ACTIVATION OF PROTEIN KINASE C INHIBITS THROMBIN AND FLUORIDE STIMULATED EICOSANOID PRODUCTION IN HUMAN PLATELETS

1987 ◽  
Author(s):  
C T Poll ◽  
P A Kyrle ◽  
J Westwick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Platelets ◽  
Free Calcium ◽  
Kinase C ◽  
Eicosanoid Production ◽  
Protein Kinase C Inhibitor ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Heart Foundation

Touqui et al (1986) have suggested that phosphorylation by protein kinase C of a 1ipomodulin-1 ike polypeptide extracted from platelets renders it inactive as an inhibitor of phospholipase A2. We have examined this suggestion by measuring thromboxane (Tx) B2 generation and cytosolic free calcium concentration ([Ca++]i) in stimulated, washed human platelets loaded with or without quin-2. Addition of thrombin (0.077, 0.23, 0.77, 2.3 and 7.7 nM) to control platelets produces a dose-related elevation of [Ca++]i (10±5, 50±7, 260±30, 550±25 and 1500±100 nM respectively) and generation of TxB2 (0, 9±4, 45±6, 194±10 and 375±30 pmoles/108 platelets respectively). Preincubation of platelets for 1 min with 1-oleoyl-2-acetyl-rac-glycerol (OAG, 22-198 μM), phorbol myristate acetate (PMA, 1.616 nM) or EGTA (2 mM) produces a marked inhibition of high and low dose thrombin (7.7 nM and 0.77 nM) or NaF (18 mM) induced elevation of [Ca++]i and TxB2 generation. Pretreatment of platelets with the protein kinase C inhibitor, H-7 (60 uM), prevented the inhibition of TxB2 formation induced by PMA (4.816 nM) or OAG (66-198 μM) in either thrombin (0.77 nM) or NaF (18 mM) stimulated platelets. When arachidonic acid (AA, 10 μM) is used as the stimulus, the Δ[Ca++]i is 190±15 nM and TxB2 generation is 35.9±2 pmoles/108 platelets. While pretreatment with 4.8 nM PMA obliterates the AA-induced Δ[Ca++]i and partially reduces (p< 0.05) the TxB2 generation to 27.8+3 pmoles/108 platelets. PMA and OAG pretreatment also inhibits TxB2 generation in thrombin-stimulated, non-quin-2-1oaded platelets. Thus, at least with intact, agonist- and NaF-stimulated platelets, activation of protein kinase C inhibits eicosanoid production.We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

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.

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.

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.

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.

Diacylglycerol kinase inhibitor R59022 and stimulated neutrophil responses

1988 ◽  
Vol 255 (5) ◽  
pp. C589-C594 ◽  
Author(s):  
J. L. Mege ◽  
W. Tao ◽  
T. F. Molski ◽  
J. Gomez-Cambronero ◽  
C. K. Huang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cytochalasin B ◽  
Kinase Inhibitor ◽  
Platelet Activating Factor ◽  
Diacylglycerol Kinase ◽  
Leukotriene B4 ◽  
Free Calcium ◽  
Kinase C ◽  
Protein Kinase C Inhibitor

The generation of phosphatidic acid in neutrophils stimulated by the chemotactic factor formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) is inhibited by the diacylglycerol kinase inhibitor R59022. Superoxide generation produced by fMet-Leu-Phe, leukotriene B4, platelet-activating factor, or phorbol 12-myristate 13-acetate can be greatly increased in neutrophils pretreated with R59022. The potentiation occurs in the presence or absence of cytochalasin B and is evident in the absence of extracellular calcium. In addition, where the superoxide generated by fMet-Leu-Phe is not inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), the increase by R59022 is diminished by this compound. Unlike cytochalasin B, R59022 does not affect the increase in cytoskeletal actin produced by fMet-Leu-Phe or platelet-activating factor nor does it decrease the basal level. Furthermore, the basal intracellular concentration of free calcium, but not the rise produced by fMet-Leu-Phe or platelet-activating factor, is elevated by R59022. The data presented here suggest that the potentiation by R59022 of the oxidative burst is most likely mediated through protein kinase C.

scholarly journals Ca2(+)-stimulatable and protein kinase C-inhibitable accumulation of inositol 1,3,4,6-tetrakisphosphate in human platelets

1990 ◽  
Vol 270 (1) ◽  
pp. 125-131 ◽  
Author(s):  
W G King ◽  
C P Downes ◽  
G D Prestwich ◽  
S E Rittenhouse
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Periodate Oxidation ◽  
Total Activity ◽  
Human Platelets ◽  
Inositol Polyphosphate ◽  
Kinase C ◽  
Strong Anion Exchange ◽  
Protein Kinase C Inhibitor ◽  
Major Route

Thrombin-stimulated (10 s) human platelets produce Ins(1,4,5)P3 and an additional inositol trisphosphate (InsP3), in approximately a 1:20 ratio. The major InsP3 co-migrates with Ins(1,3,4)P3 on strong-anion-exchange h.p.l.c. To identify this species unequivocally, we treated putative Ins(1,3,4)P3 obtained from thrombin-stimulated myo-[3H]inositol-labelled platelets with NaIO4/NaBH4 or 4-phosphomonoesterase. The products indicate that the major InsP3 is at least 90% D-Ins(1,3,4)P3. D-[3H]Ins(1,3,4)P3 added to saponin-permeabilized platelets is hydrolysed to an InsP2 (7.8%) and phosphorylated by a kinase to yield an inositol polyphosphate (0.9%) in 5 min. The phosphorylation product co-migrates with Ins(1,3,4,6)P4 on Partisphere WAX h.p.l.c. Under similar conditions, L-[3H]Ins(1,3,4)P3 is dephosphorylated but not phosphorylated. Relative phosphatase:kinase ratios are 8.7:1 (Vmax. values) and 0.86:1 (Km values) with respect to D-Ins(1,3,4)P3. The kinase activity is predominantly cytosolic (96.8% of total activity) in freeze-thaw-disrupted platelets, and the accumulation of its product is Ca2(+)-dependent. The activity is identified as a 6-kinase on the basis of its product's insensitivity to 5-phosphomonoesterase, resistance to periodate oxidation and co-migration with standard Ins(1,3,4,6)P4 on h.p.l.c. Incubation of platelets with β-phorbol dibutyrate (beta-PDBu, 76 nM), causing activation of protein kinase C, results in a 57.5% inhibition (reversible by the protein kinase C inhibitor staurosporine) of Ins(1,3,4,6)P4 accumulation. alpha-PDBu, which does not stimulate protein kinase C, has no effect. Stimulation of intact platelets with thrombin results in the production of Ins(1,3,4,6)P4 (1.4-fold rise in 30 s) and Ins(1,3,4,5)P4, with the latter being the major InsP4 species. Accumulation of Ins(1,3,4,6)P4 is slightly delayed in comparison with Ins(1,3,4)P3 and is relatively small. We propose that the major route of Ins(1,3,4)P3 metabolism in stimulated human platelets is via phosphatase action.

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