Targeting murine heart and brain: visualisation conditions for multi-pinhole SPECT with (99m)Tc- and (123)I-labelled probes.

PURPOSE: The study serves to optimise conditions for multi-pinhole SPECT small animal imaging of (123)I- and (99m)Tc-labelled radiopharmaceuticals with different distributions in murine heart and brain and to investigate detection and dose range thresholds for verification of differences in tracer uptake. METHODS: A Triad 88/Trionix system with three 6-pinhole collimators was used for investigation of dose requirements for imaging of the dopamine D(2) receptor ligand [(123)I]IBZM and the cerebral perfusion tracer [(99m)Tc]HMPAO (1.2-0.4 MBq/g body weight) in healthy mice. The fatty acid [(123)I]IPPA (0.94 +/- 0.05 MBq/g body weight) and the perfusion tracer [(99m)Tc]sestamibi (3.8 +/- 0.45 MBq/g body weight) were applied to cardiomyopathic mice overexpressing the prostaglandin EP(3) receptor. RESULTS: In vivo imaging and in vitro data revealed 45 kBq total cerebral uptake and 201 kBq cardiac uptake as thresholds for visualisation of striatal [(123)I]IBZM and of cardiac [(99m)Tc]sestamibi using 100 and 150 s acquisition time, respectively. Alterations of maximal cerebral uptake of [(123)I]IBZM by >20% (116 kBq) were verified with the prerequisite of 50% striatal of total uptake. The labelling with [(99m)Tc]sestamibi revealed a 30% lower uptake in cardiomyopathic hearts compared to wild types. [(123)I]IPPA uptake could be visualised at activity doses of 0.8 MBq/g body weight. CONCLUSION: Multi-pinhole SPECT enables detection of alterations of the cerebral uptake of (123)I- and (99m)Tc-labelled tracers in an appropriate dose range in murine models targeting physiological processes in brain and heart. The thresholds of detection for differences in the tracer uptake determined under the conditions of our experiments well reflect distinctions in molar activity and uptake characteristics of the tracers.

Pyrazolinone analgesics prevent the antiplatelet effect of aspirin and preserve human platelet thromboxane synthesis.

BACKGROUND: Anti-inflammatory analgesics, including ibuprofen and naproxen, are known to interfere with the antiplatelet effect of aspirin, presumably as a result of a drug-drug interaction at the level of platelet cyclooxygenase-1 (COX-1). OBJECTIVE: We studied whether dipyrone, which has recently been reported to inhibit COX isoforms by a mechanism different from conventional non-steroidal anti-inflammatory drugs (NSAIDs), also interferes with the antiplatelet effect of aspirin. METHODS: Arachidonic acid- and collagen-induced aggregation, as well as thromboxane formation, were measured in human platelet-rich plasma. Platelet P-selectin expression was determined by flow cytometry and cell-free COX enzyme activity was quantified by luminol-enhanced luminescence of human platelet microsomes. In addition, computerized docking was performed based on the crystal structure of COX-1. RESULTS: 4-Methylaminoantipyrine (MAA), the active metabolite of dipyrone, largely attenuated or even completely abolished the inhibition of arachidonic acid-induced platelet aggregation, thromboxane formation and P-selectin expression by aspirin. Similar results were obtained for other pyrazolinones, as well as for the conventional NSAIDs ibuprofen and naproxen. Moreover, MAA attenuated the effect of aspirin on COX activity of platelet microsomes, suggesting a competition with aspirin at the COX-1 enzyme. This was confirmed by docking studies, which revealed that MAA forms a strong hydrogen bond with serine 530 within the COX-1, thereby preventing enzyme acetylation by aspirin. CONCLUSION: This study demonstrates for the first time that dipyrone and other pyrazolinones have a high potential to attenuate or prevent the antiplatelet effect of aspirin. This should be considered if pyrazolinone analgesics are administered to patients with cardiovascular disease requiring antiplatelet aspirin therapy.

Platelet function testing: dead or alive.

Essentials Pharmacodynamic response to antiplatelet medication is heterogeneous. Platelet reactivity to dual antiplatelet therapy was analyzed by three platelet function assays. The prevalence of high and low platelet reactivity differed significantly between assays. Future trials are needed to determine the best assay to analyze platelet function. SUMMARY: Background High on-treatment platelet reactivity (HTPR) to antiplatelet medication leads to ischemic events, whereas low on-treatment platelet reactivity (LTPR) increases bleeding risk. However, various trials have failed to demonstrate superiority of tailored antiplatelet regimens (ARCTIC, ANTARCTIC, Trigger-PCI, and GRAVITAS). TROPICAL-ACS was the first study that demonstrated the benefit of tailoring antiplatelet medication according to platelet function analysis. A potential reason may be that different platelet function assays were used in these trials. Objectives To evaluate whether the results of platelet function tests are comparable. Patients/Methods We tested three commonly used assays - light transmission aggregometry (LTA), (Multiplate impedance aggregometry [MP]), and vasodilator-stimulated phosphoprotein phosphorylation assay (VASP) - in 23 patients receiving dual antiplatelet therapy with aspirin and clopidogrel. Results With LTA, HTPR occurred in 57% of patients; with VASP, it occurred in 43% of patients; and with MP, it occurred in 13% of patients. According to LTA, only 35% of patients were in the therapeutic window; according to VASP, 57% of patients were in the therapeutic window; and according to MP, 48% of patients were in the therapeutic window. With LTA, LTPR occurred in 9% of patients; with VASP, it occurred in 0% of patients; and with MP, it occurred in 39% of patients. Therefore, the prevalences of HTPR and LTPR differed significantly between assays. Remarkably, in 17% of patients, one assay showed HTPR whereas another showed LTPR. Conclusions The results of different platelet function assays differ substantially. Up to now, only TROPICAL-ACS had demonstrated a benefit of tailoring antiplatelet medication according to platelet function analysis. Future trials are needed to evaluate whether the platelet function assay used in TROPICAL-ACS is the 'correct' one and revives platelet function testing.

Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation.

Essentials Whether or not dabigatran enhances the risk of myocardial infarction is under discussion. We measured platelet reactivity and thrombin receptor expression in dabigatran patients. Platelet reactivity and thrombin receptor expression is enhanced during dabigatran treatment. This should be considered when choosing the optimal direct oral anticoagulant for individuals. SUMMARY: Background The direct oral anticoagulant (DOAC) dabigatran is a direct thrombin inhibitor. Its landmark trial, the RE-LY study, observed a trend towards a higher incidence of myocardial infarctions (MIs) in dabigatran-treated patients. Since then, there have been discussions on whether dabigatran increases the risk of MI. Objective In this study, we aimed to assess platelet reactivity and platelet thrombin receptor expression in dabigatran-treated patients. Methods We conducted a cross-sectional study in 13 hospitalized patients with planned initiation of dabigatran medication. Platelet reactivity was measured by light-transmission aggregometry and platelet thrombin receptor expression was measured by flow cytometry analysis. Results Platelet reactivity was higher after initiation of dabigatran medication as compared with baseline (baseline 44 ± 24% vs. dabigatran 70 ± 25%). Accordingly, the density of both platelet thrombin receptors (protease activated receptor [PAR]-1 and PAR-4) on platelets increased during dabigatran treatment (PAR1, baseline 63 ± 11% vs. dabigatran 70 ± 10%; PAR4, baseline 1.1 ± 0.5% vs. dabigatran 1.6 ± 0.9%). Conclusions Dabigatran increases platelet reactivity by enhancing the thrombin receptor density on platelets. This finding should be considered while choosing the optimal DOAC in individualized medicine.

Antiplatelet effects of aspirin in chronic kidney disease patients.

UNLABELLED: ESSENTIALS: Chronic kidney disease (CKD) patients have a high risk of cardiovascular events. A pharmacodynamic evaluation of the effects of aspirin in 116 patients was carried out. The antiplatelet effects of aspirin are associated with impaired renal function. The optimal antithrombotic regimen in CKD patients must be investigated on a larger scale. BACKGROUND: The pharmacodynamic response to aspirin varies significantly between individuals. Insufficient antiplatelet effects of aspirin are associated with increased risk of ischemic events. Chronic kidney disease (CKD) is suggested to affect the pharmacodynamic response to antiplatelet medication. High on-treatment platelet reactivity (HTPR) to clopidogrel has been reported to partially account for the enhanced risk of death and cardiovascular events in CKD patients. Objective To investigate the antiplatelet effects of aspirin in patients with CKD. METHODS: We conducted a cross-sectional study in 116 patients on permanent aspirin medication. The pharmacodynamic response to aspirin was determined by arachidonic acid-induced thromboxane formation. RESULTS: HTPR to aspirin was more frequent in patients with impaired renal function (47% vs. 22%; odds ratio, 3.16; 95% confidence interval [CI], 1.34-7.41; P = 0.008). The pharmacodynamic response to aspirin was impaired in patients with moderate/severe CKD (92; interquartile range [IQR], 282 ng mL(-1) ) as compared to patients with normal/mildly reduced renal function (36; IQR, 100 ng mL(-1) ; difference in medians, 57; CI, 5-110 ng mL(-1) ; P = 0.013). Bivariate Pearson analysis showed residual thromboxane formation to be correlated with glomerular filtration rate (R = -0.303; R(2) = 0.092; P = 0.001). Patients with CKD were older and more frequently female. Multivariate linear regression analysis revealed that the correlation was independent of age (R = -0.314; R(2) = 0.082; P = 0.002) and gender (R = -0.305; R(2) = 0.077; P = 0.006). CONCLUSION: Renal function is correlated with pharmacodynamic response to aspirin. Patients with CKD have an increased risk of impaired antiplatelet effects of aspirin. Larger trials are needed to assess the clinical impact of this finding and investigate the optimal antithrombotic regimen in CKD patients.

Functional and biochemical evaluation of platelet aspirin resistance after coronary artery bypass surgery.

BACKGROUND: Aspirin inhibits platelet activation and reduces atherothrombotic complications in patients at risk of myocardial infarction and stroke. However, a sufficient inhibition of platelet function by aspirin is not always achieved. The causes of this aspirin resistance are unknown. METHODS AND RESULTS: Patients undergoing coronary artery bypass grafting (CABG) have a high incidence of aspirin resistance. To evaluate functional and biochemical responses to aspirin, platelet-rich plasma was obtained before and at days 1, 5, and 10 after CABG. Thromboxane formation, aggregation, and alpha-granule secretion were effectively inhibited by 30 or 100 micromol/L aspirin in vitro before CABG, but this inhibition was prevented or attenuated after CABG. Whereas the inhibition of thromboxane formation and aggregation by aspirin in vitro partly recovered at day 10 after CABG, oral aspirin (100 mg/d) remained ineffective. The inducible isoform of cyclooxygenase in platelets, COX-2, has been suggested to confer aspirin resistance. In fact, immunoreactive COX-2 was increased 16-fold in platelets at day 5 after CABG, but the COX-2 selective inhibitor celecoxib did not alter aspirin-resistant thromboxane formation. By contrast, the combined inhibitor of thromboxane synthase and thromboxane receptor antagonist terbogrel equally prevented thromboxane formation of platelets obtained before (control) and after CABG. CONCLUSIONS: Platelet aspirin resistance involves an impairment of both in vivo and in vitro inhibition of platelet functions and is probably due to a disturbed inhibition of platelet COX-1 by aspirin.

Inhibition of antiplatelet effects of aspirin by nonopioid analgesics.

In patients undergoing coronary bypass grafting, we noticed that low-dose aspirin failed to inhibit platelet aggregation, potentially elevating the risk of thrombotic bypass occlusion. This "high on-treatment platelet reactivity" was reproducible in vitro and could be transferred with patient plasma or urine to aspirin-sensitive donor platelets, suggesting a drug/drug interaction. Loss of aspirin efficacy was associated with analgesia by dipyrone (metamizol) and initiated further study of the interaction between aspirin and other nonopioid analgesics.

Antiatherosclerotic effects of oral cicaprost in experimental hypercholesterolemia in rabbits.

The efficacy of the oral prostacyclin mimetic cicaprost in preventing atheromatous plaque formation was studied in an in vivo model of experimental hypercholesterolemia. New Zealand white rabbits were fed either standard chow or a cholesterol-enriched (1%) diet for 12 weeks. Cicaprost was added to the drinking water in a non-hypotensive dose (5 micrograms/kg/day) and withdrawn 3 days prior to studying platelet, leukocyte and endothelial function. In cholesterol-fed rabbits, oral cicaprost reduced the aortic intimal surface covered by atheromatous lesions from 84 to 63% (P < 0.05). There was no major difference in serum lipid composition between cicaprost- and vehicle-treated animals. In hyper-cholesterolemic rabbits there was a significant impairment of endothelium-dependent relaxations. Cicaprost treatment considerably improved this endothelial function but had no effect in rabbits receiving standard diet. In addition, platelet and leukocyte hyperreactivity, as seen in hypercholesterolemic rabbits, were largely reduced by cicaprost treatment. These data are the first to demonstrate marked antiatherosclerotic effects of long-term oral prostacyclin treatment. The mechanism may be related to improved endothelial function and subsequent prevention of secondary platelet and neutrophil hyperreactivity.

Antiplatelet effects of ticlopidine are reduced in experimental hypercholesterolemia.

This study determines the antiplatelet effects of oral ticlopidine (100 mg/kg x day) in experimental hypercholesterolemia. Rabbits were fed either a standard diet or a cholesterol-enriched diet (0.5% for 3 months, 1% for 1 month). In normocholesterolemic controls ADP-, but not collagen-induced platelet aggregation was inhibited by ticlopidine treatment. This was accompanied by a significantly enhanced inhibition of ADP-induced platelet aggregation and stimulation of cyclic AMP accumulation by iloprost. Hypercholesterolemia considerably attenuated the inhibition of ADP-induced aggregation by ticlopidine but did not change its effect on the iloprost-induced inhibition of platelet function and cyclic AMP formation. ADP-induced platelet-derived thromboxane formation was considerably greater in hypercholesterolemic rabbits and not reduced by ticlopidine. Ticlopidine did also not significantly influence the extent and severity of atherosclerotic plaque formation although a tendency for improvement was observed in a subgroup of animals. The data suggest that hypercholesterolemia attenuates the inhibitory effect of ticlopidine on ADP-induced platelet aggregation. This might be related to the stimulation of thromboxane formation by ADP in hypercholesterolemia. The maintained protection from ADP-induced inhibition of cAMP accumulation suggests a minor role of this mechanism in the progression of hypercholesterolemia-induced vessel disease in this model.

The contribution of tumour necrosis factor-alpha and endothelin-1 to the increase of coronary resistance in hearts from rats treated with endotoxin.

1. Inflammatory disease states predispose to myocardial infarction. Here we have investigated the effects of a systemic inflammatory response syndrome, i.e. lipopolysaccharide (LPS)-induced circulatory shock in rats, on coronary vascular tone. 2. Anaesthetized rats were given LPS (10 mg kg-1, i.v.) and the hearts excised 180 min later for isolated perfusion at constant flow by the Langendorff technique. Once the ex vivo perfusion was started, the perfusion pressure strongly increased in these hearts compared to hearts from control rats (130 +/- 3 vs. 49 +/- 3 mmHg after 10 min). This increase in coronary resistance was not associated with a reduction in endothelial cell function, for the vasodilator responses to bradykinin were unchanged. 3. When hearts were removed 30 min after the injection of LPS, the LPS-induced rise in perfusion pressure was delayed. No changes in perfusion pressure were seen when the hearts were removed 15 min after the injection of LPS. Pre-treatment with cycloheximide or an anti-tumour necrosis factor-alpha (TNF-alpha) antibody or continuous infusion in vivo and in vitro of the endothelin ETA receptor selective antagonist FR 139317, greatly decreased the increase in coronary vascular resistance induced by LPS. 4. These data suggest that TNF-alpha may induce the release of endothelin-1 (ET-1) and that this mediates at least part of the coronary vasoconstriction. This hypothesis is supported by the demonstration that LPS administration increased the circulating levels of both TNF-alpha and ET-1. 5. We conclude, therefore, that in inflammatory disease states, such as LPS-induced septic shock, there is the sequential release of TNF-alpha and endothelin-1 which leads to an increase in coronary vascular tone and so a predisposition to myocardial ischaemia. Inactivation of TNF-alpha by an antibody as well as ETA receptor blockade by a selective antagonist may effectively interfere with this pathway.

Acetylsalicylic acid enhances arrhythmogeneity in a model of local ischemia of isolated rabbit hearts.

Acetylsalicylic acid often is used in the treatment and prophylaxis of regional myocardial ischemia and infarction. However, only little is known about its electrophysiological effects and on possible proarrhythmic effects of the drug. Thus, the aim of this study was to evaluate the electrophysiological effects of acetylsalicylic acid in normal isolated saline perfused rabbit hearts and in hearts submitted to regional ischemia. Isolated saline perfused rabbit hearts were treated with increasing concentrations of acetylsalicylic acid (0.05, 0.1, 0.5 and 1 microM). The epicardial activation and repolarisation process were analysed using an epicardial mapping (256 unipolar leads). Activation and repolarisation time were determined for each electrode from which data the 'breakthrough-points' of epicardial activation were determined. At each electrode an activation vector was calculated giving the direction and velocity of the local excitation wave. The similarity of selected heart beats compared to the control was evaluated by determination of the percentage of identical breakthrough-points and of similar vectors (deviation < or = 5 degrees). At each electrode the local epicardial action potential duration was assessed as the activation recovery interval and the standard deviation of the epicardial action potential duration (of 256 leads, = dispersion) was determined. In a second series of experiments 30 min regional ischemia was induced by occlusion of the left descendent coronary artery followed by 30 min reperfusion in the absence or presence of 0.5 microM acetylsalicylic acid or 1 micro/M indomethacin. The degree of ischemia was assessed by the reduction in coronary flow, by the degree of ST-elevation and by the area in which ST-elevation was registered. Under non-ischemic conditions acetylsalicylic acid led to an increase in the epicardial action potential duration (7%), a decrease in the breakthrough-point similarity (by 10%) and vectorfield similarity (by 15%). In control hearts submitted to regional ischemia the similarity of the vectorfields and of the breakthrough-points, as well as the duration of the epicardial action potentials were markedly reduced while the dispersion was greatly increased. In the ischemic region there was a significant ST-deviation from the isoelectrical line. These changes of ST-segments were significantly enhanced by 0.5 microM acetylsalicylic acid, so that in all (7/7) acetylsalicylic acid treated hearts sustained ventricular fibrillation occurred after 20 min ischemia, whereas in the absence of acetylsalicylic acid fibrillation was found in only 2/7 hearts during reperfusion and not during ischemia. 1 microM indomethacin did not cause these changes. In all ischemia/reperfusion series of experiments the reduction in coronary flow and left ventricular pressure by ischemia was of the same degree and we did not observe significant differences in the size of ischemic area. Using 14C-acetylsalicylic acid, an accumulation of acetylsalicylic acid in the ischemic region could be observed. From these results we conclude, that acetylsalicylic acid can induce ventricular fibrillation. Thus, in acute myocardial ischemia, acetylsalicylic acid may have (besides the well known and desired antiaggregatory effects) electrophysiologic side effects which seem to be proarrhythmic in regional ischemia at least in this model.

ADP-induced second wave aggregation in platelet rich plasma from hypercholesterolemic rabbits.

Platelet aggregation, ATP secretion and thromboxane formation were measured in platelet-rich plasma (PRP) of rabbits fed a cholesterol-rich (1%) diet for 3 months. Addition of ADP (0.6-2 microM) resulted in a clear biphasic aggregation in 7/8 cholesterol-fed rabbits. This was not seen in any of the 9 control animals kept on a standard diet for the same period of time (P less than 0.01). The secondary ADP-induced aggregation was irreversible and was accompanied by enhanced ATP secretion and significant thromboxane generation. There was no ADP-induced thromboxane formation and only a minor ATP secretion at the highest ADP concentration studied (20 microM) in control rabbits. These data confirm platelet hyperreactivity in hypercholesterolemia and demonstrate for the first time the occurrence of an ADP-induced second wave aggregation and granule secretion in citrated PRP of rabbits which may be triggered by thromboxane generation.

Analysis of a porcine EP3-receptor: cloning, expression and signal transduction.

A cDNA clone, encoding a complete porcine EP3 receptor, was isolated from a porcine heart cDNA library. The deduced amino acid sequence revealed a protein of 387 amino acid residues with an estimated molecular weight of 43 kD and strongest homology to the human EP3-II receptor (84% identity on protein level). Ligand binding studies with transfected COS-7 cells, expressing the porcine receptor, showed displacement of [3H]PGE1 with the EP3-specific agonist M&B 28.767, the EP1/EP3-agonist sulprostone but not with the EP2-specific agonist butaprost. Stimulation of transfected CHO cells with M&B 28.767 resulted in inhibition of forskolin-induced cAMP formation, suggesting coupling to an inhibitory G protein. Agonist-induced translocation of the transcription factor NFkappaB into the nucleus of transfected CHO cells was demonstrated by Western blot analysis, indicating that these EP3 receptors modulate NFkappaB-dependent cellular signal transduction. Analysis of the genomic organization identified the major transcription initiation site at about 160 bp upstream of the ATG start codon. The 800-bp 5' flanking region contains a variety of putative cis-acting regulatory elements, including binding sites for AP2, SP1 and MyoD (E-box). The present data will now allow further studies on EP3 receptor-mediated signal transduction and its regulation.

Effects of candesartan and perindopril on renal function, TGF-beta1 plasma levels and excretion of prostaglandins in stable renal allograft recipients.

AIMS: Although on account of their nephroprotective effects, ACE inhibitors and angiotensin receptor antagonists appear to be advantageous for patients after renal transplantation, their use in these patients has been limited up to now. This is in part due to the risk of inducing a decrease in the glomerular filtration pressure gradient with subsequent impairment of allograft function. The aim of the present study was to investigate the effects of ACE inhibitors and angiotensin receptor antagonists on renal function, excretion of prostaglandins as a parameter of glomerular hemodynamics and TGF-beta1 plasma levels during an 8-week withdrawal phase in pretreated patients. PATIENTS AND METHODS: Sixteen patients with stable long-term allograft function undergoing therapy with candesartan (group 1) and 16 patients with stable long-term allograft function undergoing therapy with perindopril (group 2) were included in the study. Any signs of chronic allograft dysfunction were defined as exclusion criteria. Renal function, albuminuria, TGF-beta1 plasma levels as well as the excretion of thromboxane B2 and 6-keto-prostaglandin-F-1alpha were monitored during an 8-week withdrawal phase of the angiotensin receptor antagonist or ACE inhibitor, respectively. Normotension was maintained throughout the study period through adjustment of other anti-hypertensive drugs. RESULTS: Creatinine clearance as well as TGF-beta1 plasma levels and the excretion of prostaglandins remained unchanged after discontinuation of candesartan or perindopril. However, after withdrawal of the substances a significant increase in albuminuria was noted in both patient groups throughout the observation period. After 8 weeks, median albuminuria had increased by 63% in group 1 and by 163% in group 2. CONCLUSIONS: We were able to demonstrate that the use of ACE inhibitors and angiotensin receptor antagonists in patients after renal transplantation is safe. Favorable effects of both substances on albuminuria were detectable in patients who showed no signs of chronic allograft dysfunction according to the usual criteria. Therefore, a nephroprotective effect of candesartan as well as of perindopril, is highly probable in patients after renal transplantation. Further investigations regarding routine use in these patients are therefore mandatory.

Improvement of epidermal adhesion by surface modification of craniofacial abutments.

Craniofacial implants may present peri-implant inflammation because there is no close adhesion of the epithelium to abutments and because of bacteria infiltrating the subcutaneous tissue through the gap. Therefore an attempt was made to improve adhesion of epithelium to abutments. In an in vitro model, adhesion of epithelial cells (HaCat cells) to nonmodified and 3 modified Brånemark System abutment surfaces was quantified. It was found that more cells were adherent in sequence at silicone-coated surfaces, sandblasted surfaces, and collagen-coated (Types I and IV) surfaces than on nonmodified abutments. It was concluded that it is possible to improve epidermal adhesion to abutments through modification of abutment surfaces.

Mechanisms of anti-ischemic action of prostaglandin E1 in peripheral arterial occlusive disease.

The mechanisms of anti-ischemic effects of PGE1 in patients with peripheral arterial occlusive disease (PAD) are probably complex and clearly not limited to a direct vasodilator action. In addition to the known effects on blood flow, viscosity, fibrinolysis and platelet aggregation, the compound also inhibits monocyte and neutrophil function, suggesting that PGE1 will also have anti-inflammatory effects. Recent research has detected additional actions of PGE1 and prostacyclin analogs which might be relevant to its clinical efficacy. This includes inhibition of expression of adhesion molecules (E-selectin, ICAM-1, and VCAM-1), release of inflammatory cytokines (TNFalpha, MCP-1), matrix components and generation and release of growth factors (CYR61, CTGF). These actions may also contribute to the long-term effects of PGE1, particularly in more advanced stages of PAD. Gene-expression experiments with chemically stable prostacyclins and PGE1 suggest that several genes in vascular smooth muscle cells and fibroblasts are modified by prostaglandins at the transcriptional level. This includes TNFalpha-induced VCAM expression in vascular smooth muscle cells which appears to be inhibited via the prostaglandin EP2 receptor as well as IL-1-induced expression of the type-1 collagen gene in fibroblasts. Thus, regulation of gene transcription by PGE1 may contribute to tissue protection in critical ischemia of the lower limbs.

Pharmacokinetics and pharmacodynamics of acetylsalicylic acid after intravenous and oral administration to healthy volunteers.

BACKGROUND: The pharmacology of single doses of acetylsalicylic acid (ASA) administered intravenously (250 or 500 mg) or orally (100, 300, or 500 mg) was evaluated in a randomized, placebo-controlled, crossover study. METHODS: Blood and urine samples were collected before and up to 24 hours after administration of ASA in 22 healthy volunteers. Pharmacokinetic parameters and measurements of platelet aggregation were determined using validated techniques. RESULTS: A comparison between administration routes showed that the geometric mean dose-corrected peak concentrations (Cmax/D) and the geometric mean dose-corrected area under the curve (AUC0-∞/D) were higher following intravenous administration of ASA 500 mg compared with oral administration (estimated ratios were 11.23 and 2.03, respectively). Complete inhibition of platelet aggregation was achieved within 5 minutes with both intravenous ASA doses, reflecting a rapid onset of inhibition that was not observed with oral dosing. At 5 minutes after administration, the mean reduction in arachidonic acid-induced thromboxane B2 synthesis ex vivo was 99.3% with ASA 250 mg intravenously and 99.7% with ASA 500 mg intravenously. In exploratory analyses, thromboxane B2 synthesis was significantly lower after intravenous versus oral ASA 500 mg (P<0.0001) at each observed time point up to the first hour after administration. Concentrations of 6-keto-prostaglandin1α at 5 and 20 minutes after dosing were also significantly lower with ASA 500 mg intravenously than with ASA 500 mg orally. CONCLUSION: This study demonstrates that intravenous ASA provides more rapid and consistent platelet inhibition than oral ASA within the first hour after dosing.

High on treatment platelet reactivity against aspirin by non-steroidal anti-inflammatory drugs--pharmacological mechanisms and clinical relevance.

Inhibition of platelet function by aspirin results from irreversible inhibition of platelet cyclooxygenase (COX)-1. While sufficient inhibition is obtained at antiplatelet doses (75-325 mg/day) in most (≥95%) treated patients, the antiplatelet effect of aspirin and subsequent cardiovascular risk reduction is much less in clinical settings and disease-dependent. Several reasons for this "high on treatment platelet reactivity" are known. This paper reviews the evidence for an interaction between aspirin and other COX inhibitors, namely non-steroidal anti-inflammatory drugs (NSAIDs). Numerous experimental studies demonstrated a pharmacodynamic interaction between aspirin and NSAIDs. This likely occurs within the hydrophobic substrate channel of platelet COX-1 and might be explained by molecular competition between inhibitor drugs and substrate (arachidonic acid) at overlapping binding sites. This interaction is found with some compounds, notably ibuprofen and dipyrone (metamizole), but not with others, such as diclofenac and acetaminophen (paracetamol). Hence, this interaction is not a class effect of NSAIDs and/or non-steroidal analgesics but rather due to specific structural requirements which still remain to be defined. In vivo studies on healthy subjects and patients tend to confirm this type of interaction as well as large differences between NSAIDs and non-steroidal analgesics, respectively. These interactions may be clinically relevant and may increase the cardiovascular risk in long-term treatment for primary and secondary cardiovascular prevention in patients with chronic inflammation, such as rheumatoid arthritis. These patients have an elevated risk for myocardial infarctions and may require chronic antiplatelet treatment by aspirin in addition to treatment of inflammatory pain.

Release of sphingosine-1-phosphate from human platelets is dependent on thromboxane formation.

BACKGROUND: Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. OBJECTIVES: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. METHODS: S1P was detected using thin-layer chromatography in [(3)H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. RESULTS: Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 µM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets. CONCLUSIONS: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.