggest that this adhesion assay could be used as a screening device for the study of the influence of various surfaces and soluble substances on platelet adhesion.

Comparison of methods to evaluate clopidogrel-mediated platelet inhibition after percutaneous intervention with stent implantation.

A high on-treatment residual ADP-inducible platelet reactivity in light transmission aggregometry (LTA) has been associated with an increased risk of adverse outcomes after percutaneous coronary intervention (PCI). However, LTA is weakly standardized, and results obtained in one laboratory may not be comparable to those obtained in another one. We therefore sought to determine the test correlating best with LTA to estimate clopidogrel-mediated platelet inhibition in 80 patients on dual antiplatelet therapy after elective percutaneous intervention with stent implantation. We selected the VerifyNow P2Y12 assay, the vasodilator-stimulated phosphoprotein phosphorylation assay, multiple electrode platelet aggregometry and the Impact-R for comparisons with LTA. Cut-off values for residual ADP-inducible platelet reactivity were defined according to quartiles of each assay. Sensitivities and specificities of the different platelet function tests were based on the results from LTA. The results from all four assays correlated significantly with those from LTA. The VerifyNow P2Y12 assay revealed the strongest correlation (r = 0.61, p < 0.001). Sensitivities and specificities ranged from 35% to 55%, and from 78.3% to 85%, respectively. In conclusion, although all assays correlated significantly with LTA, they need to be improved to become clinically used diagnostic tests. Further, it may be too early to define the gold standard method for assessing residual ADP-inducible platelet reactivity and generally acceptable cut-off values.