A novel gold nanorod-based spectrographic method for evaluating the curcumin inhibitory action on HepG2 cells

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

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The Effect of Agonists and Antagonists of Platelet Aggregation on von Willebrand Factor-Mediated Platelet Agglutination

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648192 ◽

1991 ◽

Vol 65 (05) ◽

pp. 573-577 ◽

Cited By ~ 1

Author(s):

Jean McPherson ◽

Marjorie B Zucker ◽

Evelyn A Mauss ◽

Sandra Brownlea

Keyword(s):

Receptor Antagonist ◽

Intracellular Calcium ◽

Calcium Ions ◽

Inhibitory Action ◽

Platelet Rich Plasma ◽

Cytoskeletal Proteins ◽

A 23187 ◽

Sulphydryl Groups ◽

Affinity Receptor ◽

Adp Receptor

SummaryRistocetin-induced platelet agglutination (RIPA) in EDTA-treated citrated platelet-rich plasma was reduced to 49 ± 11% by 1.25 ΜM ADP, 41 ± 14% by 1 ΜM A 23187, and 26 ± 7% by 0.1 Μg/ml platelet activating factor (PAF). The effect of 5-110 ΜM epinephrine was not dose-dependent, but varied between donors, with RIPA from 56-100% of the control. The inhibitory effects of these agonists were not altered by prior treatment of platelets with aspirin. Prior addition of 200 ΜM ATP (an ADP receptor antagonist acting at both high and low affinity ADP receptors) prevented the inhibitory action of ADP but not that of A 23187 or PAF, suggesting that the inhibitory actions of the latter are not mediated by released ADP. As 700 ΜM 8-bromoadenosine 5-diphosphate (an ADP receptor antagonist acting mainly at the high affinity receptor) did not prevent ADP-induced inhibition of RIPA, interaction of ADP with the low affinity receptor is presumably responsible for its inhibitory action. As A 23187, but not phorbol myristate acetate (0.1 ΜM) inhibited RIPA, an increase in intracellular calcium ions rather than direct stimulation of protein kinase C appears to mediate agonist-induced inhibition. Cytochalasin B (10.5-21 ΜM), dibucaine (0.5-1 mM), and prostaglandin E1 (25 nM), added before or after the agonist, prevented or reversed ADP-, A23187-, and PAF-induced inhibition of RIPA, suggesting that the state of the platelet cytoskeleton affects inhibition. N-ethylmaleimide (0.25-0.5 mM), an agent that can penetrate cell membranes and block sulphydryl groups, prevented or reversed ADP, A 23187- and PAF-induced inhibition of RIPA, but 0.5 mM dithionitrobisbenzoic acid, a non-penetrating sulphydryl blocker, had no effect. Diamide (0.1-0.5 mM), an agent that can crosslink cytoskeletal proteins by oxidation of sulphydryl groups, reduced RIPA. Thus an increase in intracellular calcium ions with resultant cytoskeletal changes and reorganisation of intracellular sulphydryl groups may mediate the inhibitory action of agonists on RIPA.

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The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656315 ◽

1992 ◽

Vol 68 (01) ◽

pp. 040-047 ◽

Cited By ~ 3

Author(s):

C Scott Jamison ◽

Bryan F Burkey ◽

Sandra J Friezner Degen

Keyword(s):

Total Protein ◽

Hepg2 Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Response Analysis ◽

Secreted Protein ◽

Mrna Levels ◽

Vitamin K1 ◽

Maximal Increase ◽

Protein Levels ◽

Warfarin Treatment

SummaryCultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 µg vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 µg/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 µg/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 µg/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240–350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on γ-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

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Inhibition of ADP-Induced Responses in Human Platelets by Agents Elevating the Cyclic AMP Level: Comparison of Aggregation and Shape Change

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661208 ◽

1984 ◽

Vol 52 (03) ◽

pp. 333-335 ◽

Cited By ~ 4

Author(s):

Vider M Steen ◽

Holm Holmsen

Keyword(s):

Inhibitory Action ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Shape Change ◽

Inhibitory Effect ◽

Human Platelets ◽

Inhibitory Effects ◽

Early Step ◽

Stimulus Response ◽

Sequential Relationship

SummaryThe inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses.Human platelet-rich plasma was preincubated for 2 min at 37° C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors.These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.

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Mechanism of the Antiplatelet Action of Dipyridamole in Whole Blood: Modulation of Adenosine Concentration and Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661437 ◽

1986 ◽

Vol 55 (01) ◽

pp. 012-018 ◽

Cited By ~ 86

Author(s):

Paolo Gresele ◽

Jef Arnout ◽

Hans Deckmyn ◽

Jos Vermylen

Keyword(s):

Platelet Aggregation ◽

Adenosine Receptor ◽

Whole Blood ◽

Blood Cells ◽

Inhibitory Action ◽

Phosphodiesterase Inhibitor ◽

Inhibitory Effect ◽

Adenosine Concentration ◽

Pharmacological Studies

SummaryDipyridamole inhibits platelet aggregation in whole blood at lower concentrations than in plasma. The blood cells responsible for increased effectiveness in blood are the erythrocytes. Using the impedance aggregometer we have carried out a series of pharmacological studies in vitro to elucidate the mechanism of action of dipyridamole in whole blood. Adenosine deaminase, an enzyme breaking down adenosine, reverses the inhibitory action of dipyridamole. Two different adenosine receptor antagonists, 5’-deoxy-5’-methylthioadenosine and theophylline, also partially neutralize the activity of dipyridamole in blood. Enprofylline, a phosphodiesterase inhibitor with almost no adenosine receptor antagonistic properties, potentiates the inhibition of platelet aggregation by dipyridamole. An inhibitory effect similar to that of dipyridamole can be obtained combining a pure adenosine uptake inhibitor (RE 102 BS) with a pure phosphodiesterase inhibitor (MX-MB 82 or enprofylline). Mixing the blood during preincubation with dipyridamole increases the degree of inhibition. Lowering the haematocrit slightly reduces the effectiveness.Although we did not carry out direct measurements of adenosine levels, the results of our pharmacological studies clearly show that dipyridamole inhibits platelet aggregation in whole blood by blocking the reuptake of adenosine formed from precursors released by red blood cells following microtrauma. Its slight phosphodiesterase inhibitory action potentiates the effects of adenosine on platelets.

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