scholarly journals Analysis of the mechanism of anagrelide-induced thrombocytopenia in humans

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 1931-1937 ◽  
Author(s):  
EM Mazur ◽  
AG Rosmarin ◽  
PA Sohl ◽  
JL Newton ◽  
A Narendran
Keyword(s):  
Colony Size ◽  
Molecular Mechanisms ◽  
Human Peripheral Blood ◽  
Inhibitory Effect ◽  
Colony Development ◽  
Maturation Stage ◽  
Inhibitory Effects ◽  
Plasma Clot ◽  
Liquid Suspension ◽  
Normal Human Peripheral Blood

Abstract Anagrelide is a new therapeutic compound recently demonstrated to have a rapid and selective thrombocytopenic effect in humans. The effects of anagrelide were evaluated in plasma clot and liquid suspension cultures of optimally stimulated normal human peripheral blood megakaryocyte progenitors in order to determine the mechanism of its thrombocytopenic activity. In plasma clot cultures, at clinically relevant, therapeutic concentrations (5 to 50 ng/mL), anagrelide exerted no significant inhibitory effect on megakaryocyte colony numbers or colony size. Only at anagrelide concentrations of 10 to 500 times therapeutic doses did anagrelide inhibit megakaryocyte colony development: an anagrelide concentration of 5 micrograms/mL reduced colony numbers by 57% and colony size by 31%. In contrast, lower, therapeutic anagrelide concentrations exerted profound effects in liquid culture on megakaryocyte cytoplasmic maturation, size, and DNA content. When present for the entire 12-day culture duration, anagrelide induced left- shifted megakaryocyte maturation and reduced both megakaryocyte ploidy and megakaryocyte diameter. Anagrelide, at concentrations of 5 to 50 ng/mL, shifted the modal cultured megakaryocyte morphologic stage from III to II, reduced the model ploidy value from 16N to 8N, and decreased the mean megakaryocyte diameter by up to 22%, from 27.6 microns to 21.6 microns. Megakaryocyte diameter was significantly reduced in most instances, even when analyzed as a function of morphologic stage. When anagrelide was added to the cultures after 6- and 9-day delays (during the final 6 and 3 days, respectively, of culture), similar inhibitory effects on megakaryocyte maturation stage and ploidy distribution were observed. However, the magnitude of the left-shift in ploidy appeared to be less as the duration of anagrelide exposure was reduced. Conversely, megakaryocyte diameter was not significantly affected by the shorter 3- and 6-day anagrelide exposures. These data indicate that therapeutic concentrations of anagrelide influence primarily the postmitotic phase of megakaryocyte development, decreasing platelet production by reducing megakaryocyte size and ploidy, as well as by disrupting full megakaryocyte maturation. Inhibition of megakaryocyte diameter appears to require more prolonged anagrelide exposure than inhibition of maturation stage and ploidy. The molecular mechanisms responsible for the inhibitory effects of anagrelide on megakaryocytopoiesis remain to be defined.

scholarly journals Analysis of the mechanism of anagrelide-induced thrombocytopenia in humans

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 1931-1937 ◽  
Author(s):  
EM Mazur ◽  
AG Rosmarin ◽  
PA Sohl ◽  
JL Newton ◽  
A Narendran
Keyword(s):  
Colony Size ◽  
Molecular Mechanisms ◽  
Human Peripheral Blood ◽  
Inhibitory Effect ◽  
Colony Development ◽  
Maturation Stage ◽  
Inhibitory Effects ◽  
Plasma Clot ◽  
Liquid Suspension ◽  
Normal Human Peripheral Blood

Anagrelide is a new therapeutic compound recently demonstrated to have a rapid and selective thrombocytopenic effect in humans. The effects of anagrelide were evaluated in plasma clot and liquid suspension cultures of optimally stimulated normal human peripheral blood megakaryocyte progenitors in order to determine the mechanism of its thrombocytopenic activity. In plasma clot cultures, at clinically relevant, therapeutic concentrations (5 to 50 ng/mL), anagrelide exerted no significant inhibitory effect on megakaryocyte colony numbers or colony size. Only at anagrelide concentrations of 10 to 500 times therapeutic doses did anagrelide inhibit megakaryocyte colony development: an anagrelide concentration of 5 micrograms/mL reduced colony numbers by 57% and colony size by 31%. In contrast, lower, therapeutic anagrelide concentrations exerted profound effects in liquid culture on megakaryocyte cytoplasmic maturation, size, and DNA content. When present for the entire 12-day culture duration, anagrelide induced left- shifted megakaryocyte maturation and reduced both megakaryocyte ploidy and megakaryocyte diameter. Anagrelide, at concentrations of 5 to 50 ng/mL, shifted the modal cultured megakaryocyte morphologic stage from III to II, reduced the model ploidy value from 16N to 8N, and decreased the mean megakaryocyte diameter by up to 22%, from 27.6 microns to 21.6 microns. Megakaryocyte diameter was significantly reduced in most instances, even when analyzed as a function of morphologic stage. When anagrelide was added to the cultures after 6- and 9-day delays (during the final 6 and 3 days, respectively, of culture), similar inhibitory effects on megakaryocyte maturation stage and ploidy distribution were observed. However, the magnitude of the left-shift in ploidy appeared to be less as the duration of anagrelide exposure was reduced. Conversely, megakaryocyte diameter was not significantly affected by the shorter 3- and 6-day anagrelide exposures. These data indicate that therapeutic concentrations of anagrelide influence primarily the postmitotic phase of megakaryocyte development, decreasing platelet production by reducing megakaryocyte size and ploidy, as well as by disrupting full megakaryocyte maturation. Inhibition of megakaryocyte diameter appears to require more prolonged anagrelide exposure than inhibition of maturation stage and ploidy. The molecular mechanisms responsible for the inhibitory effects of anagrelide on megakaryocytopoiesis remain to be defined.

Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels in the Cardiovascular System

2004 ◽  
Vol 101 (2) ◽  
pp. 390-398 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Akira Takahashi ◽  
Yasuo Tsutsumi ◽  
Yoshinobu Tomiyama ◽  
...  
Keyword(s):  
Cardiovascular System ◽  
Adenosine Triphosphate ◽  
Local Anesthetics ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Katp Channels ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Sulfonylurea Receptor ◽  
Inhibitory Effects

Background Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. Methods The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. Results Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 microm) stereoselectively (levobupivacaine, IC50 = 168 microm; ropivacaine, IC50 = 249 microm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2 delta C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. Conclusions Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.

scholarly journals Phytohemagglutinin-stimulated normal human peripheral blood lymphocytes in folate-depleted medium: an in vitro model for megaloblastic hemopoiesis

Blood ◽  
1980 ◽  
Vol 55 (5) ◽  
pp. 863-865 ◽  
Author(s):  
RJ Hayman ◽  
MB Van Der Weyden
Keyword(s):  
Peripheral Blood ◽  
Thymidine Incorporation ◽  
Human Peripheral Blood ◽  
Inhibitory Effect ◽  
Peripheral Blood Lymphocytes ◽  
Blood Lymphocytes ◽  
Human Peripheral Blood Lymphocytes ◽  
Normal Human ◽  
Normal Human Peripheral Blood

Abstract Normal human peripheral blood lymphocytes cultured with phytohemagglutinin in folate-free RPMI 1640 medium supplemented with normal human serum exhibit megaloblastic maturation. These changes are accompanied by a decrease in intracellular folate content, and when compared to folate-replete cells, reveal increased 3H-thymidine incorporation into DNA together with a decreased inhibitory effect of deoxyuridine on 3H-thymidine incorporation. This in vitro system of cellular folate deficiency is a convenient model for analysis of biochemical events accompanying megaloblastic maturation.

scholarly journals Phytohemagglutinin-stimulated normal human peripheral blood lymphocytes in folate-depleted medium: an in vitro model for megaloblastic hemopoiesis

Blood ◽  
1980 ◽  
Vol 55 (5) ◽  
pp. 863-865
Author(s):  
RJ Hayman ◽  
MB Van Der Weyden
Keyword(s):  
Peripheral Blood ◽  
Thymidine Incorporation ◽  
Human Peripheral Blood ◽  
Inhibitory Effect ◽  
Peripheral Blood Lymphocytes ◽  
Blood Lymphocytes ◽  
Human Peripheral Blood Lymphocytes ◽  
Normal Human ◽  
Normal Human Peripheral Blood

Normal human peripheral blood lymphocytes cultured with phytohemagglutinin in folate-free RPMI 1640 medium supplemented with normal human serum exhibit megaloblastic maturation. These changes are accompanied by a decrease in intracellular folate content, and when compared to folate-replete cells, reveal increased 3H-thymidine incorporation into DNA together with a decreased inhibitory effect of deoxyuridine on 3H-thymidine incorporation. This in vitro system of cellular folate deficiency is a convenient model for analysis of biochemical events accompanying megaloblastic maturation.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Inhibition of Coagulation and Fibrinolysis by Aromatic Amidino Compounds

1971 ◽  
Vol 25 (03) ◽  
pp. 391-404 ◽  
Author(s):  
J.D Geratz
Keyword(s):  
Prothrombin Time ◽  
Human Plasma ◽  
Partial Thromboplastin Time ◽  
Inhibitory Effect ◽  
Clot Lysis ◽  
Similar Degree ◽  
Contact Activation ◽  
Plasma Clot ◽  
Human Thrombin ◽  
Canine Plasma

Summary1. Aromatic diamidines which are potent inhibitors of trypsin possess a marked inhibitory effect on the clotting activity of human thrombin and on the prothrombin time and partial thromboplastin time of human plasma. They also block the contact activation phase of the coagulation process. The strongest inhibitor among the compounds tested was M & B 4596 which was followed in second place by pentamidine.2. Pentamidine was 10 times more active than ε-ACA in impeding streptokinase-induced lysis of human plasma clots. It was 100-200 times stronger than ε-ACA in inhibiting the activation of bovine plasminogen by activators formed from the interaction between streptokinase and either human plasmin(ogen) or human plasma.3. The prothrombin time and partial thromboplastin time of canine plasma were less susceptible to inhibition by pentamidine than the same tests on human plasma. Clot lysis in the canine system was inhibited by pentamidine to a similar degree as in the human system. After intravenous injection of pentamidine in the dog there occurred the expected prolongation of the partial thromboplastin time and of the clot lysis time.

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

1984 ◽  
Vol 52 (03) ◽  
pp. 333-335 ◽  
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.

The Inhibitory Effect of Heparin and Related Glycosaminoglycans on Neutrophil Chemotaxis

1984 ◽  
Vol 52 (02) ◽  
pp. 134-137 ◽  
Author(s):  
Yaacov Matzner ◽  
Gerard Marx ◽  
Ruth Drexler ◽  
Amiram Eldor
Keyword(s):  
Specific Binding ◽  
Anticoagulant Activity ◽  
Neutrophil Migration ◽  
Inhibitory Effect ◽  
Chemotactic Factor ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
Neutrophil Chemotaxis ◽  
Inhibitory Effects ◽  
Chemotactic Factors

SummaryClinical observations have shown that heparin has antiinflammatory activities. The effect of heparin on neutrophil chemotaxis was evaluated in vitro in the Boyden Chamber. This method enabled differentiation between the direct effects of heparin on neutrophil migration and locomotion, and its effects on chemotactic factors. Heparin inhibited both the random migration and directed locomotion of human neutrophils toward zymosan-activated serum (ZAS) and F-met-leu-phe (FMLP). Inhibition was found to be dependent on the concentrations of the heparin and of the chemotactic factors. No specific binding of heparin to the neutrophils could be demonstrated, and heparin’s inhibitory effects were eliminated by simple washing of the cells. When added directly to the chamber containing chemotactic factor, heparin inhibited the chemotactic activity of ZAS but not that of FMLP, suggesting a direct inhibitory effect against C5a, the principal chemotactic factor in ZAS.Experiments performed with low-molecular-weight heparin, N-desulfated heparin, dextran sulfate, chondroitin sulfate and dextran indicated that the inhibitory effects of heparin on neutrophil chemotaxis are not related to its anticoagulant activity, but probably depend on the degree of sulfation of the heparin molecule.

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