Staphylococcal Enterotoxin B Initiates Protein Kinase C Translocation and Eicosanoid Metabolism While Inhibiting Thrombin-Induced Aggregation in Human Platelets

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

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Use of d-erythro-sphingosine as a pharmacological inhibitor of protein kinase C in human platelets

Biochemical Journal ◽

10.1042/bj2780387 ◽

1991 ◽

Vol 278 (2) ◽

pp. 387-392 ◽

Cited By ~ 19

Author(s):

W A Khan ◽

S W Mascarella ◽

A H Lewin ◽

C D Wyrick ◽

F I Carroll ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Platelet Activation ◽

Biological Activities ◽

Dose Dependence ◽

Human Platelets ◽

Kinase C ◽

Similar Dose ◽

Induced Aggregation

Sphingosine is a naturally occurring long-chain amino diol with potent inhibitory activity against protein kinase C in vitro and in cell systems. The use of sphingosine as a pharmacological tool to probe the activity of protein kinase C has been hampered by its amphiphilicity, possible contamination of its commercial preparations, and the existence of other targets for its action. To address these problems, high-purity D-erythro-sphingosine was prepared and employed to develop an approach for the use of sphingosine as a pharmacological agent. The addition of synthetic D-erythro-sphingosine to intact human platelets resulted in quick uptake and preferential partitioning into the particulate fraction. It was rapidly metabolized by intact platelets, 60% being degraded within 1 min after addition. Sphingosine was found to be a potent inhibitor of gamma-thrombin-induced aggregation and secretion of washed human platelets. Multiple criteria indicated that this effect is probably mediated through the inhibition of protein kinase C: (1) sphingosine inhibited protein kinase C activity in intact platelets with a similar dose/response to its inhibition of platelet aggregation and secretion; (2) sphingosine inhibited phorbol binding to intact platelets under identical conditions and with a similar dose-dependence; (3) exogenous dioctanoylglycerol overcame sphingosine's inhibition of platelet activation. The effectiveness of sphingosine in inhibiting platelet activation was primarily determined by the ratio of sphingosine to total number of platelets. These data are discussed in relation to a general approach for the use of sphingosine and other parameters for determining biological activities of protein kinase C.

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Synthesis of intracellular histamine in platelets is associated with activation of protein kinase C, but not with mobilization of Ca2+

Biochemical Journal ◽

10.1042/bj2730405 ◽

1991 ◽

Vol 273 (2) ◽

pp. 405-408 ◽

Cited By ~ 6

Author(s):

S P Saxena ◽

C Robertson ◽

A B Becker ◽

J M Gerrard

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Human Platelets ◽

Ionophore A23187 ◽

Dependent Manner ◽

Kinase C ◽

Histamine Synthesis ◽

Ic50 Values ◽

Induced Aggregation ◽

Pkc Activation

In previous reports, we have provided evidence indicating that newly formed histamine is an intracellular messenger in human platelets. The involvement of protein kinase C (PKC) and intracellular calcium (Ca2+i) in the synthesis of histamine was investigated. Human platelets were stimulated by phorbol 12-myristate 13-acetate (PMA), collagen and the Ca2+ ionophore A23187, with or without the PKC inhibitor staurosporine. Aggregation, histamine synthesis and phosphorylation of pleckstrin (47 kDa; P47) and myosin light chain (20 kDa; P20) proteins were monitored. Staurosporine inhibited PMA- and collagen-induced aggregation, histamine synthesis and phosphorylation of 47 kDa and 20 kDa proteins in a dose-dependent manner. For PMA, median inhibitory concentrations (IC50 values) for staurosporine inhibition of aggregation, histamine synthesis and phosphorylation were similar, suggesting that histamine synthesis induced by this agonist may be a consequence of PKC activation. Conversely, collagen-stimulated histamine synthesis was inhibited by staurosporine at concentrations significantly higher than those required to inhibit aggregation (P less than 0.005) or pleckstrin phosphorylation (P less than 0.01), indicating the possible involvement of non-PKC mechanism(s) in the synthesis of histamine induced by this agonist. A23187 failed to induce the synthesis of intracellular histamine in platelets, whereas staurosporine blocked A23187-induced aggregation and phosphorylation of the 20 kDa protein at significantly higher concentrations than those needed to inhibit PKC. When platelets were stimulated with a combination of A23187 and PMA, the increase in platelet histamine was less than that with PMA alone. The results provide evidence that the synthesis of intracellular histamine in platelets occurs as a consequence of PKC activation and may be down-regulated under conditions where there is a substantial rise in [Ca2+]i.

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Impaired activation of platelets lacking protein kinase C-θ isoform

Blood ◽

10.1182/blood-2008-07-169268 ◽

2009 ◽

Vol 113 (11) ◽

pp. 2557-2567 ◽

Cited By ~ 60

Author(s):

Bela Nagy ◽

Kamala Bhavaraju ◽

Todd Getz ◽

Yamini S. Bynagari ◽

Soochong Kim ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Arterial Occlusion ◽

Human Platelets ◽

Wild Type ◽

Functional Responses ◽

Glycoprotein Vi ◽

Kinase C ◽

Granule Secretion ◽

Induced Aggregation

Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-θ is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-θ–selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and α-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-θ, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-θ RACK peptide, which may contribute to the lower levels of granule secretion when PKC-θ function is lost. Furthermore, the level of JON/A binding to activated αIIbβ3 receptor was also significantly decreased in PKC-θ−/− mice compared with wild-type littermates. PKC-θ−/− murine platelets showed significantly lower agonist-induced thromboxane A2 (TXA2) release through reduced extracellular signal–regulated kinase phosphorylation. Finally, PKC-θ−/− mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl3 in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-θ isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors.

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Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽

10.1182/blood.v82.9.2704.bloodjournal8292704 ◽

1993 ◽

Vol 82 (9) ◽

pp. 2704-2713

Author(s):

R Vezza ◽

R Roberti ◽

GG Nenci ◽

P Gresele

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Prostaglandin E2 ◽

Platelet Activation ◽

Human Platelets ◽

Platelet Surface ◽

Kinase C ◽

Activated Platelets ◽

Induced Aggregation

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

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ENDOTOXIC LIPID A INDUCES BINDING OF FIBRINOGEN TO HUMAN PLATELETS VIA PROTEIN KINASE C PATHWAY

10.1055/s-0038-1644252 ◽

1987 ◽

Author(s):

Sheila Timmons ◽

Jadwiqa Grabarek ◽

Jack Hawiqer

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Lipid A ◽

Human Platelets ◽

Biologically Active ◽

Active Principle ◽

Gram Negative ◽

Kinase C ◽

Induced Aggregation ◽

Protein Kinase C Activation

Endotoxic Lipid A is the biologically active principle of lipopolysaccharide of Gram-negative bacteria, a most frequent cause of sepsis underlying Disseminated Intravascular Coagulation (DIC) and shock. We have shown that endotoxic Lipid A activates Protein Kinase C in human platelets. Phosphorylation of a 47kDa protein (P47), a marker for Protein Kinase C activation, was observed within the first minute of interaction of Lipid A with platelets. This was accompanied by gradual exposure of the receptor for 125I-labeled fibrinogen (F). Binding of 125I-F was saturable and specific. When Lipid X, a precursor of endotoxic Lipid A and its competitive inhibitor, was used, the binding of 125I-F was blocked with 50% inhibition at a 1:1 stoichiometry between Lipid X and Lipid A. At the same time, phosphorylation of P47 was prevented. Since Lipid X constitutes a "half molecule" of Lipid A, we interpret these results as indicative of competitive blocking of endotoxic Lipid A in terms of Protein Kinase C activation and exposure of platelet receptors for fibrinogen. Binding of fibrinogen is necessary for platelet aggregation and endotoxic Lipid A-induced aggregation was also blocked by Lipid X. Endotoxic Lipid A-induced exposure of fibrinogen receptors via the Protein Kinase C pathway can contribute to involvement of platelets in microcirculatory thrombosis observed in patients with DIC and Gram-negative sepsis

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Activation of phospholipase C and protein kinase C has little involvement in ADP-induced primary aggregation of human platelets: effects of diacylglycerols, the diacylglycerols, the diacylglycerol kinase inhibitor R59022, staurosporine and okadaic acid

Biochemical Journal ◽

10.1042/bj2900849 ◽

1993 ◽

Vol 290 (3) ◽

pp. 849-856 ◽

Cited By ~ 31

Author(s):

M A Packham ◽

A A Livne ◽

D H Ruben ◽

M L Rand

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase C ◽

Okadaic Acid ◽

Kinase Inhibitor ◽

Diacylglycerol Kinase ◽

Human Platelets ◽

Primary Phase ◽

Kinase C ◽

Induced Aggregation

The primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although the reason for this effect is unknown, it is unlikely to involve inhibition of phospholipase C, since formation of diacylglycerol appears to have little involvement in the primary phase of ADP-induced aggregation.

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