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Biomedical subjects

A A Weber

Publications and source records attributed to A A Weber.

At least 19 recordsLinked to original sources
PubMed2001

Differential inhibition of adenosine diphosphate- versus thrombin receptor-activating peptide-stimulated platelet fibrinogen binding by abciximab due to different glycoprotein IIb/IIIa activation kinetics.

The exposure of internal glycoprotein (GP) IIb/IIIa receptors has been proposed to explain the incomplete inhibition of aggregation of thrombin receptor-activating peptide (TRAP)-stimulated platelets by abciximab. However, a marked and rapid externalization of GPIIb/IIIa was also observed upon stimulation with 30 microM adenosine diphosphate (ADP). ADP-induced fibrinogen binding was completely inhibited by 10 microg/mL abciximab, 30 nM tirofiban, or 3 microg/mL eptifibatide, while fibrinogen binding induced by 100 microM TRAP was inhibited only by 50%. Interestingly, striking differences in fibrinogen binding kinetics in ADP- versus TRAP-stimulated platelets were observed. ADP-induced fibrinogen binding was much slower than that of abciximab. These differences in the fibrinogen binding rate were due to differential GPIIb/IIIa activation kinetics because the actual fibrinogen binding rate (measured by adding fibrinogen after platelet activation) was similar in ADP- and TRAP-stimulated platelets. Thus, the TRAP-induced GPIIb/IIIa activation rate would allow significant amounts of fibrinogen to occupy externalized GPIIb/IIIa receptors even in the presence of the inhibitor.

Abciximab↗
PubMed2001

Troglitazone and rosiglitazone induce apoptosis of vascular smooth muscle cells through an extracellular signal-regulated kinase-independent pathway.

Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. The peroxisome proliferator-activated receptor gamma (PPARgamma) ligands thiazolidinediones have been reported to induce apoptosis in macrophages and in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis in VSMC, cultured rat VSMC were treated with increasing doses of the thiazolidinedione analogues troglitazone (TRO) and rosiglitazone (RSG). Both ligands induced cell death in a concentration-dependent manner (EC50 12.1+/-3.3 microM and 1.43+/-0.39 microM, respectively), causing almost complete cell death at the highest concentrations (100 microM and 10 microM for TRO and RSG, respectively), along with an expected parallel decrease in [3H]thymidine uptake into cell DNA (EC50 6.7+/-2.4 microM and 0.75+/-0.19 microM, respectively). The cell count was determined by the coulter counter principle. Furthermore two apoptotic markers were measured, the caspase 3 activity and the cytoplasmic histone-associated DNA fragments, both of which were significantly increased when the aforementioned high concentrations were used. This indicates that apoptosis is involved in the TRO- and RSG-induced VSMC growth suppression. The same concentrations of TRO and RSG caused an unexpected stimulation of the extracellular signal-regulated response kinases 1 and 2 (ERK1/2) and stimulated the p38 mitogenic-activated protein (MAP) kinase as determined by Western blotting. In order to establish whether the proapoptotic effects of TRO and RSG are mediated through ERK1/2 activation, we used the selective MAP kinase kinase (MEK) inhibitor PD98059 (20 microM), which suppressed the TRO- and RSG-induced ERK1/2 activation but did not abolish their proapoptotic effects. We conclude that the thiazolidinedione analogues TRO and RSG induce cell death due to apoptosis in VSMC through an ERK1/2-independent pathway.

Animals↗
PubMed2001

Recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers.

AIMS: To study the recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers. METHODS: Ten healthy volunteers were treated with clopidogrel (75 mg day(-1)) for 7 days. CD62P expression and PAC-1 binding were measured by flow cytometry. RESULTS: Adenosine diphosphate (ADP, 30 microM)-induced platelet responses were almost completely inhibited by clopidogrel. After discontinuation of the drug, platelet function gradually increased and complete recovery was seen 7 days after the last clopidogrel dose. The mean difference (95% CI) for ADP-induced PAC-1 binding (fluorescence intensity) between baseline and 7 days after the last dose was 0.01 (0.61, -0.59). Single cell analysis provides direct evidence for an irreversible mode of action of clopidogrel. CONCLUSIONS: This is the first report to directly demonstrate irreversibility of clopidogrel action in humans.

Adenosine Diphosphate↗
PubMed2001

Effectiveness of a caries preventive program in pregnant women and new mothers on their offspring.

OBJECTIVE: The medical centre at Almirante Nef Naval Hospital, Vina del Mar, Chile, operates a mother and child preventive dental program (PDP) which includes women from their fourth month of pregnancy and mothers with their offspring. The aim was to evaluate the effectiveness of this prenatal and postnatal prevention program after the first four years. METHODS: The prevalence of early childhood caries of 180 1- to 3.5-year-old children enrolled in the PDP was compared with 180 non-participating children. The control group was randomly selected from the rolls of the healthy child system of of the Valparaiso-San Antonio Health Service and was comparable by age, socio-economic status and level of fluoride in the drinking water (1.0 p.p.m. F) with the PDP group. Clinical caries examinations were conducted by two calibrated examiners using the WHO visual criteria. RESULTS: In the PDP group, 97% of the children were caries free compared with the 77% in the control group. The dft (mean +/- SD) of the PDP children was 0.11 +/- 0.78 versus 0.66 +/- 1.55 for the control children, a difference of 83.3%. The differences between the groups in caries-free status and caries prevalence were both statistically significant (P < 0.05). CONCLUSIONS: The preventive dental program was effective in inhibiting caries in pre-school children, even in a population already receiving the benefits of community water fluoridation.

Chi-Square Distribution↗
PubMed2001

Troglitazone and rosiglitazone inhibit the low density lipoprotein-induced vascular smooth muscle cell growth.

Troglitazone (TRO) and rosiglitazone (RSG) belong to the thiazolidinedione class (insulin-sensitizing agents) and exert many of their metabolic effects as peroxisome proliferator-activated receptor gamma (PPARgamma) ligands. In the present study we examined the effects of TRO and RSG on LDL-induced VSMC growth. Pretreatment of VSMC with 1 microM TRO or 0.1 microM RSG completely blocked the LDL-induced cell proliferation as measured by [3H]thymidine incorporation into DNA and by determination of the cell number. We then examined with Western blotting whether these growth suppressing effects are mediated through the mitogen-activated protein kinase (MAPK) pathway, a common signaling pathway activated by growth factors. TRO and RSG had no effect on the LDL-induced stimulation of the MAP kinases ERK1/2, p38 and SAP/JNK. We conclude that thiazolidinediones are potent inhibitors of LDL-induced VSMC growth acting downstream of the cytoplasmic activation of MAPK.

Animals↗
PubMed2001

Platelet CD40 ligand (CD40L)--subcellular localization, regulation of expression, and inhibition by clopidogrel.

This study compares the subcellular localization and the regulation of expression of the platelet activation markers CD62P and CD63 with CD40 ligand (CD40L) on the surface of washed human platelets. CD40L was expressed upon stimulation with a wide range of platelet activators. However, quantitative flow cytometry demonstrated that, as compared with CD62P and CD63, CD40L expression was low. Upon stimulation with thrombin receptor-activating peptide (TRAP-6), all activation markers were expressed. In contrast, upon stimulation with low concentrations of collagen (1-3 microg/ml), CD40L, but not the granule proteins (CD62P, CD63), were expressed. Using immunofluorescence microscopy, a cytoplasmic staining was observed for CD40L, and cytoplasmic localization of CD40L was verified by Western blotting of subcellular platelet fractions. The staining of CD40L was different from that of filamentous actin and only little association of CD40L with platelet cytoskeleton was found. Surface expression of CD40L was dependent on internal Ca2+ stores and protein kinase C, while the mitogen-activated protein kinases (ERK, p38) or tyrosine kinases were not involved. ADP (30 microM)-induced CD40L expression was not inhibited by aspirin. In contrast, clopidogrel treatment completely abolished ADP-induced expression of CD40L. Finally, the expression level of CD40L was shown to be upregulated by phorbol myristate acetate (PMA) in the promegakaryocytic cell line MEG-01.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2000

Validated electrospray liquid chromatographic-mass spectrometric assay for the determination of the mushroom toxins alpha- and beta-amanitin in urine after immunoaffinity extraction.

Specific detection of amanitins in body fluids is necessary for an early diagnosis of an intoxication with amanita mushrooms. In this paper, a liquid chromatographic-mass spectrometric assay after immunoaffinity extraction (IAE-LC-MS) is described for the determination of alpha- and beta-amanitin in urine. The method has been validated according to the criteria established by the Journal of Chromatography B. The assay was found to be selective. The calibration curves for alpha- and beta-amanitin were linear from 5 to 75 ng/ml. Intra- and inter-day accuracy and precision were inside the required limits. Amatoxins in frozen urine samples or immunoaffinity extracts were stable for more than 6 months, and the IAE columns could be used more than fifty times without remarkable loss in performance. LOD for alpha- and beta-amanitin was 2.5 ng/ml and LOQ for both was 5.0 ng/ml. The absolute recoveries of alpha- and beta-amanitin were 63% and 58% for the low quality control and 61% and 57% for the high quality control. The absolute recovery for the internal standard gamma-amanitin methyl ether at 25 ng/ml was 60%. The analysis of 5 authentic urine samples from patients intoxicated by amanita mushrooms showed a good correlation between the results measured by radioimmunoassay and the IAE-LC-MS assay. A partial validation showed that the assay was also suitable for plasma analysis.

Agaricales↗
PubMed2000

Studies on the metabolism and toxicological detection of the amphetamine-like anorectic fenproporex in human urine by gas chromatography-mass spectrometry and fluorescence polarization immunoassay.

Studies on the metabolism and the toxicological analysis of fenproporex (R,S-3-[(1-phenyl-2-propyl)-amino]-propionitrile, FP) using GC-MS and fluorescence polarization immunoassay are described. The metabolites were identified in urine samples of volunteers by GC-MS after cleavage of conjugates, extraction and acetylation. Besides unchanged FP, fourteen metabolites, including amphetamine, could be identified. Two partially overlapping metabolic pathways could be postulated: ring degradation by one- and two-fold aromatic hydroxylation followed by methylation and side chain degradation by N-dealkylation to amphetamine (AM). A minor pathway leads via beta-hydroxylation of AM to norephedrine. For GC-MS detection, the systematic toxicological analysis procedure including acid hydrolysis, extraction at pH 8-9 and acetylation was suitable (detection limits 50 ng/ml for FP and 100 ng/ml for AM). Excretion studies showed, that only AM but neither FP nor its specific metabolites were detectable 30-60 h after ingestion of 20 mg of FP. Therefore, misinterpretation can occur. The Abbott TDx FPIA amphetamine/methamphetamine II gave positive results up to 58 h. All the positive immunoassay results could be confirmed by the described GC-MS procedure.

Acetylation↗
PubMed2000

Habitual smoking causes an abnormality in platelet thromboxane A2 metabolism and results in an altered susceptibility to aspirin effects.

The present study investigates the effects of aspirin (100 mg every second day for 14 days) on platelet function in nine healthy non-smokers and in nine healthy habitual smokers. There was a significantly (P < 0.05) stronger inhibition of collagen (0.6 microgram/ml)- and ADP (2 microM)-induced platelet aggregation by aspirin in smokers as compared to non-smokers. This difference occurred in the presence of an almost complete (> 95%) inhibition of thromboxane A2 (TXA2) synthesis in both groups. The platelet capacity to generate TXA2 in vitro was significantly reduced in smokers, urinary excretion of TXA2, however, was significantly increased. Thus, the better susceptibility of smokers to anti-aggregatory effects of aspirin is very likely to be related to a chronic smoking-induced alteration of platelet TXA2 system. Cessation of smoking should, therefore, be encouraged.

Adenosine Diphosphate↗
PubMed1999

Platelet surface membranes are highly mitogenic for coronary artery smooth muscle cells--A novel mechanism for sustained proliferation after vessel injury?

The effects of isolated platelet surface membranes on DNA synthesis and proliferation of bovine coronary artery smooth muscle cells (SMC) were studied. Platelet membranes were very potent mitogens for SMC. The potency was about 10-fold higher than the maximum effects of platelet-derived growth factor-BB (PDGF). Platelet membrane-induced mitogenesis was inhibited by rapamycin, wortmannin or heating for 15 min at 70 degrees C but not by the PDGF receptor antagonist SCH 13.929 or by neutralizing PDGF antibodies. Only a partial (30%) inhibition was seen with PD 98059. In contrast, PDGF-induced SMC mitogenesis was heat-stable but sensitive to SCH 13. 929, PDGF antibodies, and PD 98059. These findings provide evidence for a novel mechanism for platelet-induced SMC proliferation that is independent of PDGF secretion. Platelet membranes, attached to or incorporated into the vessel wall, could maintain sustained SMC proliferation following injury.

Androstadienes↗
PubMed1999

Cyclooxygenase-2 expression in human esophageal carcinoma.

On the basis of epidemiological observations that nonsteroidal antiinflammatory drugs reduce the risk of esophageal carcinoma, we studied the expression of cyclooxygenase-2 (COX-2) in esophageal squamous cell carcinomas (SCCs; n = 172) and in esophageal adenocarcinomas (ADCs; n = 27). Using immunohistochemistry, we observed COX-2 expression in 91% of the SCCs and in 78% of the ADCs. Western blot analysis showed enhanced expression of the COX-2 protein in some tumors as compared with normal esophageal squamous epithelium, whereas similar amounts of the COX-1 protein were found in normal and cancerous tissues. COX expression was also studied in two esophageal cancer cell lines (OSC-1 and OSC-2) to evaluate the functional relevance of COX-2-derived prostaglandins (PGs). OSC-2 cells expressed COX-2 but not COX-1, whereas OSC-1 cells expressed high levels of COX-1 but showed only a very weak COX-2 expression. Accordingly, PGE2 synthesis was 600 times higher in the OSC-2 cells as compared with the OSC-1 cells. Treatment of OSC-2 cells with the selective COX-2 inhibitors flosulide and NS-398 concentration dependently suppressed PGE2 synthesis and proliferation and also induced apoptosis. In contrast, no effect of the COX-2 inhibitors was seen in OSC-1 cells. Our data demonstrate that COX-2 is expressed in the majority of esophageal SCCs and ADCs and that COX-2-derived PGs play an important role in the regulation of proliferation and apoptosis of esophageal tumor cells. It is concluded that inhibition of COX-2 may be useful in the therapy of esophageal cancer.

Carcinoma↗
PubMed1999

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro.

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.

Adenosine Diphosphate↗
PubMed1999

cAMP is an important messenger for ADP-induced platelet aggregation.

In rat platelets, basal cAMP levels were not changed upon stimulation with ADP and it was concluded that cAMP is not an important messenger for ADP-induced aggregation (Savi et al., Blood Coagul Fibrinolysis, 1996; 7: 249-52). In the present study, the effects of prostaglandin E(1) (PGE(1)) and ADP on human platelet aggregation, cAMP generation and VASP phosphorylation were studied. Phosphorylation of the protein kinase A (PKA) substrate VASP and inhibition of platelet aggregation by PGE(1) occurred without measurable changes in cellular cAMP levels. In addition, a marked inhibition of basal VASP phosphorylation by ADP was observed. It is concluded that cAMP determinations do not necessarily detect a possible activation or inhibition of the cAMPPKA pathway in platelets. Thus, cAMP might well be an important second messenger for ADP-induced platelet aggregation.

Journal Article↗
PubMed1999

The significance of platelet-derived growth factors for proliferation of vascular smooth muscle cells.

Platelets are important in acute thrombotic occlusion of injured vessels, e.g., subsequent to angioplasty. In contrast to these acute events of thrombus formation, much less is known about the significance of platelets for the control of smooth muscle cell (SMC) proliferation. A body of experimental and clinical evidence indicates an involvement of platelets in the pathology of atherosclerosis and restenosis. However, the precise role of platelet-derived growth factors for SMC proliferation in atherosclerotic and restenotic vessels is not clear and many questions remain unresolved. Platelet-dependent SMC mitogenesis is determined by a coordinate action of several classes of mitogenic factors which are either released from storage pools or generated upon platelet activation. Although platelet-derived growth factor (PDGF) is considered to be the most important platelet mitogen it is very likely that yet unknown factors and mechanisms are involved. Differential (stimulatory or inhibitory) effects on SMC growth and differentiation have been reported for different platelet-derived growth factors. Thus, for the overall response, complex interactions between multiple factors need to be considered. In addition, multicellular interactions, e.g., between platelets and endothelial cells may modulate the effects of platelet-derived factors on SMC mitogenesis. Taken together, the mechanisms of platelet-dependent SMC proliferation need to be reevaluated. The assessment of the precise role of platelet mitogens in the complex proliferative repair mechanisms of an injured vessel wall clearly requires further studies.

Journal Article↗