Exploring the potential cost-effectiveness of a novel platelet assay for guiding dual antiplatelet therapy duration in acute coronary syndrome patients following percutaneous coronary intervention.

OBJECTIVE: Duration of dual antiplatelet therapy (DAPT) after percutaneous coronary intervention (PCI) influences ischemic and bleeding events. Platelet expression of constant fragment of immunoglobulin, low affinity IIa, receptor (FcγRIIa) independently predicts risk of ischemic complications and is proposed as a tool to guide individualized care. METHODS: We used a Markov model to predict lifetime ischemic and bleeding events and healthcare costs in acute myocardial infarction (MI) patients treated with PCI and DAPT and to project cost-effectiveness of platelet FcγRIIa-assay-guided care (30:3 months DAPT for patients at high: low ischemic risk) versus current standard care (12 months DAPT) from the perspective of the US healthcare system. Model inputs included assay sensitivity and specificity, ischemic and bleeding event rates, and impacts on quality of life, mortality, and costs. Assay cost was $90. Sensitivity analyses were conducted over a range of plausible clinical and cost assumptions. RESULTS: Under base case assumptions, platelet FcγRIIa-assay-guided DAPT duration was projected to increase lifetime costs by $19 versus standard care, with an associated incremental cost-effectiveness ratio (ICER) of $436 per quality-adjusted life-year (QALY) gained. Assay-guided DAPT duration was consistent with high-value care (ICER < $50 000/QALY gained) over a broad range of alternative assumptions. CONCLUSION: Based on a decision-analytic model, for patients with MI treated with PCI, the additional costs of the platelet FcγRIIa assay for guiding DAPT duration would be largely offset by reductions in downstream event-related costs, and assay-guided care would be highly cost-effective by current standards. These findings require confirmation in prospective studies and in a randomized clinical trial of assay-guided versus nonassay-guided DAPT duration.

Assessment of Cardiovascular Risk by the Combination of Clinical Risk Scores Plus Platelet Expression of FcγRIIa.

Platelet expression of FcγRIIa was quantified after myocardial infarction (MI) and we found that patients with high platelet FcγRIIa expression (>11,000/platelet) had a fourfold greater risk of subsequent MI, stroke, and death. This analysis of the original cohort of 197 patients was designed to determine whether platelet expression of FcγRIIa could be used in combination with clinical risk scores (GRACE [Global Registry of Acute Coronary Events] and DAPT [Dual Antiplatelet Therapy]) to refine cardiovascular risk assessment. Platelet expression of FcγRIIa quantified with the use of flow cytometry was broadly distributed in patients stratified into high and low risk groups based on clinical risk scores. In patients identified as high risk by the GRACE score, 62% had high platelet FcγRIIa expression. Similarly, in patients identified as high risk by DAPT, 55% had high platelet FcγRIIa expression. High platelet FcγRIIa expression discriminated high and low risk cohorts in patients with high cardiovascular risk defined by either the GRACE score (high platelet FcγRIIa 18.9% vs low platelet FcγRIIa 0%; odds ratio = 15.7, p = 0.06) or the DAPT score (high platelet FcγRIIa 15.4% vs low platelet FcγRIIa 3.7%; odds ratio = 5.6, p = 0.03) assessment. Platelet expression of FcγRIIa merits additional study to determine whether low platelet FcγRIIa expression can be used to guide early transition to aspirin monotherapy and high platelet FcγRIIa expression can be used to guide continuation of DAPT.

Change of energy expenditure from physical activity is the most powerful determinant of improved insulin sensitivity in overweight patients with coronary artery disease participating in an intensive lifestyle modification program.

The objective was to evaluate the determinants of change (Δ) in insulin sensitivity in overweight coronary artery disease male patients without diabetes after an intensive lifestyle intervention. All patients received nutritional counseling and performed 4 months of exercise training (ET) according to 1 of 2 protocols: aerobic ET (65%-70% of peak aerobic capacity [VO(2)]) 25 to 40 minutes 3 times a week (n = 30) or walking (50%-60% of peak VO(2)) 45 to 60 minutes at least 5 times a week (n = 30). Data from participants of both ET groups were pooled, and post-intensive lifestyle intervention results were compared with baseline data. The primary outcome was Δ insulin sensitivity (m-value) assessed by the criterion standard technique, the euglycemic-hyperinsulinemic clamp. Changes in weight, body mass index, total and percentage fat mass (by dual-energy x-ray absorptiometry scan), waist circumference, total abdominal and visceral fat (by computed tomographic scan), high-sensitivity C-reactive protein, peak VO(2), daily energy intake, and physical activity energy expenditure (PAEE) (by doubly labeled water technique) were also assessed. Daily energy intake decreased by 335 kcal, and PAEE increased by 482 kcal/d (all P < .0001). The mean weight loss was 6.4 kg, and the mean improvement in m-value was 1.6 mg/kg fat-free mass per minute. Univariate determinants of Δ m-value were low baseline PAEE, walking protocol, Δ weight, Δ body mass index, Δ total and percentage fat mass, Δ waist circumference, Δ total abdominal and visceral fat, and Δ PAEE (all P < .05). In multivariate analysis, the only significant determinant of Δ m-value was Δ PAEE (P < .02). In this analysis, the most powerful determinant of improved insulin sensitivity in overweight coronary artery disease patients is the change in PAEE.

High-calorie-expenditure exercise: a new approach to cardiac rehabilitation for overweight coronary patients.

BACKGROUND: More than 80% of patients entering cardiac rehabilitation (CR) are overweight, and >50% have metabolic syndrome. Current CR exercise protocols result in little weight loss and minimal changes in cardiac risk factors. We sought to design an exercise protocol that would lead to greater weight loss and risk factor change. METHODS AND RESULTS: We performed a randomized controlled clinical trial to evaluate the effect of high-calorie-expenditure exercise (3000- to 3500-kcal/wk exercise-related energy expenditure) compared with standard CR exercise (7 to 800 kcal/wk) on weight loss and risk factors in 74 overweight patients with coronary heart disease. Both groups were counseled for weight loss and taking evidence-based preventive medications. High-calorie-expenditure exercise resulted in double the weight loss (8.2+/-4 versus 3.7+/-5 kg; P<0.001) and fat mass loss (5.9+/-4 versus 2.8+/-3 kg; P<0.001) and a greater waist reduction (-7+/-5 versus -5+/-5 cm; P=0.02) than standard CR exercise at 5 months. High-calorie-expenditure exercise reduced insulin resistance, measured with the euglycemic hyperinsulinemic clamp, along with the ratio of total to high-density lipoprotein cholesterol and components of the metabolic syndrome, more than standard CR exercise (each P<0.01). Overall, fat mass loss best predicted improved metabolic risk, and the prevalence of metabolic syndrome decreased from 59% to 31%. Changes in cardiac risk factors included decreased insulin resistance, increased high-density lipoprotein cholesterol, and decreased measures of insulin, triglycerides, blood pressure, plasminogen activator inhibitor-1, and the ratio of total to high-density lipoprotein cholesterol (each P<0.05). Significant weight loss was maintained at 1 year. CONCLUSIONS: High-calorie-expenditure exercise promotes greater weight loss and more favorable cardiometabolic risk profiles than standard CR for overweight coronary patients.

Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads.

Bacteria that survive under variable conditions possess an assortment of genetic regulators to meet these challenges. The group IV or extracytoplasmic function (ECF) sigma factors regulate gene expression in response to specific environmental signals by altering the promoter specificity of RNA polymerase. We have undertaken a study of PvdS, a group IV sigma factor encoded by Pseudomonas syringae pv. tomato DC3000 (DC3000), a plant pathogen that is likely to encounter variations in nutrient availability as well as plant host defences. The gene encoding PvdS was previously identified by sequence similarity to the Pseudomonas aeruginosa orthologue, which directs transcription of genes encoding the biosynthesis of pyoverdine, a siderophore involved in iron acquisition, and is responsible for the characteristic fluorescence of the pseudomonads. We identified 15 promoters regulated by PvdS in DC3000 and characterized the promoter motif using computational analysis. Mutagenesis of conserved nucleotides within the motif interfered with promoter function and the degree of the effect was different depending on which region of the motif was mutated. Hidden Markov models constructed from alignments of sequence motifs extracted from DC3000 and PAO1 were used to query genomes of DC3000 and other fluorescent pseudomonads for similar motifs. We conclude that the role of PvdS as a regulator of pyoverdine synthesis is conserved among the fluorescent pseudomonads, but the promoters recognized by PvdS orthologues may differ subtly from species to species.

Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A.

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.

Efficiency in clinical research: assessment in vitro of potential anti-thrombotic drug interactions.

OBJECTIVE: To determine whether testing in vitro of combinations of anti-thrombotic agents can identify potentially important interactions, we evaluated the combination of rNAPc2 with antagonists of platelet GP IIb-IIIa to identify potentially altered anticoagulant properties, antiplatelet effects, or both. METHODS: Blood was obtained from healthy subjects who were taking aspirin (325 mg/day). Selected concentrations of rNAPc2, enoxaparin, and GP IIb-IIIa inhibitors were added in vitro. Platelet function was assessed with the use of flow cytometry. RESULTS: No effect on clotting or platelet inhibition was apparent when abciximab was added to the combination of aspirin, enoxaparin, and rNAPc2 at concentrations up to 250 ng/ml. A modest (less than 10%, P <0.02) effect on the time to clot assessed with the activated clotting time was demonstrated when either eptifibatide or tirofiban was combined with aspirin and enoxaparin plus rNAPc2. rNAPc2 did not alter antiplatelet effects of eptifibatide. By contrast, a modest, approximately 10%, increase in the inhibition of fibrinogen binding (P <0.01) was seen when rNAPc2 was added to the combination of aspirin, enoxaparin, and tirofiban. CONCLUSIONS: The lack of an exaggerated effect on clotting and platelet function when GP IIb-IIIa inhibitors were combined with rNAPc2, aspirin, and enoxaparin suggests that no substantial increment in the incidence of bleeding would be observed when concentrations of rNAPc2 up to 250 ng/ml were to be used in clinical studies. More extensive use of testing in vitro in advance of large-scale clinical trials of anti-thrombotic agents and regimens is likely to enhance their design and implementation.

Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor.

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N(epsilon)-(indole-3-acetyl)-l-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.