Ni2+ impairs thrombin-induced signal transduction by acting on the agonist and/or receptor in human platelets

1993 ◽  
Vol 265 (6) ◽  
pp. C1681-C1688 ◽  
Author(s):  
F. J. Azula ◽  
R. Alonso ◽  
A. Marino ◽  
M. Trueba ◽  
J. M. Macarulla
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Myristate Acetate ◽  
Kinase C ◽  
Induced Signal ◽  
Dose Dependent ◽  
Protein Kinase C Activation

We have investigated the effect of NiCl2 on platelet activation induced by thrombin, phorbol 12-myristate 13-acetate, and calcium ionophores. Besides blocking Ca2+ influx, NiCl2 inhibited platelet aggregation, intracellular Ca2+ mobilization, and phospholipase C activation induced by thrombin in a dose-dependent manner. In contrast to ionomycin, NiCl2 completely blocked the platelet aggregation and intracellular Ca2+ mobilization induced by A23187. A23187 was not able to translocate Ni2+ across the plasma membrane. Ni2+ also inhibited phorbol myristate acetate-induced platelet aggregation. The results with staurosporine and low NiCl2 concentrations are in agreement in that increases in intracellular Ca2+ concentration and protein kinase C activation are necessary for full platelet activation mediated by thrombin.

Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcγRIIA and αIIbβ3

2005 ◽  
Vol 93 (05) ◽  
pp. 932-939 ◽  
Author(s):  
Caroline Pampolina ◽  
Archibald McNicol
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Lag Phase ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Streptococcus Sanguis

SummaryThe low-affinity IgG receptor, FcγRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcγRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017–78 caused the tyrosine phosphorylation of FcγRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCγ2. These early events were dependent on Src family kinases but independent of either TxA2 or the engagement of the αIIbβ3 integrin. During the lag phase prior to platelet aggregation, FcγRIIA, Syk, LAT, and PLCγ2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017–78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcγRIIA, Syk, LAT, and PLCγ2 were rephosphorylated in an RGDS-sensitive, and therefore αIIbβ3-dependent, manner. Additionally, TxA2 release, 5-hydro-xytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited αIIbβ3-mediated re-phosphorylation. Therefore, S.sanguis-bound IgG cross links FcγRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of αIIbβ3 leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA2 release and consequent platelet aggregation.

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.

The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3904-3904
Author(s):  
Samantha Baldassarri ◽  
Alessandra Bertoni ◽  
Paolo Lova ◽  
Stefania Reineri ◽  
Chiara Sarasso ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Cannabinoid Receptors ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Dependent Manner ◽  
Cb2 Receptor ◽  
Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

scholarly journals Studies on the Biological Effects of Ozone: 10. Release of Factors from Ozonated Human Platelets

1999 ◽  
Vol 8 (4-5) ◽  
pp. 205-209 ◽  
Author(s):  
G. Valacchi ◽  
Velio Bocci
Keyword(s):  
Platelet Aggregation ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Platelet Rich Plasma ◽  
Biological Effects ◽  
Transforming Growth Factor Β1 ◽  
Human Platelets ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Tgf Β1

In a previous work we have shown that heparin, in the presence of ozone (O3), promotes a dose-dependent platelet aggregation, while after Ca2+chelation with citrate, platelet aggregation is almost negligible. These results led us to think that aggregation may enhance the release of platelet components. We have here shown that indeed significantly higher amount of platelet-derived growth factor (PDGF), transforming growth factor β1 (TGF-β1) and interleukin-8(IL-8) are released in a dose-dependent manner after ozonation of heparinised platelet-rich plasma samples. These findings may explain the enhanced healing of torpid ulcers in patients with chronic limbischemia treated with O3autohaemoteraphy (O3-AHT).

Multiple Signal-Transduction Pathways Synergize in Platelet Activation

Physiology ◽  
1991 ◽  
Vol 6 (2) ◽  
pp. 51-56 ◽  
Author(s):  
W Siess
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
The Other ◽  
Signal Transduction Pathways ◽  
Protein Activation ◽  
The Third ◽  
Kinase C ◽  
Gi Protein ◽  
Protein Kinase C Activation ◽  
Mobilization Protein

At least three signal-transduction pathways are present in platelets which synergize with each other: calcium mobilization, protein kinase C activation, and an unknown pathway triggered by Gi protein activation. Each pathway can synergize with the other independently of the third pathway in the induction of platelet aggregation.

STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of Its Transcription Factor Activity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 157-157
Author(s):  
Zhou Zhou ◽  
Francisca C. Gushiken ◽  
Angela Bergeron ◽  
Vinod K. Vijayan ◽  
Rolando Rumbaut ◽  
...  
Keyword(s):  
Shear Stress ◽  
Transcription Factor ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Transcription Factor Activity ◽  
Factor Activity ◽  
Dose Dependent

Abstract Abstract 157 Signal Transducer and Activator of Transcription 3 (STAT3) serves as a transcription factor activated by cytokine-induced intracellular signals, which are critical in megakaryopoiesis. This signaling pathway may also be active in anucleated platelets that are primed by proinflammatory cytokines, suggesting that STAT3 plays a role in platelet hyperactivity associated with inflammation. We have recently found that three different classes of STAT3 inhibitors each selectively inhibited collagen-induced aggregation of human platelets by ∼50%. They also blocked thrombus formation (∼80%) on immobilized collagen under an arterial shear stress of 62.5 dyn/cm2. These STAT3 inhibitors also blocked platelet aggregation induced by collagen-related peptide, suggesting that they acted on GP VI-mediated intracellular signaling in platelets. These in vitro results were further verified in two sets of experiments in mouse models. First, an oligonucleotide G-quartet STAT3 inhibitor (1 mg/ml) or a scrambled control oligonucleotide were infused into C57/BJ6 mice daily for three days. Collagen-induced platelet aggregation was then induced and found to be reduced by up to 60% in mice infused with the STAT3 inhibitor, but not with the control oligonucleotide. Photochemical injury-induced thrombosis in the cremaster arterioles was also significantly delayed in the inhibitor-infused mice as compared to control mice. Second, infusing STAT3 inhibitor could result in platelet inhibitor indirectly by acting endothelial cells. To address this concern, we have generated platelet-specific STAT3 null mice that have developed normally and have normal platelet counts. The collagen-, but not TRAP-induced platelet aggregation in the platelet STAT3 KO mice was reduced as compared to their littermates. Platelets from the platelet-specific STAT3 KO mice were also significantly defective in thrombus formation on immobilized collagen under 62.5 dyn/cm2 of fluid shear stress that was generated in a parallel-plate flow chamber system. Consistent with results from these functional assays, collagen induced rapid (peaked at 5 min after stimulation) and dose-dependent tyrosine phosphorylation of STAT3, but not of STAT1 or STAT5 in washed human platelets. The phosphorylation was blocked dose-dependently by two STAT3 inhibitors. Syk inhibitors also blocked collagen-induced STAT3 phosphorylation in a dose-dependent manner, but STAT3 inhibitors had no effect on Syk phosphorylation, suggesting that Syk acts upstream of STAT3. Furthermore, STAT3 inhibitors also dose-dependently reduced collagen-induced tyrosine phosphorylation of PLCγ2, which is a known substrate of Syk. Consistent with this temporal interaction among STAT3, Syk and PLCγ2, activated STAT3 co-immunoprecipitated phosphorylated Syk and PLCγ2 in collagen-activated human platelets. The tri-molecular complex was also immunoprecipitated by an antibody to PLCγ2. Taken together, these data suggest that STAT3 regulates collagen-induced platelet aggregation, independent of its transcription factor activity. The regulation is potentially achieved by STAT3 serving as a protein scaffold linking the kinase Syk with its substrate PLCγ2 to enhance the signal relay in collagen-activated platelets. This cross-talk between collagen and cytokine signaling pathways provides a mechanism for how proinflammatory mediators could prime platelets for activation by hemostatic ligands. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Intracellular Ca2+ rise in human platelets induced by polymorphonuclear-leucocyte-derived cathepsin G

1992 ◽  
Vol 288 (3) ◽  
pp. 741-745 ◽  
Author(s):  
M Molino ◽  
M Di Lallo ◽  
G de Gaetano ◽  
C Cerletti
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Platelets ◽  
Polymorphonuclear Leucocyte ◽  
Cation Channel ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Bivalent Cation ◽  
Platelet Suspension

Cathepsin G, a serine protease released by polymorphonuclear-leucocyte azurophilic granules upon stimulation, activates human platelets, inducing an increase in intra-platelet Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (50-200 nM). The [Ca2+]i rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca2+ transient. Higher (100-200 nM) cathepsin G concentrations induced a monophasic increase in intracellular Ca2+. Acetylsalicylic acid, nordihydroguaiaretic acid and ketanserin did not affect platelet activation by cathepsin G, whereas the ADP-scavenger system phosphocreatine/creatine kinase significantly decreased Ca2+ mobilization, platelet aggregation and 5-hydroxytryptamine secretion by cathepsin G. Preventing cathepsin G-induced platelet aggregation with the synthetic peptide RGDSP (Arg-Gly-Asp-Ser-Pro) did not significantly affect cathepsin G-induced Ca2+ transients. Ni2+ (4 mM), a bivalent-cation-channel inhibitor, decreased the cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either decreasing Ni2+ or increasing cathepsin G concentration. Preventing Ca2+ influx across the plasma membrane with 4 mM-EGTA totally abolished Ca2+ transients. However, EGTA also strongly decreased catalytic activity of cathepsin G, which is essential for platelet activation. Evidence of a rapid and sustained bivalent-cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 s or 3 min after cathepsin G. No accumulation of InsP3 could be detected when platelets were stimulated with cathepsin G. All these data indicate that cathepsin G induces a [Ca2+]i increase mainly through an influx across the plasma membrane. This massive Ca2+ entry is probably due to opening of receptor-operated channels and is amplified by endogenous ADP release.

scholarly journals The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin

1996 ◽  
Vol 313 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Mustapha SI-TAHAR ◽  
Patricia RENESTO ◽  
Hervé FALET ◽  
Francine RENDU ◽  
Michel CHIGNARD
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Inositol Phosphates ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Functional Responses ◽  
Kinase C

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 μM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 μM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.

Resveratrol Attenuates Adenosine Diphosphate-Induced Platelet Activation by Reducing Protein Kinase C Activity

2008 ◽  
Vol 36 (03) ◽  
pp. 603-613 ◽  
Author(s):  
Yu-Min Yang ◽  
Xing-Xiang Wang ◽  
Jun-Zhu Chen ◽  
Shi-Jun Wang ◽  
Hu Hu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Adenosine Diphosphate ◽  
Dependent Manner ◽  
Chinese Medicinal Herb ◽  
Concentration Dependent Manner ◽  
Kinase C

Inappropriate platelet activation is the key point of thrombogenesis. The aim of the present study was to investigate the effects of resveratrol (RESV), a compound extracted from the Chinese medicinal herb Polygonum cuspidatum sieb et Zucc, on the platelet activation induced by adenosine diphosphate (ADP) and its possible mechanism. The percentage of platelet aggregation and surface P-selectin-positive platelets, and the activity of protein kinase C (PKC) of platelet were observed with platelet aggregometer, flow cytometry and phosphorimaging system, respectively. RESV at 25, 50 and 100 μM showed anti-platelet aggregation and inhibition of surface P-selectin-positive platelets in a concentration-dependent manner. RESV (50 μM) inhibited the activity of PKC in the membrane fraction of platelets and decreased the percentage of membrane associated PKC activity in total PKC activity. Moreover, DL-erythro-1,3-Dihydroxy-2-aminooctadecane, an elective protein kinase C inhibitor (PKCI), and RESV had additive effects of inhibiting the percentage of platelet aggregation and surface P-selectin-positive platelets. It is suggested that RESV may inhibit platelet aggregation, the percentage of surface P-selectin-positive platelets and subsequent thrombus formation. The mechanisms may be partly relative to the decrease of the activity of PKC of platelets.

The Gas6 Receptor Axl Enhances Platelet Activation Responses through Stimulation of PI3kinase- and PLC-Dependent Signaling Pathways.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 630-630
Author(s):  
Weston R. Gould ◽  
Sangita Baxi ◽  
Lisa A. Perrin ◽  
Robert J. Leadley
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Intracellular Signaling ◽  
Human Platelets ◽  
Dense Granule ◽  
Calcium Mobilization ◽  
Dependent Manner ◽  
Induced Aggregation ◽  
Granule Release ◽  
Stimulation Of

Abstract At the site of vascular injury, platelet activation is paramount in supporting formation of a platelet plug and generating a functional surface for the protein elements of coagulation. Recently, we demonstrated that the receptors for the α-granule constituent Gas6, support and enhance platelet aggregation and dense-granule release. The current study examined additional affects of Gas6 signaling in human platelets and sought to decipher intracellular signaling mechanisms initiated by stimulation of Axl, a Gas6 platelet receptor. Flow cytometry analyses indicated that all three Gas6 receptors, Axl, Sky, and Mer were present on the platelet surface. Blockade of Gas6, Sky, or Mer by specific antibodies not only inhibited TRAP- and ADP-induced platelet aggregation and dense granule release, but also prevented thrombin mediated clot retraction by as much as 55%. Furthermore, intracellular calcium mobilization in response to TRAP activation was greater than 80% inhibited in the presence of each of these blocking antibodies. A highly specific antibody directed toward Axl (< 2% cross reactivity with Sky and Mer) activated Axl leading to an enhancement of TRAP and ADP induced aggregation and degranulation. Stimulation of human platelets by this Axl agonist led to a modest, but sustained increase in calcium mobilization suggesting that Axl signaling incorporated activation of PLC. The increase in calcium mobilization was sensitive to wortmannin, demonstrating that PLC activation occurred concurrent with or downstream of PI3K. Indeed, additional experiments to ascertain the intracellular mediators of Axl activity identified a two-fold increase in specific phosphorylation of Akt downstream of PI3K as well as a similar increase in phosphorylation of PLCγ. TRAP stimulation of human platelets also increased the phosphorylation levels of Akt and PLCγ in a Gas6 dependent manner as a Gas6 blocking antibody reduced the levels of Akt and PLCγ phosphorylation by 50%. Overall, these studies suggest that Gas6 enhancement of human platelet activation occurs through the low-level stimulation of the intracellular signaling molecules Akt and PLCγ, serving at the juncture of several mediators of platelet activation. These events also increase levels of cytoplasmic calcium, further supporting an enhancement of activation observed in response to low levels of known platelet agonists. Thus, platelet Gas6 functions to support platelet activation at the very early stages of the hemostatic response to injury.

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