EXOGENOUSLY ADDED DIACYLGLYCEROL (DAG) AND ELEVATED INTRACELLULAR Ca2+ LEVELS CANNOT SUBSTITUTE FOR PROSTAGLANDIN END0PER0XIDES/ THROMBOXANE ki IN MEDIATING WEAK AGONIST-INDUCED PLATELET SECRETION

1987 ◽  
Author(s):  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Human Platelets ◽  
Similar Degree ◽  
Low Concentration ◽  
Kinase C ◽  
Platelet Secretion

We have compared the abilities of exogenously added U46619, the PG endoperoxide analogue and, sn-l-oleoyl 2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (diCg), the membrane-permeant DAG analogues, at restoring weak agonist-induced secretion in indomethacin (10μM)-treated platelets (I-PL) in the absence of endogenous PG/Tx synthesis. [14C]-5HT secretion from pre-loaded, washed human platelets was correlated with the levels of [Ca2+]i, using platelets loaded with quin 2. Concentrations of OAG (62-125μM) and diCg (15-30μM), which have previously been shown to be fully effective at activating protein kinase C, failed to significantly enhance [14C]-5HT secretion in combination with ADP (10μM), adrenaline (10μM) or PAF (0.2μM) although they potentiated platelet aggregation, when added 10-30 sec after these agonists to I-PL. eg ADP-0%, 30jiM diCg-9.8%, ADP+diCg-11.9%, 5HT release (p>O.05). In contrast, a low concentration of U46619 (0.2μM), that induced no aggregation, [14C]-5HT secretion or rise in [Ca2+]i levels on its own, was able to synergize strongly at potentiating secretion in combination with all three weak agonists examined, as well as in combination with OAG and diCg (U46619-0%, ADP+U46619-20.4%, U46619+30μM diC8-48% 5HT release) . The greater effectiveness of U46619 at potentiating secretion in combination with the weak agonists was not related to different degrees of [Ca2+]i mobilisation, as ADP and PAF-induced rise in [Ca2+]i occurred to a similar degree in the presence of U46619 and diCg. At a higher concentration of U46619 (0.6μM), which was maximally effective at inducing secretion and elevating [Ca2+]i levels on its own, addition of the weak agonists or OAG or diCg, along with U46619, resulted in a further enhancement of secretion which was independent of changes in [Ca2+]i levels. The results demonstrate that U46619 but not OAG or diCg, is able to fully restore weak agonist-induced secretion in indomethacin-treated platelets, suggesting that the actions of endogenously formed PG endoperoxides/TxA2 cannot be substituted by DAG and raised [Ca2+]i levels and, may be mediated via a mechanism additional to that involving these mediators.

Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on αIIbβ3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2

2000 ◽  
Vol 347 (2) ◽  
pp. 561-569 ◽  
Author(s):  
Tsukasa OHMORI ◽  
Yutaka YATOMI ◽  
Naoki ASAZUMA ◽  
Kaneo SATOH ◽  
Yukio OZAKI
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Human Platelets ◽  
Sh2 Domain ◽  
Kinase C ◽  
Tyrosine Kinase 2 ◽  
Αiibβ3 Integrin

Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKβ or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on αIIbβ3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for αIIbβ3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N-terminal SH3 domain of Grb2 and that the phosphotyrosine residue of Shc binds to the SH2 domain of Grb2. Although Pyk2 and FAK have been reported to be differentially regulated in many cell types, our results suggest that, in human platelets, the mode of Pyk2 activation is mostly similar to that of FAK, in terms of αIIbβ3 integrin-dependent and PKC-dependent tyrosine phosphorylation. Furthermore, Pyk2, as well as FAK, might have one or more important roles in post-aggregation tyrosine phosphorylation events, in association with the cytoskeleton and through interaction with adapter proteins including Grb2 and Shc.

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
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

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.

Mechanisms Involved in the Antiplatelet Activity of Midazolam in Human Platelets

2002 ◽  
Vol 96 (3) ◽  
pp. 651-658 ◽  
Author(s):  
Joen R. Sheu ◽  
George Hsiao ◽  
Hsiung N. Luk ◽  
Yi W. Chen ◽  
Ta L. Chen ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Antiplatelet Activity ◽  
Phosphoinositide Breakdown ◽  
Kinase C ◽  
Inhibitory Mechanisms ◽  
Intracellular Ca

Background Midazolam is widely used as a sedative and anesthetic induction agent. The aim of this study was to systematically examine the inhibitory mechanisms of midazolam in platelet aggregation. Methods The inhibitory mechanisms of midazolam in platelet aggregation were explored by means of analysis of the platelet glycoprotein IIb-IIIa complex, phosphoinositide breakdown, intracellular Ca+2 mobilization, measurement of membrane fluidity, thromboxane B2 formation, and protein kinase C activity. Results In this study, midazolam dose-dependently (6-26 microm) inhibited platelet aggregation in human platelets stimulated by agonists. Midazolam also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Midazolam (6-26 mum) significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, midazolam (15 and 26 mum) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by collagen (2 microg/ml). This phosphorylation was markedly inhibited by midazolam (26 microm). Conclusions These results indicate that the antiplatelet activity of midazolam may be involved in the following pathways: the effects of midazolam may initially be caused by induction of conformational changes in platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein.

scholarly journals Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein

1990 ◽  
Vol 266 (2) ◽  
pp. 527-535 ◽  
Author(s):  
R C Carroll ◽  
R E Worthington ◽  
C Boucheix
Keyword(s):  
Monoclonal Antibody ◽  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Surface Membrane ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Kinase C ◽  
Autocrine Stimulation

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5′(6′)-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1′,7(3H)-isobenzofuran]-3′-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab′)2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab′)2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.

scholarly journals Calpain I activation is not correlated with aggregation in human platelets

1989 ◽  
Vol 261 (3) ◽  
pp. 1039-1042 ◽  
Author(s):  
J S Elce ◽  
L Sigmund ◽  
M J Fox
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Necessary Conditions ◽  
Human Platelets ◽  
Actin Binding ◽  
Calpain Activation ◽  
Calmodulin Binding ◽  
Kinase C ◽  
Hydrolysis Of

Calpain-catalysed hydrolysis of platelet substrates such as cytoskeletal and calmodulin-binding proteins, and of protein kinase C, is assumed to contribute to platelet aggregation. We have measured calpain I activation by immunoblotting, and [Ca2+]i (cytoplasmic Ca2+ concn.) by fura-2 fluorescence, in parallel with measurement of aggregation, in stirred human platelets treated at different [Ca2+]ext (extend Ca2+ concns.) with A23187, leupeptin, phorbol ester and thrombin. Hydrolysis of actin-binding protein, and [3H]5-hydroxytryptamine release, were also measured in some cases. A rise in [Ca2+]i, platelet aggregation and calpain activation often occurred together. With some combinations of agonists and [Ca2+]ext, however, this correlation was clearly not maintained. It was shown: (a) that activation of calpain and its hydrolysis of platelet substrates were not strictly necessary conditions for platelet secretion and aggregation; (b) conversely, that calpain activation could occur without aggregation.

Inhibitory Effects of Resveratrol on Platelet Activation Induced by Thromboxane A2Receptor Agonist in Human Platelets

2011 ◽  
Vol 39 (01) ◽  
pp. 145-159 ◽  
Author(s):  
Yumin Yang ◽  
Xiaoling Wang ◽  
Li Zhang ◽  
Huiping An ◽  
Zhigao Zao
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Red Wine ◽  
Human Platelets ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Signal Transduction System ◽  
Concentration Dependent Manner ◽  
Kinase C

Resveratrol (RSVL), a polyphenolic compound found in red wine is believed to be a contributor in decreasing the incidence of coronary heart disease. Although its primary target is unknown, it blocks platelet aggregation by an ill-defined mechanism. Protein kinase C (PKC), which would redistribute from the cytosol to the platelet membrane upon platelet stimulation, plays a key role in the signal transduction system of platelets in human. In this study, we investigated the effect of RSVL and a PKC inhibitor (DL-erythro-1,3-Dihydroxy-2-aminooctadecane, PKCI) on platelet aggregation induced by a thromboxane A2receptor agonist (U46619, 9,11-Dideoxy-11α, 9α-epoxymethanoprostaglandin F2α) using a platelet aggregometer. We also studied the platelet membranebound fibrinogen (PFig) content and the activity of protein kinase C (PKC) in platelets from healthy volunteers using flow cytometry, and a phosphorimaging system, respectively. Our results showed that RSVL blocked platelet aggregation and PFig content induced by U46619 in a concentration-dependent manner. PKCI and RSVL had an additive effect in inhibiting platelet aggregation and PFig content. Furthermore, RSVL (final concentration 50 μM) remarkably depressed the activity of PKC in the membrane of platelets and the percentage of membrane PKC activity in total PKC activity. Taken together, these results suggested that RSVL suppressed U46619-induced platelet aggregation and PFig content partially through the inhibition of the activity of PKC in platelets.

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
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.

Comparison of the modes of action of Ca2+ ionophore A23187 and thrombin in protein kinase C activation in human platelets

FEBS Letters ◽  
1985 ◽  
Vol 192 (1) ◽  
pp. 4-8 ◽  
Author(s):  
Kimihiko Sano ◽  
Hajime Nakamura ◽  
Tamotsu Matsuo ◽  
Yasuhiro Kawahara ◽  
Hisashi Fukuzaki ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Modes Of Action ◽  
Kinase C ◽  
Protein Kinase C Activation
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