scholarly journals Src family kinase–mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3410-3414 ◽  
Author(s):  
Analia Garcia ◽  
Todd M. Quinton ◽  
Robert T. Dorsam ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Kinase Inhibitor ◽  
Thromboxane A2 ◽  
Receptor Activation ◽  
Src Family Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Src Family Kinase

AbstractThe binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non–aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.

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 Hydrogen Peroxide Is Involved in Collagen-Induced Platelet Activation

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 484-490 ◽  
Author(s):  
Pasquale Pignatelli ◽  
Fabio M. Pulcinelli ◽  
Luisa Lenti ◽  
Pier Paolo Gazzaniga ◽  
Francesco Violi
Keyword(s):  
Hydrogen Peroxide ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Acid Metabolism ◽  
Thromboxane A2 ◽  
Arachidonic Acid Metabolism ◽  
H2o2 Production ◽  
High Concentrations

Abstract In this study, we investigated whether (1) collagen-induced platelet aggregation is associated with a burst of H2O2, (2) this oxidant species is involved in the activation of platelets, and (3) the pathways of platelet activation are stimulated by H2O2. Collagen-induced platelet aggregation was associated with production of H2O2, which was abolished by catalase, an enzyme that destroys H2O2. H2O2 production was not observed when ADP or thrombin were used as agonists. Catalase inhibited dose-dependently thromboxane A2 production, release of arachidonic acid from platelet membrane, and Inositol 1,4,5P3 (IP3) formation. In aspirin-treated platelets stimulated with high concentrations of collagen, catalase inhibited platelet aggregation, calcium mobilization, and IP3 production. This study suggests that collagen-induced platelet aggregation is associated with a burst of H2O2 that acts as a second messenger by stimulating the arachidonic acid metabolism and phospholipase C pathway.

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.

HUMAN PLATELET ACTIVATION BY BACTERIAL PHOSPHOLIPASE C: MECHANISM OF INHIBITION BY FLURAZEPAM

1987 ◽  
Author(s):  
Huzoor Akbar ◽  
David Wallace ◽  
Khursheed Anwer
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Calcium Ionophore ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free System ◽  
Serotonin Secretion

We have shown earlier that flurazepam inhibits human platelet aggregation and serotonin secretion induced by bacterial phospholipase C (BPLC, Thromb. Res. 38, 361-374, 1985). This study was conducted to examine the mechanism(s) of inhibitory action of flurazepam. Only 15 uM and 11 uM flurazepam were required to inhibit platelet aggregation and serotonin secretion by 50%. In a platelet free system, BPLC hydrolyzed 14C-phosphatidylcholine (14C-PC> in a time- and concentration-dependent manner in the presence of calcium ions. Flurazepam had no effect on BPLC-induced hydrolysis of 14C-PC. Incubation of 14C-arachidonic acid labelled platelets with BPLC produced diacylglycerol(DAG) in a time- and concentration-dependent manner. Flurazepam did not inhibit DAG production by BPLC. However, prostaglandin E1 and paranitrophenolphosphorylcholine inhibited DAG production by 20% and 75% respectively. Platelet cytosolic fraction,containing phosphatidylinositol-specific PLC (PI-PLC), hydrolyzed 3H -phosphatidylinositol (3H-PI) in a concentration-dependent manner. Flurazepam did not inhibit hydrolysis:of 3H-PI by PI-PLC. BPLC caused phosphorylation of 47,000 Dalton protein (P47) in 32P-labelled platelets. Flurazepam did not inhibit phosphorylation of P47 in the first five minutes of incubation. However, flurazapam completely blocked phosphorylation of P47 by seven minutes. In Other experiments, flurazepam inhibited platelet aggregation induced by ionomycion, a calcium ionophore, in a concentration-dependent manner. These data lead us to suggest that flurazapam does not inhibit BPLC-ihduced platelet activation by inhibiting the action of BPLC or PI-PLC on platelet phospholipids or DAG production. However, the ability of flurazepam to inhibit ionomycin-induced platelet aggregation indicates that it may be blocking BPLC-induced platelet aggreagtion by interfering with the influx, of calcium ions into platelets. (Supported in part by the American Osteopathic Association, The Baker Award from Ohio University and the OUCOM).

Activation of PI3K-Akt pathway mediates antiapoptotic effects of β-adrenergic agonist in airway eosinophils

2005 ◽  
Vol 288 (5) ◽  
pp. L860-L867 ◽  
Author(s):  
Kentaro Machida ◽  
Hiromasa Inoue ◽  
Koichiro Matsumoto ◽  
Miyuki Tsuda ◽  
Satoru Fukuyama ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Survival Signal ◽  
Eosinophil Survival ◽  
Mitogen Activated Protein ◽  
Allergen Provocation ◽  
Eosinophil Apoptosis

β-Adrenoceptor agonists reportedly decrease spontaneous apoptosis of peripheral blood eosinophils; however, its signaling pathway is unknown. Survival signals can be elicited by the activation of phosphatidylinositol 3-kinase (PI3K) and Akt, both of which are known to be potent regulators of apoptosis, and Akt in turn inactivates Forkhead transcription factors, including FKHR (Forkhead in rhabdomyosarcoma). We have investigated the effect of β-agonists on apoptosis of local eosinophils isolated from the airways and the involvement of PI3K, Akt, and FKHR in its survival signal. Eosinophils obtained from immunized mice by bronchoalveolar lavage after allergen provocation underwent apoptosis in a time-dependent manner. Incubation of eosinophils with isoproterenol or formoterol dose-dependently inhibited both spontaneous eosinophil apoptosis and apoptosis induced by Fas receptor activation. Incubation with cAMP or forskolin also inhibited eosinophil apoptosis. The PI3K inhibitors wortmannin and LY-294002 and an Akt inhibitor, 1-l-6-hydroxymethyl- chiro-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate, but not a mitogen-activated protein kinase kinase inhibitor PD-98059, blocked isoproterenol-mediated eosinophil survival. Wortmannin also inhibited cAMP-mediated eosinophil survival. Isoproterenol rapidly induced phosphorylation of Akt and FKHR in eosinophils in a PI3K-dependent manner. These findings indicate that the PI3K-Akt-FKHR pathway conveys a critical survival signal induced by β-agonists in airway eosinophils.

Dextran sulphate induces fibrinogen receptor activation through a novel Syk-independent PI-3 kinase-mediated tyrosine kinase pathway in platelets

2013 ◽  
Vol 109 (06) ◽  
pp. 1131-1140 ◽  
Author(s):  
Todd M. Getz ◽  
Bhanu Manne ◽  
Lorena Buitrago ◽  
Yingying Mao ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Akt Phosphorylation ◽  
Glycoprotein Vi ◽  
Fibrinogen Receptor ◽  
Adp Receptor Antagonists ◽  
Kinase Pathway

SummaryIn our attempt to find a physiological agonist that activates PAR3 receptors, we screened several coagulation proteases using PAR4 null platelets. We observed that FXIIa and heat inactivated FXIIa, but not FXII, caused platelet aggregation. We have identified a contaminant activating factor in FXIIa preparation as dextran sulfate (DxS), which caused aggregation of both human and mouse platelets. DxS-induced platelet aggregation was unaffected by YM254890, a Gq inhibitor, but abolished by pan-Src family kinase (SFK) inhibitor PP2, suggesting a role for SFKs in this pathway. However, DxS-induced platelet aggregation was unaffected in FcRγ-chain null murine platelets, ruling out the possibility of glycoprotein VI-mediated events. More interesting, OXSI-2 and Go6976, two structurally unrelated inhibitors shown to affect Syk, had only a partial effect on DxS-induced PAC-1 binding. DxS-induced platelet aggregation and intracellular calcium increases were abolished by the pan PI-3 kinase inhibitor LY294002, or an isoform-specific PI-3 kinase β inhibitor TGX-221. Pretreatment of platelets with Syk inhibitors or ADP receptor antagonists had little effect on Akt phosphorylation following DxS stimulation. These results, for the first time, establish a novel tyrosine kinase pathway in platelets that causes fibrinogen receptor activation in a PI-3 kinase-dependent manner without a crucial role for Syk.

Abstract 384: The Prothrombotic Profile of BCR-ABL Inhibitors Ponatinib, Nilotinib and Imatinib

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Cassandra P Loren ◽  
Rachel A Rigg ◽  
Joseph E Aslan ◽  
Laura D Healy ◽  
Andras Gruber ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Kinase ◽  
Platelet Activation ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Thrombotic Complications ◽  
Platelet Spreading

Background: Thrombotic complications observed in patients treated with the tyrosine kinase inhibitor ponatinib resulted in the temporary suspension of the drug by the FDA in late 2013. Ponatinib is a pan-BCR-ABL inhibitor designed for treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Ponatinib has been shown to inhibit a subset of the class III/IV family of receptor tyrosine kinases. The pathogenic mechanism underlying the prothrombotic phenotype associated with ponatinib remains ill-defined. Hypothesis: We tested the hypothesis that BCR-ABL inhibitors regulate platelet activation, spreading, and aggregation. Methods & Results: Our results show that treatment of platelets with ponatinib (1 μM) abrogated platelet spreading on fibrinogen (50 μg/ml) or collagen (100 μg/ml). Both nilotinib and imatinib inhibited platelet spreading, although not to the same extent as ponatinib at equimolar concentrations. We next examined the effects of BCR-ABL inhibitors on platelet activation and aggregation in response to the GPVI-agonist, CRP, which mediates platelet activation in a receptor tyrosine kinase-dependent manner. Intriguingly, our results show that ponatinib abrogated platelet aggregation in response to CRP (1 μg/ml), whereas equimolar concentrations of nilotinib or imatinib had minimal effects on CRP-induced platelet aggregation. Conclusions: While tyrosine kinase inhibitors are generally associated with bleeding diathesis, thrombotic complications have been observed in patients treated with ponatinib. Our results indicate that ponatinib, nilotinib, and imatinib inhibit platelet spreading and aggregation, with ponatinib having the most significant effect, suggesting that ponatinib may act as a platelet antagonist. Our future work will be focused on identifying platelet proteins affected by the BCR-ABL inhibitors as well as the pathogenic mechanisms underlying the prothrombotic phenotype associated with ponatinib.

scholarly journals Insulin-like growth factor-1 regulates platelet activation through PI3-Kα isoform

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4206-4213 ◽  
Author(s):  
Soochong Kim ◽  
Analia Garcia ◽  
Shaun P. Jackson ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Growth Factor ◽  
Platelet Activation ◽  
Shape Change ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Concentration Dependent Manner ◽  
Induce Platelet Aggregation

Platelets release insulin-like growth factor-1 (IGF-1) from α granules upon activation. We have investigated the regulation of IGF-1 in Gi-dependent pathways leading to Akt activation and the role of IGF-1 in platelet activation. IGF-1 alone failed to induce platelet aggregation, but IGF-1 potentiated 2-MeSADP–induced platelet aggregation in a concentration-dependent manner. IGF-1 triggered platelet aggregation in combination with selective P2Y1 receptor activation. IGF-1 also caused platelet aggregation without shape change when combined with selective Gz stimulation by epinephrine, suggesting the role of IGF-1 in platelet aggregation by supplementing Gi pathways. The potentiating effect of IGF-1 was not affected by intracellular calcium chelation. Importantly, IGF-1 was unable to potentiate platelet aggregation by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, suggesting a critical regulation by PI3-K. Moreover, the potentiating effect of IGF-1 was abolished by the presence of PI3-K p110α inhibitor PIK-75. Stimulation of platelets with IGF-1 resulted in phosphorylation of Akt, a downstream effector of PI3-K, which was completely inhibited by wortmannin. IGF-1-induced Akt phosphorylation was abolished by PIK-75 suggesting the contribution of PI3-K p110α for activation of Akt by IGF-1. These results demonstrate that IGF-1 plays a role in potentiating platelet aggregation by complementing Gi- but not Gq-signaling pathways via PI3-K p110α.

scholarly journals Hydrogen Peroxide Is Involved in Collagen-Induced Platelet Activation

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 484-490 ◽  
Author(s):  
Pasquale Pignatelli ◽  
Fabio M. Pulcinelli ◽  
Luisa Lenti ◽  
Pier Paolo Gazzaniga ◽  
Francesco Violi
Keyword(s):  
Hydrogen Peroxide ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Acid Metabolism ◽  
Thromboxane A2 ◽  
Arachidonic Acid Metabolism ◽  
H2o2 Production ◽  
High Concentrations

In this study, we investigated whether (1) collagen-induced platelet aggregation is associated with a burst of H2O2, (2) this oxidant species is involved in the activation of platelets, and (3) the pathways of platelet activation are stimulated by H2O2. Collagen-induced platelet aggregation was associated with production of H2O2, which was abolished by catalase, an enzyme that destroys H2O2. H2O2 production was not observed when ADP or thrombin were used as agonists. Catalase inhibited dose-dependently thromboxane A2 production, release of arachidonic acid from platelet membrane, and Inositol 1,4,5P3 (IP3) formation. In aspirin-treated platelets stimulated with high concentrations of collagen, catalase inhibited platelet aggregation, calcium mobilization, and IP3 production. This study suggests that collagen-induced platelet aggregation is associated with a burst of H2O2 that acts as a second messenger by stimulating the arachidonic acid metabolism and phospholipase C pathway.

Mitochondrial ROS initiate phosphorylation of p38 MAP kinase during hypoxia in cardiomyocytes

2002 ◽  
Vol 282 (6) ◽  
pp. L1324-L1329 ◽  
Author(s):  
Andre Kulisz ◽  
Ningfang Chen ◽  
Navdeep S. Chandel ◽  
Zuohui Shao ◽  
Paul T. Schumacker
Keyword(s):  
Kinase Inhibitor ◽  
Anion Channel ◽  
P38 Map Kinase ◽  
Proximal Region ◽  
Mitogen Activated Protein Kinase ◽  
Ros Generation ◽  
Dependent Manner ◽  
Adaptive Responses ◽  
Mitochondrial Ros ◽  
Mitogen Activated Protein

The p38 mitogen-activated protein kinase (MAPK) is phosphorylated in response to oxidative stress. Mitochondria in cardiomyocytes increase their generation of reactive oxygen species (ROS) during hypoxia (1–5% O2). These ROS participate in signal transduction pathways involved in adaptive responses, including ischemic preconditioning and gene transcription. The present study therefore tested the hypothesis that hypoxia induces p38 MAPK phosphorylation by augmenting mitochondrial ROS generation. In cardiomyocytes, phosphorylation of p38 was observed in a Po 2-dependent manner during hypoxia. This response was inhibited by rotenone, thenoyltrifluoroacetone, and myxothiazol, inhibitors of mitochondrial complexes I, II, and III, respectively. A similar inhibition was observed in the cells pretreated with anion channel inhibitor DIDS, which may block ROS release from mitochondria. During normoxia, increases in mitochondrial ROS elicited by azide (1–2 mM) or by the mitochondrial inhibitor antimycin A caused increased phosphorylation of p38. Brief treatment with exogenous H2O2 during normoxia also induced phosphorylation of p38 as hypoxia, but this effect was not abolished by myxothiazol or DIDS. The antioxidant N-acetyl-cysteine abolished the p38 response to hypoxia, presumably by scavenging H2O2, but the mitogen extracellular receptor kinase inhibitor PD-98059 did not inhibit p38 phosphorylation during hypoxia. Thus physiological hypoxia leads to p38 phosphorylation through a mechanism that requires electron flux in the proximal region of the mitochondrial electron transport chain, which suggests that either H2O2 or superoxide participates in activating that process.

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