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1.  Effect of common single-nucleotide polymorphisms in acetylsalicylic acid metabolic pathway genes on platelet reactivity in patients with diabetes 
Background
Platelet reactivity in patients on acetylsalicylic acid (ASA) therapy can be influenced by physiological or pathological conditions affecting ASA pharmacokinetics or pharmacodynamics. The mechanism of such variability in the therapeutic response to ASA, particularly in diabetic patients, is poorly understood. The rate of elimination of ASA and its metabolite, salicylic acid (SA), is likely a major factor determining drug efficacy. The objective of this study was to investigate the effect of genetic polymorphisms in the selected candidate genes within the ASA metabolic pathway on the platelet reactivity and concentration of ASA and thromboxane A2 (TxA2) metabolites in a population of patients with type 2 diabetes mellitus (T2DM).
Material/Methods
The study cohort consisted of 287 Caucasians with T2DM who had been taking ASA tablets at the dose of 75 mg per day for at least 3 months. Platelet reactivity analyses were performed using VerifyNow Aspirin and PFA-100 assays. The measured ASA metabolite included salicylic acid (ASA), and TxA2 metabolites included serum TxB2 and urinary 11-dh-TxB2. Genotyping for the selected 18 single-nucleotide polymorphisms (SNPs) within 5 genes of the ASA metabolic pathway was performed using a Sequenom iPLEX platform.
Results
No statistically significant association was observed between the investigated SNPs genotypes, platelet reactivity, and measured metabolites in the investigated cohort of patients.
Conclusions
The results of our study failed to confirm that the selected variants in the genes within the ASA metabolic pathway might contribute to platelet reactivity in a diabetic population treated with ASA.
doi:10.12659/MSM.883922
PMCID: PMC3670858  PMID: 23715170
acetylsalicylic acid; cyclooxygenase-1; diabetes mellitus; single-nucleotide polymorphisms; thromboxane; salicylic acid
2.  Tailored antiplatelet therapy can overcome clopidogrel and aspirin resistance - The BOchum CLopidogrel and Aspirin Plan (BOCLA-Plan) to improve antiplatelet therapy 
BMC Medicine  2011;9:3.
Background
Dual antiplatelet therapy using acetylsalicylic acid (ASA, aspirin) and clopidogrel is of great importance following coronary stenting. However, the variable platelet inhibitory effectiveness compromises the antithrombotic advantages provided by dual antiplatelet therapy. The aim of this single-center prospective study was to reduce the low response incidence of dual antiplatelet therapy with ASA and clopidogrel according to a prespecified therapy algorithm.
Methods
Platelet function testing using whole blood aggregometry (Chronolog 590) was performed 48 hours following coronary stenting (for either acute coronary syndromes or stable coronary artery disease) on 504 patients. The antiplatelet therapy included a loading dose of 600 mg clopidogrel and 500 mg ASA, followed by 75 mg clopidogrel and 100 mg ASA once daily. Clopidogrel low responders (CLR: >5 ohm; adenosine diphosphate (ADP) 5 μM) and/or ASA low responders (ALR: >0 ohm; arachidonic acid 10 μM) were treated according to a structured therapy plan: in the case of CLR, the maintenance + dose was doubled (repeated loading dose followed by 150 mg daily), and when still ineffective ticlopidine or prasugrel, if available and not contraindicated, were used. ALR was treated by increasing the dose to 300 mg in a first step or to 500 mg ASA when the first modification did not take effect sufficiently. In addition, ADP receptor antagonist 2-methylthioadenosine 5'-monophosphate triethylammonium salt (MeSAMP) testing and ASA incubation were performed to rule out either a platelet ADP-receptor defect or an ASA pharmacokinetic resistance.
Results
Of the total cohort of 504 patients, we detected 30.8% clopidogrel low-responders and 19.4% aspirin low-responders. For ALR, with a dose adjustment of 300 mg ASA daily, 94.6% of ALR were effectively treated and the residual 5.4% by administration of daily dosages of 500 mg ASA. This means that after modification of the ASA maintenance dose, all initial ALRs had an adequate antiplatelet response.
The results for clopidogrel revealed that 69% of the CLR were treated effectively by increasing the clopidogrel dose to 150 mg daily. When prasugrel was not available or contraindicated, 12.7% of the remaining low responders showed an adequate result after being switched to ticlopidine. Consequently, by applying the therapy algorithm, we were able to reduce the CLR prevalence by 86.6%. On including prasugrel in the therapy plan, we were finally able to eliminate thienopyridine low response. In addition, no ADP receptor defect was found in this study as a potential reason for CLR.
We identified the following factors associated with both CLR and ALR status: acute coronary syndromes, positive troponin values as well as diabetes mellitus and elevated HbA1C values and a higher platelet count. Furthermore, our data revealed for CLR elevated C-reactive protein values and a high PREDICT-score (including an age >65 years, acute coronary syndrome, diabetes mellitus, renal failure, and reduced left ventricular function) as risk factors. The following factors correlated with the risk of ASA low response: patients with elevated hemoglobin, serum creatinine and C-reactive protein values. In addition, medication with nitrates reduced the risk of being CLR. As also holds true for CLR, we found the PREDICT-score to be correlated to the risk of being ALR. However, by far the strongest risk factor for CLR or ALR was the fact of dual resistance.
Conclusion
Following a structured therapy plan based on a "test and treat" strategy, the prevalence of clopidogrel or aspirin low response can be significantly reduced and the risk of inadequate dual antiplatelet therapy minimized.
Trial Registration
NCT01212302 (Clinicaltrials.gov)
doi:10.1186/1741-7015-9-3
PMCID: PMC3033359  PMID: 21226927
3.  Challenging the FDA Black Box Warning for High Aspirin Dose With Ticagrelor in Patients With Diabetes 
Diabetes  2013;62(3):669-671.
Ticagrelor, a novel reversible antiplatelet agent, has a Food and Drug Administration (FDA) black box warning to avoid maintenance doses of aspirin (ASA) >100 mg/daily. This restriction is based on the hypothesis that ASA doses >100 mg somehow decreased ticagrelor’s benefit in the Platelet Inhibition and Patient Outcomes (PLATO) U.S. cohort. However, these data are highly postrandomized, come from a very small subgroup in PLATO (57% of patients in the U.S. site), and make no biological sense. Moreover, the ticagrelor-ASA interaction was not significant by any multivariate Cox regression analyses. The Complete Response Review for ticagrelor indicates that for U.S. PLATO patients, an ASA dose >300 mg was not a significant interaction for vascular outcomes. In the ticagrelor-ASA >300 mg cohort, all-cause and vascular mortality were not significantly increased (hazard ratio [HR] 1.27 [95% CI 0.84–1.93], P = 0.262 and 1.39 [0.87–2.2], P = 0.170), respectively. Furthermore, for major adverse cardiovascular events (MACEs), 30-day all-cause mortality, and 30-day vascular mortality, the strongest interaction is the diabetes-ASA interaction. That is, patients who had diabetes had significantly fewer MACEs through study end (0.49 [0.34–0.63], P < 0.0001), significantly less 30-day all-cause mortality (0.33 [0.20–0.56], P < 0.0001), and significantly less 30-day vascular mortality (0.35 [0.22–0.55], P < 0.0001), respectively, when given high-dose (300–325 mg) ASA, regardless of treatment (clopidogrel or ticagrelor) assignment. The black box warning for the use of maintenance ASA doses >100 mg with ticagrelor is inappropriate for patients with diabetes and not evidence based.
doi:10.2337/db12-0746
PMCID: PMC3581224  PMID: 23431005
4.  The effect of clopidogrel on platelet activity in patients with and without type-2 diabetes mellitus: a comparative study 
Background
Although antiplatelet therapy involving clopidogrel is a standard treatment for preventing cardiovascular events after coronary stent implantation, patients can display differential responses. Here, we assessed the effectiveness of clopidogrel on platelet function inhibition in subjects with and without type-2 diabetes and stable coronary artery disease. In addition, we investigated the correlation between platelet function and routine clinical parameters.
Methods
A total of 64 patients with stable coronary heart disease were enrolled in the study. Among these, 32 had known type-2 diabetes, whereas the remaining 32 subjects were non-diabetics (control group). A loading dose of 300 mg clopidogrel was given to clopidogrel-naïve patients (13 patients in the diabetes group and 14 control patients). All patients were given a daily maintenance dose of 75 mg clopidogrel. In addition, all patients received 100 mg ASA per day. Agonist-induced platelet aggregation measurements were performed on hirudin-anticoagulated blood using an impedance aggregometer (Multiple Platelet Function Analyzer, Dynabyte, Munich, Germany). Blood samples were drawn from the antecubital vein 24 h after coronary angiography with percutaneous coronary intervention. The platelets were then stimulated with ADP alone or ADP and prostaglandin-E (ADP and ADP-PGE tests, respectively) in order to evaluate clopidogrel-mediated inhibition of platelet function. The effectiveness of ASA was measured by stimulation with arachidonic acid (ASPI test). In addition, maximal platelet aggregation was assessed via stimulation with thrombin receptor-activating peptide (TRAP test).
Results
Patients with diabetes exhibited significantly less inhibition of platelet function than patients without diabetes (ADP-PGE test p = 0.003; ASPI test p = 0.022). Administering a clopidogrel loading dose of 300 mg did not result in a lower level of ADP-PGE-induced platelet reactivity in comparison to the use of a 75 mg maintenance dose. Moreover, we observed that ADP-PGE-induced platelet inhibition was positively correlated with fasting blood glucose and HbA1c (p < 0.01).
Conclusions
Patients with type-2 diabetes exhibited increased platelet reactivity compared to patients without diabetes despite combined treatment with clopidogrel and ASA. Using a loading dose of clopidogrel rather than small daily doses was not sufficient for adequately overcoming increased platelet reactivity in patients with type-2 diabetes, highlighting the need for more effective anti-platelet drugs for such patients.
doi:10.1186/s12933-015-0182-7
PMCID: PMC4324649  PMID: 25645908
Clopidogrel; Diabetes; Platelet function; Ccoronary heart disease; Percutaneous coronary intervention
5.  Effects of P2Y1 receptor antagonism on the reactivity of platelets from patients with stable coronary artery disease using aspirin and clopidogrel 
British Journal of Pharmacology  2012;166(1):221-231.
BACKGROUND AND PURPOSE
P2Y1 is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy.
EXPERIMENTAL APPROACH
Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day−1) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y1 inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed.
KEY RESULTS
Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and αIIbβ3 activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited αIIbb3 activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s−1), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y12 inhibitor 2-MeSAMP.
CONCLUSIONS AND IMPLICATIONS
Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y1 in the blood of patients with CAD appears to play a more important role than previously anticipated.
doi:10.1111/j.1476-5381.2011.01683.x
PMCID: PMC3415650  PMID: 21950486
platelets; P2Y1; P2Y12; clopidogrel; MRS2179; aspirin; coronary artery disease
6.  Platelet activation following intravenous injection of a conventional heparin: absence of effect with a low molecular weight heparinoid (Org 10172). 
1. The effects of an intravenous injection of a conventional high molecular weight heparin (HMWH) were compared with those of a low molecular weight heparinoid (Org 10172). A bolus injection of HMWH (5000 iu) was associated with: (a) a small but significant prolongation of bleeding time (BT); (b) a significant fall in PRP platelet count; (c) significantly enhanced platelet aggregation; and (d) significantly increased platelet thromboxane A2 (TXA2) release. These changes were not observed following the intravenous injection of Org 10172 (3200 anti Xa U). 2. These experiments were repeated following the oral administration of acetylsalicylic acid (ASA). HMWH again caused some enhancement of platelet aggregation despite the ASA-mediated inhibition of platelet aggregation and TXA2 release. Administration of Org 10172 to subjects taking ASA did not alter any of the platelet function indices. 3. In additional control experiments the injection of 5000 iu of HMWH was associated with a significant fall in PRP but not whole blood platelet counts. This finding suggests that the fall in PRP platelet count is a methodological artefact. 4. The HMWH also induced a significantly greater increase in serum non-esterified fatty acid (NEFA) concentrations than Org 10172. 5. The present findings indicate that Org 10172 is a less potent stimulator of platelet aggregation and lipolysis than HMWH. 6. The minor prolongation of the BT after HMWH is not compatible with enhanced aggregation but may be a consequence of alterations in the activity of coagulation factors and vascular-platelet interactions or of ongoing platelet activation accompanied by granule depletion. 7. The different effects of the two anticoagulants assessed suggests a therapeutic advantage in favour of Org 10172, especially in patients with hyperactive platelets.
PMCID: PMC1386302  PMID: 3689624
7.  Platelet Content of Nitric Oxide Synthase 3 Phosphorylated At Serine1177 Is Associated with the Functional Response of Platelets to Aspirin 
PLoS ONE  2013;8(12):e82574.
Objective
To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO).
Patients/Methods
Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100).
Results
ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position −786 (T−786→C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets.
Conclusions
Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser1177.
doi:10.1371/journal.pone.0082574
PMCID: PMC3869699  PMID: 24376548
8.  Aspirin and Extended-Release Dipyridamole versus Clopidogrel for Recurrent Stroke 
The New England journal of medicine  2008;359(12):1238-1251.
BACKGROUND
Recurrent stroke is a frequent, disabling event after ischemic stroke. This study compared the efficacy and safety of two antiplatelet regimens — aspirin plus extendedrelease dipyridamole (ASA–ERDP) versus clopidogrel.
METHODS
In this double-blind, 2-by-2 factorial trial, we randomly assigned patients to receive 25 mg of aspirin plus 200 mg of extended-release dipyridamole twice daily or to receive 75 mg of clopidogrel daily. The primary outcome was first recurrence of stroke. The secondary outcome was a composite of stroke, myocardial infarction, or death from vascular causes. Sequential statistical testing of noninferiority (margin of 1.075), followed by superiority testing, was planned.
RESULTS
A total of 20,332 patients were followed for a mean of 2.5 years. Recurrent stroke occurred in 916 patients (9.0%) receiving ASA–ERDP and in 898 patients (8.8%) receiving clopidogrel (hazard ratio, 1.01; 95% confidence interval [CI], 0.92 to 1.11). The secondary outcome occurred in 1333 patients (13.1%) in each group (hazard ratio for ASA–ERDP, 0.99; 95% CI, 0.92 to 1.07). There were more major hemorrhagic events among ASA–ERDP recipients (419 [4.1%]) than among clopidogrel recipients (365 [3.6%]) (hazard ratio, 1.15; 95% CI, 1.00 to 1.32), including intracranial hemorrhage (hazard ratio, 1.42; 95% CI, 1.11 to 1.83). The net risk of recurrent stroke or major hemorrhagic event was similar in the two groups (1194 ASA–ERDP recipients [11.7%], vs. 1156 clopidogrel recipients [11.4%]; hazard ratio, 1.03; 95% CI, 0.95 to 1.11).
CONCLUSIONS
The trial did not meet the predefined criteria for noninferiority but showed similar rates of recurrent stroke with ASA–ERDP and with clopidogrel. There is no evidence that either of the two treatments was superior to the other in the prevention of recurrent stroke.
doi:10.1056/NEJMoa0805002
PMCID: PMC2714259  PMID: 18753638
9.  Microfluidic Assay of Platelet Deposition on Collagen by Perfusion of Whole Blood from Healthy Individuals Taking Aspirin 
Clinical chemistry  2013;59(8):1195-1204.
BACKGROUND
Microfluidic devices can create hemodynamic conditions for platelet assays. We validated an 8-channel device in a study of interdonor response to acetylsalicylic acid (ASA, aspirin) with whole blood from 28 healthy individuals.
METHODS
Platelet deposition was assessed before treatment or 24 h after ingestion of 325 mg ASA. Whole blood (plus 100 μmol/L H-D-Phe-Pro-Arg-chloromethylketone to inhibit thrombin) was further treated ex vivo with ASA (0–500 μmol/L) and perfused over fibrillar collagen for 300 s at a venous wall shear rate (200 s−1).
RESULTS
Ex vivo ASA addition to blood drawn before aspirin ingestion caused a reduction in platelet deposition [half-maximal inhibitory concentration (IC50) approximately 10–20 μmol/L], especially between 150 and 300 s of perfusion, when secondary aggregation mediated by thromboxane was expected. Twenty-seven of 28 individuals displayed smaller deposits (45% mean reduction; range 10%–90%; P < 0.001) from blood obtained 24 h after ASA ingestion (no ASA added ex vivo). In replicate tests, an R value to score secondary aggregation [deposition rate from 150 to 300 s normalized by rate from 60 to 150 s] showed R < 1 in only 2 of 28 individuals without ASA ingestion, with R > 1 in only 3 of 28 individuals after 500 μmol/L ASA addition ex vivo. At 24 h after ASA ingestion, 21 of 28 individuals displayed poor secondary aggregation (R < 1) without ex vivo ASA addition, whereas the 7 individuals with residual secondary aggregation (R > 1) displayed insensitivity to ex vivo ASA addition. Platelet deposition was not correlated with platelet count. Ex vivo ASA addition caused similar inhibition at venous and arterial wall shear rates.
CONCLUSIONS
Microfluidic devices quantified platelet deposition after ingestion or ex vivo addition of aspirin.
doi:10.1373/clinchem.2012.198101
PMCID: PMC4119612  PMID: 23592503
10.  Younger age, higher body mass index and lower adiponectin concentration predict higher serum thromboxane B2 level in aspirin-treated patients with type 2 diabetes: an observational study 
Background
Evidence from the literature suggests diminished acetylsalicylic acid (ASA) treatment efficacy in type 2 diabetes (DM2). High on-aspirin platelet reactivity (HAPR) in DM2 has been linked to poor glycemic and lipid control. However, there are no consistent data on the association between HAPR and insulin resistance or adipose tissue metabolic activity. The aim of this study was to assess the relationship between laboratory response to ASA and metabolic control, insulin resistance and adipokines in DM2.
Methods
A total of 186 DM2 patients treated with oral antidiabetic drugs and receiving 75 mg ASA daily were included in the analysis. Response to ASA was assessed by measuring serum thromboxane B2 (TXB2) concentration and expressed as quartiles of TXB2 level. The achievement of treatment targets in terms of glycemic and lipid control, insulin resistance parameters (including Homeostatic Model Assessment-Insulin Resistance, HOMA-IR, index), and serum concentrations of high-molecular weight (HMW) adiponectin, leptin and resistin, were evaluated in all patients. Univariate and multivariate logistic regression analyses were performed to determine the predictive factors of serum TXB2 concentration above the upper quartile and above the median.
Results
Significant trends in age, body mass index (BMI), HOMA-IR, HMW adiponectin concentration, C-reactive protein concentration and the frequency of achieving target triglyceride levels were observed across increasing quartiles of TXB2. In a multivariate analysis, only younger age and higher BMI were independent predictors of TXB2 concentration above the upper quartile, while younger age and lower HMW adiponectin concentration were predictors of TXB2 concentration above the median.
Conclusions
These results suggest that in DM2, the most important predictor of HAPR is younger age. Younger DM2 patients may therefore require total daily ASA doses higher than 75 mg, preferably as a twice-daily regimen, to achieve full therapeutic effect. Higher BMI and lower HMW adiponectin concentration were also associated with less potent ASA effect. This is the first study to demonstrate an association of lower adiponectin concentration with higher serum TXB2 level in patients treated with ASA.
doi:10.1186/s12933-014-0112-0
PMCID: PMC4149275  PMID: 25123549
Aspirin; Platelet aggregation; Diabetes mellitus; Insulin resistance; Adipokines
11.  Selective Cumulative Inhibition of Platelet Thromboxane Production by Low-dose Aspirin in Healthy Subjects 
Journal of Clinical Investigation  1982;69(6):1366-1372.
Acetylation of platelet cyclooxygenase by oral aspirin is dose dependent and cumulative with repeated administration. However, no single dose of aspirin has been found to be completely selective of platelet thromboxane (TX) synthesis inhibition in man. We determined the dose dependence, cumulative nature and selectivity of aspirin effects on platelet TXB2 and renal prostaglandin (PG) and prostacyclin (PGI2) production. We measured, by radioimmunoassay, serum TXB2 levels after whole blood clotting and urinary excretion of PGE2, PGF2α, and 6-keto-PGF1α, before and after single or repeated oral aspirin doses given to 46 healthy subjects. Single doses of 6-100 mg aspirin resulted in a linear (r = 0.92, P < 0.01) inhibition of platelet TXB2 production, ranging from 12 to 95% after 24 h. A daily dose of 0.45 mg/kg given for 7 d produced a cumulative and virtually complete inhibition of platelet TXB2 production, without significantly reducing the urinary excretion of PGE2, PGF2α, and 6-keto-PGF1α in both healthy men and women. The platelet inhibitory effect of this regimen was maintained unaltered throughout 1 mo of therapy, with no evidence of cumulative inhibition of renal PG-synthesis. Moreover, furosemide-induced renal PGI2 synthesis and renin release were unaffected by chronic low-dose aspirin. Following cessation of aspirin therapy, platelet TXB2 production returned toward control values at a similar rate as after a single higher dose.
We conclude that in healthy subjects: (a) aspirin causes a dose-dependent inhibition of platelet TXA2 production, with no obvious sex-related difference; (b) the inhibitory effect of daily low-dose aspirin is cumulative on platelet TXA2 but not on renal PG-synthesis; (c) during chronic low-dose aspirin therapy, renal PGI2-producing cells are readily activable by furosemide at a time of virtually complete suppression of platelet cyclooxygenase activity.
PMCID: PMC370209  PMID: 7045161
12.  Platelet thromboxane (11-dehydro-Thromboxane B2) and aspirin response in patients with diabetes and coronary artery disease 
World Journal of Diabetes  2014;5(2):115-127.
Aspirin (ASA) irreversibly inhibits platelet cyclooxygenase-1 (COX-1) leading to decreased thromboxane-mediated platelet activation. The effect of ASA ingestion on thromboxane generation was evaluated in patients with diabetes (DM) and cardiovascular disease. Thromboxane inhibition was assessed by measuring the urinary excretion of 11-dehydro-thromboxane B2 (11dhTxB2), a stable metabolite of thromboxane A2. The mean baseline urinary 11dhTxB2 of DM was 69.6% higher than healthy controls (P = 0.024): female subjects (DM and controls) had 50.9% higher baseline 11dhTxB2 than males (P = 0.0004), while age or disease duration had no influence. Daily ASA ingestion inhibited urinary 11dhTxB2 in both DM (71.7%) and controls (75.1%, P < 0.0001). Using a pre-established cut-off of 1500 pg/mg of urinary 11dhTxB2, there were twice as many ASA poor responders (ASA “resistant”) in DM than in controls (14.8% and 8.4%, respectively). The rate of ASA poor responders in two populations of acute coronary syndrome (ACS) patients was 28.6 and 28.7%, in spite of a significant (81.6%) inhibition of urinary 11dhTxB2 (P < 0.0001). Both baseline 11dhTxB2 levels and rate of poor ASA responders were significantly higher in DM and ACS compared to controls. Underlying systemic oxidative inflammation may maintain platelet function in atherosclerotic cardiovascular disease irrespective of COX-1 pathway inhibition and/or increase systemic generation of thromboxane from non-platelet sources.
doi:10.4239/wjd.v5.i2.115
PMCID: PMC3990310  PMID: 24748925
Diabetes; Cardiovascular disease; Platelets; Thromboxane; Aspirin
13.  Synthesis of Prostacyclin from Platelet-derived Endoperoxides by Cultured Human Endothelial Cells 
Journal of Clinical Investigation  1980;66(5):979-986.
We have previously shown that aspirin-treated endothelial cells synthesize prostacyclin (PGI2) from the purified prostaglandin endoperoxide PGH2 (1978. J. Biol. Chem.253: 7138). To ascertain whether aspirin-treated endothelial cells produce PGI2 from endoperoxides released by stimulated platelets, [3H]arachidonic acid-prelabeled platelets were reacted in aggregometer cuvettes with the calcium ionophore A 23187, thrombin, or collagen in the presence of aspirin-treated endothelial cell suspensions. This procedure permitted thin-layer radiochromatographic quantitation of [3H]PGI2 as [3H]6-keto-PGF1α and [3H]thromboxane A2 (TXA2) as [3H]TXB2, as well as analysis of platelet aggregation responses in the same sample. In the presence of aspirin-treated endothelial cells, platelet aggregation in response to all three agents was inhibited. [3H]6-keto-PGF1α was recovered from the supernates of the combined cell suspensions after stimulation by all three agents. The order of PGI2 production initiated by the stimuli was ionophore > thrombin > collagen. The amounts of platelet [3H]TXB2 recovered were markedly reduced by the addition of aspirin-treated endothelial cells. In separate experiments, 6-keto-PGF1α and TXB2 were quantitated by radioimmunoassay; the results paralleled those obtained with the use of radiolabeling. The quantity of 6-keto-PGF1α measured by radioimmunoassay represented amounts of PGI2 sufficient to inhibit platelet aggregation. These results were obtained when 200,000 platelets/μl were combined with 3,000-6,000 aspirin-treated endothelial cells/μl. At higher platelet levels the proportion of 6-keto-PGF1α to TXB2 decreased and platelet aggregation occurred. Control studies indicated that aspirin-treated endothelial cells could not synthesize PGI2 from exogenous radioactive or endogenous arachidonate when stimulated with thrombin. Therefore the endothelial cell suspensions could only have used endoperoxides from stimulated platelets.
Thus, under our experimental conditions, production by endothelial cells of PGI2 from endoperoxides derived from activated platelets could be demonstrated by two independent methods. These experimental conditions included: (a) enhanced platelet-endothelial cell proximity, as attainable in stirred cell suspensions; (b) use of increased endothelial cell/platelet ratios; and (c) utilization of arachidonate of high specific activity in radiolabeling experiments. Furthermore, when a mixture of platelets and endothelial cells that were not treated with aspirin was stimulated with thrombin, more than twice as much 6-keto-PGF1α was formed than when endothelial cells were stimulated alone. These results indicate that endothelial cells can utilize platelet endoperoxides for PGI2 formation to a significant extent.
PMCID: PMC371534  PMID: 6776148
14.  NO-Donating Aspirin and Aspirin Partially Inhibit Age-Related Atherosclerosis but Not Radiation-Induced Atherosclerosis in ApoE Null Mice 
PLoS ONE  2010;5(9):e12874.
Background
We previously showed that irradiation to the carotid arteries of ApoE−/− mice accelerated the development of macrophage-rich, inflammatory atherosclerotic lesions, prone to intra-plaque hemorrhage. In this study we investigated the potential of anti-inflammatory and anti-coagulant intervention strategies to inhibit age-related and radiation-induced atherosclerosis.
Methodology/Principal Findings
ApoE−/− mice were given 0 or 14 Gy to the neck and the carotid arteries and aortic arches were harvested at 4 or 30 weeks after irradiation. Nitric oxide releasing aspirin (NCX 4016, 60 mg/kg/day) or aspirin (ASA, 30 or 300 mg/kg/day) were given continuously in the chow. High dose ASA effectively blocked platelet aggregation, while the low dose ASA or NCX 4016 had no significant effect on platelet aggregation. High dose ASA, but not NCX 4016, inhibited endothelial cell expression of VCAM-1 and thrombomodulin in the carotid arteries at 4 weeks after irradiation; eNOS and ICAM-1 levels were unchanged. After 30 weeks of follow-up, NCX 4016 significantly reduced the total number of lesions and the number of initial macrophage-rich lesions in the carotid arteries of unirradiated mice, but these effects were not seen in the brachiocephalic artery of the aortic arch (BCA). In contrast, high dose ASA lead to a decrease in the number of initial lesions in the BCA, but not in the carotid artery. Both high dose ASA and NCX 4016 reduced the collagen content of advanced lesions and increased the total plaque burden in the BCA of unirradiated mice. At 30 weeks after irradiation, neither NCX 4016 nor ASA significantly influenced the number or distribution of lesions, but high dose ASA lead to formation of collagen-rich “stable” advanced lesions in carotid arteries. The total plaque area of the irradiated BCA was increased after ASA, but the plaque burden was very low compared with the carotid artery.
Conclusions/Significance
The development and characteristics of radiation-induced atherosclerosis varied between different arteries but could not be circumvented by anti-inflammatory and anti-coagulant therapies. This implicates other underlying mechanistic pathways compared to age-related atherosclerosis.
doi:10.1371/journal.pone.0012874
PMCID: PMC2943480  PMID: 20877628
15.  Low dose of acetylsalicylic acid and oxidative stress-mediated endothelial dysfunction in diabetes: a short-term evaluation 
Acta Diabetologica  2014;52(2):249-256.
Current guidelines suggest the use of low doses of acetylsalicylic acid (ASA) for patients with diabetes mellitus (DM) in primary prevention. However, the evidences demonstrating the beneficial effect of ASA in primary prevention are conflicting. In this pilot study, we evaluated in a group of diabetic patients, in primary prevention, the impact of ASA treatment on oxidative stress and vascular function. We enrolled 22 newly diagnosed diabetic patients, without any previous clinical evidence of cardiovascular disease, to receive, in primary prevention, ASA (100 mg/daily). We tested, in basal condition, after 4 weeks of ASA administration and after 4 weeks of pharmacological washout, the impact of ASA treatment on endothelial function, assessed by a semipletysmographic method, measuring the main oxidative stress parameters related to it. As expected, after 4 weeks of treatment, ASA induced a significant reduction of plasma thromboxane-A2, as a consequence of cyclooxygenase-1 inhibition. By contrast, ASA significantly increased the plasma and urine 8-iso-PGF2α, a well-known prothrombotic molecule, parallel to an increase of plasma NOX2 levels. The enhancement of this oxidative pathway is associated with a significant impairment of endothelial vasodilation, assessed by reactive hyperemia index (RHI). The pharmacological washout reverted all parameters to basal condition. Our findings suggest that ASA utilization for primary prevention in diabetic patients causes a significant increase of oxidative stress burden impairing the vascular function. Present data, if confirmed on a larger population, could permanently discourage the use of the ASA for the primary prevention in patients with DM.
doi:10.1007/s00592-014-0629-4
PMCID: PMC4374120  PMID: 25091345
Aspirin; Endothelial dysfunction; Oxidative stress; Diabetes mellitus
16.  Platelet Isoprostane Overproduction in Diabetic Patients Treated With Aspirin 
Diabetes  2012;61(6):1626-1632.
Aspirin modestly influences cardiovascular events in patients with type 2 diabetes mellitus (T2DM), but the reason is unclear. The aim of the study was to determine whether in T2DM patients aspirin enhances platelet isoprostanes, which are eicosanoids with proaggregating properties derived from arachidonic acid oxidation by platelet NOX2, the catalytic subunit of reduced NAD phosphate oxidase. A cross-sectional study was performed comparing T2DM patients, treated (n = 50) or not treated (n = 50) with 100 mg/day aspirin, with 100 nondiabetic patients, matched for age, sex, atherosclerosis risk factors, and aspirin treatment. A short-term (7 days) treatment with 100 mg/day aspirin also was performed in 36 aspirin-free diabetic and nondiabetic patients. Higher platelet recruitment, platelet isoprostane, and NOX2 activation was found in diabetic versus nondiabetic patients and in aspirin-treated diabetic patients versus nontreated patients (P < 0.001). Platelet thromboxane (Tx) A2 (P < 0.001) was inhibited in all aspirin-treated patients. In the interventional study, aspirin similarly inhibited platelet TxA2 in diabetic and nondiabetic patients (P < 0.001). Platelet recruitment, isoprostane levels, and NOX2 activation showed a parallel increase in diabetic patients (P < 0.001) and no changes in nondiabetic patients. These findings suggest that in aspirin-treated diabetic patients, oxidative stress–mediated platelet isoprostane overproduction is associated with enhanced platelet recruitment, an effect that mitigates aspirin-mediated TxA2 inhibition.
doi:10.2337/db11-1243
PMCID: PMC3357260  PMID: 22427378
17.  APT102, a Novel ADPase, Cooperates With Aspirin to Disrupt Bone Metastasis in Mice 
Journal of cellular biochemistry  2008;104(4):1311-1323.
Platelets contribute to the development of metastasis, the most common cause of mortality in cancer patients, but the precise role that anti-platelet drugs play in cancer treatment is not defined. Metastatic tumor cells can produce platelet aIIbb3 activators, such as ADP and thromboxane A2 (TXA2). Inhibitors of platelet b3 integrins decrease bone metastases in mice but are associated with significant bleeding. We examined the role of a novel soluble apyrase/ ADPase, APT102, and an inhibitor of TXA2 synthesis, acetylsalicylic acid (aspirin or ASA), in mouse models of experimental bone metastases. We found that treatment with ASA and APT102 in combination (ASA + APT102), but not either drug alone, significantly decreased breast cancer and melanoma bone metastases in mice with fewer bleeding complications than observed with aIIbb3 inhibition. ASA + APT102 diminished tumor cell induced platelet aggregation but did not directly alter tumor cell viability. Notably, APT102 + ASA treatment did not affect initial tumor cell distribution and similar results were observed in b3−/− mice. These results show that treatment with ASA + APT102 decreases bone metastases without significant bleeding complications. Anti-platelet drugs such as ASA + APT102 could be valuable experimental tools for studying the role of platelet activation in metastasis as well as a therapeutic option for the prevention of bone metastases.
doi:10.1002/jcb.21709
PMCID: PMC2763643  PMID: 18260128
ADPase; aspirin; bone metastasis; platelets
18.  Cilostazol Reduces PAC-1 Expression on Platelets in Ischemic Stroke 
Background and Purpose
Cilostazol, a phosphodiesterase III inhibitor, is known to be a useful antiplatelet agent that inhibits the progression of atherosclerosis in ischemic stroke. This study investigated the effects of combining cilostazol with aspirin on the expressions of P-selectin and PAC-1 on activated platelets in acute ischemic stroke.
Methods
We analyzed 70 patients with acute ischemic stroke (<72 hrs of an ischemic event). The daily intake was 100 mg of aspirin in 37 patients and 100 mg of aspirin plus 200 mg of cilostazol in 33 patients. The expressions of P-selectin and PAC-1 on activated platelets were measured on the day of admission and 5 days later. We also evaluated the clinical progression using the National Institutes of Health Stroke Scale (NIHSS) at the same times.
Results
After 5 days the extent of PAC-1 expression on activated platelets was significantly lower for combined aspirin and cilostazol treatment (61.0±19.3%, p=0.008; mean±standard deviation) than the baseline level (70.9±12.9%), but did not differ between aspirin alone (66.0 ±19.0%) and baseline (70.1±15.7%). The expression of P-selectin did not differ between combined aspirin and cilostazol treatment and baseline. The clinical progression did not differ between the two groups, as indicated by the absence of significant changes on the NIHSS in the acute period.
Conclusions
This study found that the combined regimen of aspirin and cilostazol exerts the beneficial effect of reducing PAC-1 activity on activated platelets in acute ischemic stroke. However, the clinical outcome of this regimen was no better than that of the aspirin-only regimen. Therefore, further detailed studies of the possible clinical benefits of cilostazol in acute ischemic stroke are needed.
doi:10.3988/jcn.2008.4.4.148
PMCID: PMC2686855  PMID: 19513289
cilostazol; acute ischemic stroke
19.  Celecoxib Does Not Attenuate the Antiplatelet Effects of Aspirin and Clopidogrel in Healthy Volunteers 
Korean Circulation Journal  2010;40(7):321-327.
Background and Objectives
The prevalence of arthritis, which is often treated with celecoxib, is high in patients with coronary artery disease. Furthermore, celecoxib has been reported to reduce restenosis after coronary stenting by inhibiting expression of the proto-oncogene Akt. A concern is that celecoxib increases thrombogenicity by inhibiting the synthesis of prostacyclin in endothelial cells. However, it is not known whether the administration of celecoxib will attenuate the antiplatelet effects of aspirin and clopidogrel, which are used after stenting. We addressed this gap in our knowledge.
Subjects and Methods
We recruited healthy volunteers (n=40) and randomized them into five subgroups (n=8 for each group: aspirin, celecoxib, aspirin+celecoxib, aspirin+clopidogrel, and aspirin+clopidogrel+celecoxib). Each subject received their medications for 6 days and blood samples were taken on day 0 and day 7. Celecoxib (200 mg twice a day), and/or aspirin (100 mg daily), and/or clopidogrel (75 mg daily) were administered. We compared platelet function among subgroups using light transmittance aggregometry and arachidonic acid metabolite assays.
Results
Celecoxib treatment alone did not significantly affect platelet aggregation. The reduction in adenosine diphosphase (ADP)-induced platelet aggregation by aspirin+clopidogrel was not affected by addition of celecoxib (31.3±6.9% vs. 32.4±12.2%, p=0.83). Inhibition of collagen-induced platelet aggregation by aspirin+clopidogrel was not affected by addition of celecoxib (47.6±13.4% vs. 51.6±3.7%, p=0.69). Drug-induced changes in prostacyclin and thromboxane levels did not differ among treatment groups.
Conclusion
Celecoxib treatment does not interfere with the antiplatelet effects of aspirin or clopidogrel, suggesting that celecoxib can be safely administered in combination with dual antiplatelet therapy in patients with coronary stenting without increased thrombogenicity.
doi:10.4070/kcj.2010.40.7.321
PMCID: PMC2910288  PMID: 20664740
Celecoxib; Platelet aggregation inhibitors; Thrombosis
20.  Effects of the selective inhibition of platelet thromboxane synthesis on the platelet-subendothelium interaction 
British Journal of Pharmacology  2002;137(7):1082-1088.
Drugs that inhibit TxA2 synthesis are used to reduce platelet aggregation. The aim of this study was to compare the effects of a cyclo-oxygenase (COX) inhibitor (acetylsalicylic acid, ASA), a thromboxane synthetase (TxS) inhibitor (dazoxiben) and a dual TxS inhibitor and TxA2 receptor blocker (DT-TX 30) on platelet aggregation and the platelet-subendothelium interaction in flow conditions.The techniques used in this in vitro study were platelet aggregometry in whole blood, and measurement of platelet thromboxane B2 and prostaglandin E2 production and leucocyte production of 6-keto-PGF1α. The platelet-subendothelium interaction was evaluated in rabbit aorta subendothelium preparations exposed to flowing blood at a shear stress of 800 s−1. Morphometric methods were used to calculate the percentage of subendothelium occupied by platelets.The 50% inhibitory concentration (IC50) of DT-TX 30 in whole blood was in the range of 10−7 μM (induced with collagen or arachidonic acid) to 10−5 μM (induced with thrombin) or 10−4 (induced with ADP). IC50 values under all experimental conditions were lower with DT–TX 30 than with ASA. For thromboxane B2 the IC50 were: ASA 0.84±0.05 μM, dazoxiben 765±54 μM, DT–TX 30 8.54±0.60 μM. Prostaglandin E2 was inhibited only by ASA (IC50 1.21±0.08 μM). Leucocyte 6-keto-PGF1α was inhibited by ASA (IC50 6.58±0.76 μM) and increased by dazoxiben and DT–TX 30. The greatest reduction in percentage subendothelial surface occupied by platelets after blood perfusion was seen after treatment with DT–TX 30 in the range of concentrations that inhibited collagen-induced platelet aggregation (control group: 31.20±3.8%, DT-TX 30 at 0.1 μM: 10.71±0.55%, at 1.0 μM: 6.53±0.44%, at 5.0 μM; 1.48±0.07%). All three drugs reduced thrombus formation, although ASA (unlike dazoxiben or DT–TX 30) increased the percentage surface occupied by adhesions.In conclusion, the effect of specific blockage of TxS together with blockage of membrane receptors for TxA2 can surpass the effect of ASA in inhibiting the platelet-subendothelium interaction in flow conditions.
doi:10.1038/sj.bjp.0704963
PMCID: PMC1573583  PMID: 12429581
Thromboxane A2; acetylsalicylic acid; dazoxiben; thromboxane synthetase inhibitors; thromboxane receptor antagonists; subendothelium
21.  Efficacy and Safety of Three Antiretroviral Regimens for Initial Treatment of HIV-1: A Randomized Clinical Trial in Diverse Multinational Settings 
PLoS Medicine  2012;9(8):e1001290.
Thomas Campbell and colleagues report findings of a randomized trial conducted in multiple countries regarding the efficacy of antiretroviral regimens with simplified dosing.
Background
Antiretroviral regimens with simplified dosing and better safety are needed to maximize the efficiency of antiretroviral delivery in resource-limited settings. We investigated the efficacy and safety of antiretroviral regimens with once-daily compared to twice-daily dosing in diverse areas of the world.
Methods and Findings
1,571 HIV-1-infected persons (47% women) from nine countries in four continents were assigned with equal probability to open-label antiretroviral therapy with efavirenz plus lamivudine-zidovudine (EFV+3TC-ZDV), atazanavir plus didanosine-EC plus emtricitabine (ATV+DDI+FTC), or efavirenz plus emtricitabine-tenofovir-disoproxil fumarate (DF) (EFV+FTC-TDF). ATV+DDI+FTC and EFV+FTC-TDF were hypothesized to be non-inferior to EFV+3TC-ZDV if the upper one-sided 95% confidence bound for the hazard ratio (HR) was ≤1.35 when 30% of participants had treatment failure.
An independent monitoring board recommended stopping study follow-up prior to accumulation of 472 treatment failures. Comparing EFV+FTC-TDF to EFV+3TC-ZDV, during a median 184 wk of follow-up there were 95 treatment failures (18%) among 526 participants versus 98 failures among 519 participants (19%; HR 0.95, 95% CI 0.72–1.27; p = 0.74). Safety endpoints occurred in 243 (46%) participants assigned to EFV+FTC-TDF versus 313 (60%) assigned to EFV+3TC-ZDV (HR 0.64, CI 0.54–0.76; p<0.001) and there was a significant interaction between sex and regimen safety (HR 0.50, CI 0.39–0.64 for women; HR 0.79, CI 0.62–1.00 for men; p = 0.01). Comparing ATV+DDI+FTC to EFV+3TC-ZDV, during a median follow-up of 81 wk there were 108 failures (21%) among 526 participants assigned to ATV+DDI+FTC and 76 (15%) among 519 participants assigned to EFV+3TC-ZDV (HR 1.51, CI 1.12–2.04; p = 0.007).
Conclusion
EFV+FTC-TDF had similar high efficacy compared to EFV+3TC-ZDV in this trial population, recruited in diverse multinational settings. Superior safety, especially in HIV-1-infected women, and once-daily dosing of EFV+FTC-TDF are advantageous for use of this regimen for initial treatment of HIV-1 infection in resource-limited countries. ATV+DDI+FTC had inferior efficacy and is not recommended as an initial antiretroviral regimen.
Trial Registration
www.ClinicalTrials.gov NCT00084136
Please see later in the article for the Editors' Summary.
Editors' Summary
Background
Despite the enormous gains in reducing HIV-related illness and death over the past decade, there are still considerable challenges to meeting the global goal of universal access to highly active antiretroviral treatment—a combination of effective drugs that attack the HIV virus in various ways—to everyone living with HIV/AIDS who could benefit from treatment. In recognition of the related financial, technical, and system obstacles to providing universal access to HIV treatment, in 2010 the UN agency responsible for HIV/AIDS—UNAIDS—launched an ambitious plan called Treatment 2.0, which aims to simplify the way HIV treatment is currently provided. One of the main focuses of Treatment 2.0 is to simplify drug regimes for the treatment of HIV and to make treatment regimes less toxic. In line with Treatment 2.0, the World Health Organization currently recommends that antiretroviral regimens for the initial treatment of HIV should include two nucleoside reverse transcriptase inhibitors (zidovudine or tenofovir disoproxil fumarate [DF] with lamivudine or emtricitabine) and a non-nucleoside reverse transcriptase inhibitor (efavirenz or nevirapine.)
Why Was This Study Done?
Most of the evidence about the safety and effectiveness of clinical trials come from clinical trials in high-income countries and thus is not generally representative of the majority of people with HIV. So in this study, the researchers conducted a randomized controlled trial in diverse populations in many different settings to investigate whether antiretroviral regimens administered once daily were as effective as twice-daily regimens and also whether a regimen containing the drug atazanavir administered once daily was as safe and effective as a regimen containing efavirenz—data from previous studies have suggested that atazanavir has characteristics, such as its side effect profile, which may make it more suitable for low income settings.
What Did the Researchers Do and Find?
The researchers recruited eligible patients from centers in Brazil, Haiti, India, Malawi, Peru, South Africa, Thailand, the United States, and Zimbabwe—almost half (47%) were women. Then the researchers randomly assigned participants to one of three regimens: efavirenz 600 mg daily plus co-formulated lamivudine-zidovudine 150 mg/300 mg twice daily (EFV+3TC-ZDV); or atazanavir 400 mg once daily, plus didanosine-EC 400 mg once daily, plus emtricitabine 200 mg once daily (ATV+DDI+FTC); or efavirenz 600 mg once daily plus coformulated emtricitabine-tenofovir-DF 200 mg/300 mg once daily (EFV+FTC-TDF). During the study period ATV+DDI+FTC was found to be inferior to EFV+3TC-ZDV, so the Multinational Data Safety Monitoring Board ordered this arm of the trial to stop. Then a year later, due to the low number of treatment failures (deaths, severe HIV disease, or serious opportunistic infections) in the remaining two arms, the board advised the trial to stop early. So the researchers analyzed the data obtained up to this point and pooled the results from all of the centers.
The researchers found that during an average of 184 weeks of follow-up, there were 95 treatment failures (18%) among 526 participants taking EFV+FTC-TDF compared to 98 failures among 519 participants taking EFV+3TC-ZDV. During an average 81 weeks follow-up, there were 108 failures (21%) among 526 participants assigned to ATV+DDI+FTC and 76 (15%) among 519 participants assigned to EFV+3TC-ZDV. As for safety, 243 (46%) participants assigned to EFV+FTC-TDF reached a safety endpoint (grade 3 disease, abnormal lab measurement, or the need to change drug) compared to 313 (60%) in the EFV+3TC-ZDV group. Importantly, the researchers found that there was greater risk of safety events for women assigned to EFV+3TC-ZDV and also that the atazanavir-based regimen had a higher relative efficacy in women compared to men.
What Do These Findings Mean?
These findings suggest that in diverse populations, EFV+FTC-TDF is as effective as EFV+3TC-ZDV but importantly, the once-daily dosing of EFV+FTC-TDF makes this regimen useful for the initial treatment of HIV, especially in low-income countries. Therefore, as per the guidance in Treatment 2.0, EFV+FTC-TDF in a single combination tablet that can be taken once a day is an attractive option. These findings also indicate that as ATV+DDI+FTC was found to be inferior to the other regimens, this combination should not be used in the initial treatment of HIV. These findings also add to the evidence that antiretroviral efficacy and safety can differ between women and men and support further development of sex-specific recommendations for antiretroviral regimen options.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001290.
The UNAIDS website has more information about Treatment 2.0; and the WHO website provides technical information
For an introduction to the treatment of HIV/AIDS see http://www.avert.org/treatment.htm; the AVERT site also has personal stories from women living with HIV/AIDS
AIDSmap provides information for individuals and communities affected by HIV/AIDS
The ACTG website provides information about research to improve treatment of HIV and related complications
doi:10.1371/journal.pmed.1001290
PMCID: PMC3419182  PMID: 22936892
22.  The platelet-independent release of thromboxane A2 by Paf-acether from guinea-pig lungs involves mechanisms distinct from those for leukotriene. 
British Journal of Pharmacology  1984;82(3):565-575.
Intra-arterial injections of platelet-activating factor (Paf-acether, 10-300 ng) to the perfused guinea-pig lung induced a dose-related bronchoconstriction, followed by contraction of the rat aorta superfused with the lung effluent, indicating the release of thromboxane A2 (TXA2) activity. These effects were matched with injections of bradykinin (Bk) at 100-1000 ng, leukotriene C4(LTC4) at 10-300 ng or arachidonic acid (AA) at 30-300 micrograms. Repeated doses of Paf-acether led to a specific desensitization of the release of TXA2, under conditions where Bk, LTC4 and arachidonic acid retained their ability to release TXA2. Bronchoconstriction and the release of TXA2 induced by Paf-acether were suppressed when the lungs were perfused with acetylsalicylic acid, but not with salicylic acid. The phospholipase A2 inhibitor, p-bromophenacyl bromide suppressed the release of TXA2 by Bk, but did not interfere with its formation from AA, nor with its release with Paf-acether and LTC4. The lipoxygenase inhibitor, nordihydroguaiaretic acid, inhibited to a similar extent the release of TXA2 by Bk, LTC4 and Paf-acether but also reduced directly the formation of TXA2 from arachidonic acid, invalidating its use as a specific antilipoxygenase agent. The leukotriene C4/D4 antagonist, FPL 55712, suppressed the TXA2 releasing effects of LTC4, and was completely inactive against Paf-acether, Bk or arachidonic acid. The aerosol of Paf-acether was tested in the anaesthetized guinea-pig and resulted in bronchoconstriction, unaccompanied by thrombocytopenia. Unlike bronchoconstriction induced by intravenous Paf-acether, which is refractory to cyclo-oxygenase inhibitors, the effects of the aerosol were suppressed by aspirin. Platelet depletion, which blocks the intravenous effects of Paf-acether, failed to interfere with those of the aerosol. Paf-acether induced a marked contraction of the superfused guinea-pig isolated parenchyma lung strip, which was followed by total and irreversible desensitization to itself. The contractile effect was not inhibited by aspirin or indomethacin, atropine, mepyramine, methysergide, phenoxybenzamine or propranolol, indicating that cyclo-oxygenase products, cholinergic stimuli, histamine, 5-hydroxytryptamine and catecholamine mechanisms are not involved. Our results indicate that Paf-acether interacts with pulmonary sites distinct from those for Bk, LTC4 or AA, since no cross-desensitization between Paf-acether and the other agonists was noted, p-bromophenacyl bromide inhibited Bk only and FPL 55712 inhibited only LTC4.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC1987001  PMID: 6430375
23.  Changes in membrane glycoproteins of circulating platelets after coronary stent implantation. 
Heart  1996;76(2):166-172.
OBJECTIVES: To evaluate platelet function in patients with coronary stents. DESIGN: A non-randomised control trial in 30 patients who had immediate implantation of Palmaz-Schatz coronary stents because of a suboptimal angioplasty result. All patients received a standardised anticoagulation regimen including intravenous heparin (activated partial thromboplastin time (APTT) 80 to 120 s), oral vitamin K antagonist (target international normalised ratio (INR) of 3.5), and 100 mg aspirin twice daily. Platelet surface expression of glycoprotein IIb-IIIa, activated fibrinogen receptor, and P-selectin as well as binding of von Willebrand factor and fibrinogen were determined by flow cytometry in peripheral venous blood samples collected before the intervention and then daily for 4 days after it. The results were compared with those in 30 patients undergoing elective coronary balloon angioplasty. SETTING: University hospital. RESULTS: After coronary stenting surface expression of the activated fibrinogen receptor significantly increased, peaking at day 2 (P < 0.001). Similar results were found for von Willebrand factor binding and P-selectin surface expression, with a maximum at day 2 to 4 after stenting (von Willebrand factor, P < 0.001; P-selectin, P < 0.001). The changes in platelet membrane glycoproteins coincided with a significant drop in peripheral platelet count after stent placement (P < 0.01). No significant change in fibrinogen receptor activity, von Willebrand factor binding, P-selectin surface expression, or platelet count was seen in the control group. CONCLUSIONS: The present study shows that current anticoagulation treatment is inefficient in suppressing platelet activation in patients with coronary stents and, therefore, might not be the best treatment for reducing the incidence of subacute stent thrombosis.
PMCID: PMC484467  PMID: 8795482
24.  Reduced Platelet Thromboxane Formation in Uremia. EVIDENCE FOR A FUNCTIONAL CYCLOOXYGENASE DEFECT 
Journal of Clinical Investigation  1983;71(3):762-768.
A qualitative platelet abnormality and a bleeding tendency are frequently associated with renal failure and uremia. We demonstrated previously that uremic patients display an abnormal platelet aggregation to arachidonic acid and reduced malondialdehyde production in response to thrombin and arachidonic acid. The objectives of this investigation were: (a) to compare platelet prostaglandin (PG) and thromboxane (TX) production in whole blood and in platelet-rich plasma (PRP) of 21 uremic patients and 22 healthy subjects; (b) to evaluate the concentration and activity of platelet PG- and TX-forming enzymes; (c) to assess the functional responsiveness of the platelet TXA2/PGH2 receptor; (d) to explore the hemostatic consequences of partially reduced TXA2 production.
Platelet immunoreactive TXB2 production during whole blood clotting was significantly reduced, by ∼60%, in uremic patients as compared to age- and sex-matched controls. Exogenous thrombin (5-30 IU/ml) failed to restore normal TXB2 production in uremic platelets. Uremic PRP produced comparable or slightly higher amounts of TXB2 than normal PRP at arachidonate concentrations 0.25-1 mM. However, when exposed to substrate concentrations >2 mM, uremic PRP produced significantly less TXB2 than normal PRP. To discriminate between reduced arachidonic acid oxygenation and altered endoperoxide metabolism, the time course of immunoreactive TXB2 and PGE2 production was measured during whole blood clotting. The synthesis and release of both cyclooxygenase-derived products was slower and significantly reduced, at all time intervals considered. Furthermore, PGI2 production in whole blood, as reflected by serum immunoreactive 6-keto-PGF1α concentrations, was significantly reduced in uremic patients as compared with healthy subjects. PGH synthase levels, as determined by an immunoradiometric assay, were not significantly different in platelets from uremic patients as compared to control platelets. A single 40-mg dose of aspirin given to five healthy volunteers reduced their serum TXB2 to levels found in uremic patients. This was associated with a significant increase of threshold aggregating concentrations of ADP and arachidonic acid and prolongation of bleeding time. Substantially similar threshold concentrations of U46619, a TXA2 agonist, induced aggregation of normal and uremic platelets. Prostacyclin induced a significant elevation of uremic platelet cyclic AMP, which was suppressed by U46619, further suggesting normal responsiveness of the TXA2/PGH2 receptor.
We conclude that: (a) an abnormality of platelet arachidonic acid metabolism exists in uremia, leading to a reduced TXA2 production; (b) the characteristics of this abnormality are consistent with a functional cyclooxygenase defect; (c) reduced TXA2 production may partially explain the previously described abnormality of platelet function in uremia.
PMCID: PMC436927  PMID: 6298281
25.  Antiplatelet effect of the anaesthetic drug propofol: influence of red blood cells and leucocytes 
British Journal of Pharmacology  1999;128(7):1538-1544.
The present study was designed to investigate the mechanism of the antiplatelet action of the anaesthetic propofol in vitro.Human whole blood was incubated with different concentrations of propofol and its solvent Intralipid®. We determined, platelet aggregometry in whole blood, platelet-enriched plasma (PRP), PRP plus red blood cells (RBC), and PRP plus leucocytes (LC); platelet production of thromboxane B2 (TxB2), ATP release by platelet dense granules, adenosine uptake by RBC, intraplatelet levels of cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP), and LC production of nitric oxide (NO).Propofol-induced inhibition of platelet aggregation was greater in whole blood (IC50 80–136 μM) than in PRP (IC50>600 μM), except when aggregation was induced by arachidonic acid, in which case the antiaggregatory effect of the anaesthetic was similar in both media (IC50 72–85 μM). Inhibition of platelet aggregation correlated significantly with inhibition of TxB2 synthesis (r2=0.83). Propofol also inhibited granular ATP release; this effect was greatest in whole blood.The presence of RBC or LC increased the antiaggregatory effect of propofol, mainly when collagen was used as aggregating agent. Intralipid inhibited the uptake of adenosine by RBC, however this effect probably does not contribute significantly to its antiaggregatory effect.The anaesthetic potentiated the NO-cyclic GMP pathway, mainly by increasing the synthesis of NO by LC. Intralipid had no effect on the NO-cyclic GMP pathway in the LC-platelet interaction.Propofol inhibited platelet aggregation in human whole blood, possibly through the sum of the effects of Intralipid on the platelet-RBC interaction and the increased synthesis of NO by LC in the platelet-LC interaction.
doi:10.1038/sj.bjp.0702927
PMCID: PMC1571776  PMID: 10602334
Nitric oxide; leucocytes; platelets; propofol; thromboxane

Results 1-25 (1156002)