scholarly journals Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin, collagen, phorbol ester and exogenous diacylglycerol

1984 ◽  
Vol 222 (3) ◽  
pp. 833-836 ◽  
Author(s):  
T J Rink ◽  
A Sanchez
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Human Platelets ◽  
Free Calcium ◽  
Prostaglandin I2 ◽  
Kinase C ◽  
Inositol Lipids ◽  
Hydrolysis Of ◽  
Cytoplasmic Free Calcium

Cytoplasmic free calcium ([Ca2+]i) and secretion of ATP were measured in quin2-loaded human platelets. In certain conditions thrombin and collagen cause secretion while [Ca2+]i remains at basal concentrations, a response attributed to activation of protein kinase by diacylglycerol formed by hydrolysis of inositol lipids. This secretion evoked by thrombin could be totally suppressed by prostaglandin I2 or forskolin, as expected from the known ability of cyclic AMP to inhibit phospholipase C. The secretory response evoked by collagen at basal [Ca2+]i and that evoked by exogenous diacylglycerol or phorbol ester, direct activators of protein kinase-C, were much less affected by these inhibitors, suggesting that thrombin and collagen may promote formation of diacylglycerol by different mechanisms.

scholarly journals Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets

1996 ◽  
Vol 318 (1) ◽  
pp. 207-212 ◽  
Author(s):  
Angelika G. BÖRSCH-HAUBOLD ◽  
Ruth M. KRAMER ◽  
Steve P WATSON
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Substrate Inhibition ◽  
Human Platelets ◽  
Mitogen Activated Protein Kinase ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2´-amino-3´-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol ester-stimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 µM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST–MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.

High Density Lipoproteins Enhance the Na+/H+ Antiport in Human Platelets

1996 ◽  
Vol 75 (04) ◽  
pp. 635-641 ◽  
Author(s):  
Jerzy-Roch Nofer ◽  
Martin Tepel ◽  
Beate Kehrel ◽  
Michael Walter ◽  
Udo Seedorf ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Platelets ◽  
Covalent Modification ◽  
High Density ◽  
High Density Lipoproteins ◽  
Free Calcium ◽  
Sodium Propionate ◽  
Cytosolic Ph ◽  
Kinase C

SummaryIn the present study, we investigated the effect of high density lipoproteins 3 (HDL3) on Na+/H+ exchanger activity and cytosolic pH (pHi) in human platelets. HDL3 alone failed to affect pHi? but preincubation with HDL3 significantly enhanced the Na+/H+ antiport activation brought about by acidification with 100 mM sodium propionate or stimulation with 0.05 U/ml thrombin. The stimulatory effect of HDL3 was unaffected by indomethacin excluding a role for cyclooxygenase products. The HDL3 effect was not mediated by Ca2+/calmodulin-dependent protein kinase as HDL3 failed to increase cytosolic free calcium concentration. However, the potentiating effect of HDL3 was completely blocked in the presence of the protein kinase C inhibitor, bisindoylmaleimide and the phosphatidylcholine-specific phospholi-pase C inhibitor, D609. Furthermore, the effect of HDL3 was abolished after covalent modification of HDL3 with dimethylsuberimidate and was not observed in platelets from Glanzmann thrombasthenia type 1 which do not express GP IIb/IIIa, as well as in platelets preincubated with anti-GP Ilb/IIIa polyclonal antibodies. We conclude that HDL3 enhances the sodium propionate- and thrombin-induced Na+/H+ antiport activity in human platelets via binding to GP Ilb/IIIa and activation of protein kinase C and phosphatidylcholine-specific phospholipase C.

Influence of vasoactive agents on cytoplasmic free calcium in vascular endothelial cells

1988 ◽  
Vol 65 (5) ◽  
pp. 2221-2227 ◽  
Author(s):  
U. S. Ryan ◽  
P. V. Avdonin ◽  
E. Y. Posin ◽  
E. G. Popov ◽  
S. M. Danilov ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Pulmonary Artery ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Platelet Activating Factor ◽  
Free Calcium ◽  
Kinase C ◽  
Cytoplasmic Free Calcium

The regulation of cytoplasmic free calcium concentration [( Ca2+]i) in endothelial cells (EC) derived from human umbilical vein, aorta, and pulmonary artery, or from bovine pulmonary artery, was studied by means of the fluorescent Ca2+ indicator indo-1. Histamine and thrombin caused a rapid transient elevation in [Ca2+]i in the EC of all the human blood vessels tested. In aortic EC, [Ca2+]i also rose in response to ATP and bradykinin. It was shown that in bovine pulmonary artery EC [Ca2+]i rises in response to platelet-activating factor (PAF) and thrombin. For a more detailed investigation of the receptor-mediated mechanism of [Ca2+]i increase in EC we used histamine as a stimulating agent. Histamine effects were seen at concentrations ranging from 5 X 10(-7) to 10(-4) M [50% effective dose (ED50) approximately 2-4 microM)] and were mediated by H1-receptors. The histamine-induced increase in [Ca2+]i was not markedly diminished when the extracellular calcium was bound by excess ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The data obtained indicate that the histamine effect is best explained by Ca2+ mobilization from intracellular stores. The histamine-induced increase in [Ca2+]i was not influenced by elevating the intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or cyclic guanylic acid (cGMP) by use of isobutylmethylxanthine and forskolin or by nitroprusside preincubation, respectively. However, the protein kinase C stimulator, phorbol myristate acetate (PMA), strongly inhibits [Ca2+]i elevation. It is assumed that a negative feedback mechanism that blocks receptor-mediated [Ca2+]i increase is triggered as a result of the activation of protein kinase C.

scholarly journals Activation of Na+/H+ exchange in human platelets stimulated by thrombin and a phorbol ester

1987 ◽  
Vol 241 (1) ◽  
pp. 301-303 ◽  
Author(s):  
W Siffert ◽  
G Siffert ◽  
P Scheid
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fluorescent Probe ◽  
Phorbol Ester ◽  
Human Platelets ◽  
Cytoplasmic Ph ◽  
Kinase C ◽  
Stimulation Of

We have investigated changes in cytoplasmic pH (pHi) in activated human platelets, using the fluorescent probe 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. Stimulation of platelets by thrombin or 12-O-tetradecanoylphorbol 13-acetate increased pHi by about 0.11 pH unit above the resting value. This increase in pHi depended on the presence of external Na+ and was inhibited by ethylisopropylamiloride. The data suggest that protein kinase C mediates Na+/H+ exchange in human platelets.

Role of Protein Kinase C in the Anti-Aggregatory Effects of Endothelin-1 on Human Platelets

1995 ◽  
Vol 88 (3) ◽  
pp. 277-283 ◽  
Author(s):  
R. M. Touyz ◽  
E. L. Schiffrin
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Concentration ◽  
Human Platelets ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Endothelin 1 ◽  
Kinase C ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

1. Endothelin-1 has anti-aggregatory properties, but the mechanism underlying this inhibitory action is unknown. This in vitro study investigates effects of endothelin-1 on thrombin-stimulated aggregation and intracellular free calcium concentration in human platelets and assesses the role of protein kinase C in the interactions between endothelin-1 and thrombin. Aggregation was measured turbidometrically and the intracellular free calcium concentration was determined with the fluorescent indicator fura 2-acetoxymethyl ester. 2. Endothelin-1 at concentrations from 10−11 to 10−6 mol/l had no effect on platelet aggregation or intracellular free calcium concentration but inhibited in a dose-dependent manner aggregation induced by 0.05 unit/ml thrombin (pD2 for inhibition by endothelin = 8.1 ± 0.12). 3. Endothelin-1 at 10−9 mol/l significantly decreased (P<0.01) thrombin-stimulated aggregation from 81.4 ± 1.5% (in the absence of endothelin-1) to 53.5 ± 1.1% (in the presence of endothelin) and thrombin-stimulated intracellular free calcium concentration from 179 ± 1.7 nmol/l to 140 ± 1.8 nmol/l. 4. Preincubation of platelets with 10−7 mol/l staurosporine (protein kinase C inhibitor), calphostin C (highly selective protein kinase C inhibitor) or 5-(N,N-hexamethylene) amiloride (highly selective Na+-H+ exchange blocker) significantly inhibited (P < 0.01) thrombin-stimulated platelet responses and suppressed the inhibitory effect of endothelin-1 on thrombin-induced aggregation and intracellular free calcium concentration. 5. In conclusion, endothelin-1 decreases the aggregatory response of human platelets to thrombin by mechanisms that probably involve protein kinase C and Na+-H+ linked pathways.

Duality of plasmin effect on cytosolic free calcium in human platelets

1995 ◽  
Vol 268 (4) ◽  
pp. C958-C967 ◽  
Author(s):  
K. Nakamura ◽  
M. Kimura ◽  
J. W. Fenton ◽  
T. T. Andersen ◽  
A. Aviv
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Free Calcium ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Small Rise

Plasmin caused a modest and gradual increase in platelet cytosolic Ca2+, mediated through both Ca2+ mobilization and external Ca2+ entry. This response was associated with accelerated Ca2+ extrusion and protein tyrosine phosphorylation. Plasmin-enhanced external Ca2+ entry and Ca2+ extrusion (but not Ca2+ mobilization) were attenuated by the tyrosine kinase inhibitor, genistein. Plasmin inhibited the thrombin-evoked increase in cytosolic Ca2+ and also inhibited the Ca2+ response to the tethered peptide TRAP-6 of the thrombin receptor. Furthermore, plasmin inhibited the binding of 125I-labeled alpha-thrombin to platelets. The inhibitory effect of plasmin on the thrombin response shared some characteristics with the effect of protein kinase C stimulators but was not reversed by protein kinase C inhibitors. Plasmin did not change platelet cyclic nucleotides. These results suggest a dual effect of plasmin. Plasmin produces a small rise in platelet cytosolic Ca2+ and a tyrosine kinase-dependent enhancement of Ca2+ turnover (external Ca2+ influx and Ca2+ efflux). However, it also attenuates the thrombin-evoked cytosolic Ca2+ response by blocking Ca2+ mobilization and slowing the rate of external Ca2+ influx. The latter feature would result in a plasmin-induced inhibition of thrombogenesis.

Phorbol ester-induced activation of human platelets is associated with protein kinase C phosphorylation of myosin light chains

Nature ◽  
1983 ◽  
Vol 306 (5942) ◽  
pp. 490-492 ◽  
Author(s):  
Michiko Naka ◽  
Masakatsu Nishikawa ◽  
Robert S. Adelstein ◽  
Hiroyoshi Hidaka
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Human Platelets ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Kinase C
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