scholarly journals P2X1-mediated activation of extracellular signal-regulated kinase 2 contributes to platelet secretion and aggregation induced by collagen

Blood ◽  
2002 ◽  
Vol 100 (7) ◽  
pp. 2499-2505 ◽  
Author(s):  
Cécile Oury ◽  
Emese Toth-Zsamboki ◽  
Jos Vermylen ◽  
Marc F. Hoylaerts
Keyword(s):  
Platelet Aggregation ◽  
Ion Channel ◽  
High Performance ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Hek293 Cells ◽  
Extracellular Signal Regulated Kinase ◽  
Dense Granules ◽  
Extracellular Signal ◽  
Erk2 Activation

Adenosine triphosphate (ATP) and its stable analog, α,β-methylene ATP, activate the platelet P2X1 ion channel, causing a rapid Ca++ influx. Here, we show that, in washed apyrase-treated platelets, α,β-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca++- and protein kinase C–dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. α,β-Methylene ATP also activated the ERK2 pathway in P2X1-transfected HEK293 cells but not in cells expressing mutated P2X1delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X1-mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X1 with ADP or desensitization of this ion channel with α,β-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (≤ 1 μg/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca++ chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collagen-induced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 μg/mL), platelet aggregation and secretion no longer depended on P2X1 or ERK2 activation, as shown by the lack of their inhibition by α,β-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X1-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed.

scholarly journals The response of platelets to epinephrine in storage pool deficiency-- evidence pertaining to the role of adenosine diphosphate in mediating primary and secondary aggregation

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1717-1725 ◽  
Author(s):  
HJ Weiss ◽  
B Lages
Keyword(s):  
Platelet Aggregation ◽  
Receptor Agonist ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Dense Granule ◽  
Binding Studies ◽  
Dense Granules ◽  
Storage Pool ◽  
Receptor Sites

Abstract Aggregation responses and thromboxane (Tx) formation in ten patients with storage pool deficiency (SPD) specific to the dense granules (delta-SPD) were studied to assess further the role of dense granule adenosine diphosphate (ADP) in mediating platelet aggregation by epinephrine. The ability of epinephrine to elicit secondary aggregation (SA) responses was highly variable in delta-SPD when tested at 5 mumol/L epinephrine, but was consistently abnormal when tested over a range of concentrations. The occurrence of SA in both delta-SPD patients and normal subjects was correlated with the magnitude of the rate of primary aggregation (PA). This PA rate was normal, on average, for the entire patient group but was greater in patients with more consistent SA responses. The PA findings were related to the Kd value obtained in binding studies with 3H-yohimbine, but not with the number of alpha 2-receptor sites. Studies on Tx production (assessed by radioimmunoassay of TxB2) showed that the ability to synthesize Tx from arachidonate was not impaired in delta-SPD, and that there was an absolute positive correlation between epinephrine-induced SA and Tx production. Aggregation in delta-SPD platelets in response to the Tx receptor agonist U44069 was consistently decreased, but could be corrected by addition of ADP. The results of the study suggest that dense granule-derived ADP is not required for PA by epinephrine, but mediates SA as a synergistic agonist with TxA2. This role of ADP in SA may be elucidated more precisely by further studies on platelet activation processes in delta-SPD.

scholarly journals The response of platelets to epinephrine in storage pool deficiency-- evidence pertaining to the role of adenosine diphosphate in mediating primary and secondary aggregation

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1717-1725 ◽  
Author(s):  
HJ Weiss ◽  
B Lages
Keyword(s):  
Platelet Aggregation ◽  
Receptor Agonist ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Dense Granule ◽  
Binding Studies ◽  
Dense Granules ◽  
Storage Pool ◽  
Receptor Sites

Aggregation responses and thromboxane (Tx) formation in ten patients with storage pool deficiency (SPD) specific to the dense granules (delta-SPD) were studied to assess further the role of dense granule adenosine diphosphate (ADP) in mediating platelet aggregation by epinephrine. The ability of epinephrine to elicit secondary aggregation (SA) responses was highly variable in delta-SPD when tested at 5 mumol/L epinephrine, but was consistently abnormal when tested over a range of concentrations. The occurrence of SA in both delta-SPD patients and normal subjects was correlated with the magnitude of the rate of primary aggregation (PA). This PA rate was normal, on average, for the entire patient group but was greater in patients with more consistent SA responses. The PA findings were related to the Kd value obtained in binding studies with 3H-yohimbine, but not with the number of alpha 2-receptor sites. Studies on Tx production (assessed by radioimmunoassay of TxB2) showed that the ability to synthesize Tx from arachidonate was not impaired in delta-SPD, and that there was an absolute positive correlation between epinephrine-induced SA and Tx production. Aggregation in delta-SPD platelets in response to the Tx receptor agonist U44069 was consistently decreased, but could be corrected by addition of ADP. The results of the study suggest that dense granule-derived ADP is not required for PA by epinephrine, but mediates SA as a synergistic agonist with TxA2. This role of ADP in SA may be elucidated more precisely by further studies on platelet activation processes in delta-SPD.

scholarly journals Different Protein Tyrosine Kinases Are Required for B Cell Antigen Receptor–mediated Activation of Extracellular Signal–Regulated kinase, c-Jun NH2-terminal Kinase 1, and p38 Mitogen-activated Protein Kinase

1998 ◽  
Vol 188 (7) ◽  
pp. 1297-1306 ◽  
Author(s):  
Aimin Jiang ◽  
Andrew Craxton ◽  
Tomohiro Kurosaki ◽  
Edward A. Clark
Keyword(s):  
B Cell ◽  
P38 Mapk ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinases ◽  
B Cell Antigen Receptor ◽  
Mapk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Erk2 Activation

B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor protein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal–regulated kinase (ERK)2, c-jun NH2-terminal kinase (JNK)1, and p38 MAPK. To dissect the functional roles of these PTKs in MAPK signaling, activation of MAPKs was examined in various PTK-deficient DT40 cells. BCR-mediated activation of ERK2, although maintained in Lyn-deficient cells, was abolished in Syk-deficient cells and partially inhibited in Btk-deficient cells, indicating that BCR-mediated ERK2 activation requires Syk and that sustained ERK2 activation requires Btk. BCR-mediated JNK1 activation was maintained in Lyn-deficient cells but abolished in both Syk- and Btk-deficient cells, suggesting that JNK1 is activated via a Syk- and Btk-dependent pathway. Consistent with this, BCR-mediated JNK1 activation was dependent on intracellular calcium and phorbol myristate acetate–sensitive protein kinase Cs. In contrast, BCR-mediated p38 MAPK activation was detected in all three PTK-deficient cells, suggesting that no single PTK is essential. However, BCR-mediated p38 MAPK activation was abolished in Lyn/Syk double deficient cells, demonstrating that either Lyn or Syk alone may be sufficient to activate p38 MAPK. Our data show that BCR-mediated MAPK activation is regulated at the level of the PTKs.

scholarly journals Antimelanogenic Effects of Polygonum tinctorium Flower Extract from Traditional Jeju Fermentation via Upregulation of Extracellular Signal-Regulated Kinase and Protein Kinase B Activation

2018 ◽  
Vol 19 (10) ◽  
pp. 2895 ◽  
Author(s):  
You Chung ◽  
Ji-Hye Ko ◽  
Hyun-Kyu Kang ◽  
Seoyeon Kim ◽  
Choon Kang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
High Performance ◽  
Protein Kinase B ◽  
Skin Irritation ◽  
Akt Inhibitor ◽  
Western Blot Assay ◽  
Extracellular Signal Regulated Kinase ◽  
Flower Extract ◽  
Extracellular Signal ◽  
Polygonum Tinctorium

This study was carried out to investigate the antimelanogenic effects of a Polygonum tinctorium flower extract obtained using red nuruk, a traditional Jeju barley-based fermentation starter. We also studied the mechanism of action of the P. tinctorium fermented flower extract (PTFFE) in mouse melanoma cells (B16F10). Cells were treated with various concentrations (62.5, 125 and 250 μg/mL) of PTFFE and the results showed that PTFFE significantly decreased the melanin content and tyrosinase activity without being cytotoxic. In addition, PTFFE strongly inhibited the expression of tyrosinase and tyrosinase-related protein 2 by decreasing the expression of the microphthalmia-associated transcription factor, as shown by a western blot assay. Furthermore, PTFFE inhibited melanogenesis via upregulation of the phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B, also known as AKT. We also used inhibitors such as PD98059 (a specific ERK inhibitor) or LY294002 (an AKT inhibitor) to determine whether the signaling pathways are involved. High-performance liquid chromatography fingerprinting showed the presence of a quercetin glucoside (isoquercitrin) and quercetin in PTFFE. To test the potential for PTFFE application as a cosmetic material, we also performed a primary skin irritation test on human skin. In this assay, PTFFE did not induce any adverse reactions at the treatment dose. Based on these results, we suggest that PTFFE may be considered a potential antimelanogenesis candidate for topical applications.

The Effects of Glycoprotein IIb/IIIa Antagonist RGDS on Platelet Aggregation and Release Reaction In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3908-3908
Author(s):  
Shuangfeng Xie ◽  
Songmei Yin ◽  
Danian Nie ◽  
Yiqing Li ◽  
Xiuju Wang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Adenosine Diphosphate ◽  
Platelet Glycoprotein ◽  
High Concentration ◽  
Release Reaction ◽  
Dense Granules ◽  
Negative Effect ◽  
Platelet Release

Abstract Platelet activation, including platelet adhesion, platelet aggregation and platelet release reaction, played an important role in thrombogenesis. We all knew that Platelet glycoprotein IIb/IIIa antagonist was the most effective drug for anti-aggregation, while we don’t know clearly its effect on platelet release reaction and the relations between its effects on platelet aggregation and release reaction. Platelet release reactions included α-granules and dense granules releasing. When α-granules were released, its membrane glycoprotein CD62p was expressed in the platelet membrane. We used the CD62p expression as the index of platelet release reaction. In the current study, the 4-peptides RGDS (Arg-Gly-Asp-Ser) was used as glycoprotein IIb/IIIa antagonist. We detected the effects of RGDS on platelet aggregation and CD62p expression induced by adenosine diphosphate (ADP) (finial concentration, 5μmol/L) in vitro. 50, 100, 200, 400 and 800μmol/L RGDS were used separately in the test. RGDS of each concentration could significantly inhibited maximal platelet aggregation (PAG(M)) induced by ADP, the 50% inhibiting concentration was approximately 200μmol/L. 800μmol/L RGDS could inhibited PAG(M) by 80.48±8.18%. Only ≥200μmol/L RGDS could significantly inhibited platelet CD62p expression. 800μmol/L RGDS could inhibit platelet CD62p expression by 27.31±9.74%. The inhibiting effect of RGDS on PAG(M) and platelet CD62p expression had significantly correlation (r =0.976, P<0.05). These results indicated that RGDS in low concentration (<200μmol) had little negative effect on platelet release reaction induced by ATP, while in relatively high concentration (≥200μmol) RGDS could inhibit platelet release reaction. When RGDS concentrations were same its effect on platelet release reaction was much less than that on platelet aggregation, which indicated that platelet glycoprotein IIb/IIIa compound could only partly participated in the platelet release reaction but fully participated in platelet aggregation induced by ADP.

scholarly journals Platelet malondialdehyde production and aggregation responses induced by arachidonate, prostaglandin-G2, collagen, and epinephrine in 12 patients with storage pool deficiency

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 27-33
Author(s):  
HJ Weiss ◽  
B Lages
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Synthetic Pathway ◽  
Dense Granules ◽  
Storage Pool ◽  
Increased Sensitivity

We assessed the integrity of the prostaglandin synthetic pathway by measuring malondialdehyde (MDA) production and studied platelet aggregation responses to arachidonic acid and PGG2 in 12 patients with storage pool deficiency (SPD). Eight patients were deficient only in dense granules (delta-SPD) and four were deficient in both dense and alpha-granules (alpha delta-SPD). Production of MDA in response to arachidonic acid (AA), epinephrine, and collagen suggested that the transformation of AA to prostaglandin metabolites was normal in delta- SPD but abnormal in alpha delta-SPD and that the liberation of AA from phospholipids were abnormal in the majority of patients with SPD. Since the content of secretable adenosine diphosphate (ADP) is diminished in SPD platelets, the aggregation responses of these platelets to AA and PGG2 were studied to help answer the question whether these agents aggregate platelets directly or through release of endogenous ADP. Among patients with delta-SPD, aggregation by both AA and PGG2 was decreased in four albinos whose platelets were markedly deficient in ADP. In contrast, normal, or less strikingly abnormal, responses were observed in patients whose platelets either contained higher levels of platelet ADP or showed increased sensitivity to ADP. The more marked impaired responses to AA and PGG2 in patients with alpha delta-SPD suggest that substances derived from alpha-granules may also play a role in platelet aggregation by these agents. The aggregation responses in these patients with various types of SPD is consistent with a theory that granule-derived ADP mediates platelet aggregation by AA and PGG2.

scholarly journals Adenosine Diphosphate (ADP) and ADP Receptor Play a Major Role in Platelet Activation/Aggregation Induced by Sera From Heparin-Induced Thrombocytopenia Patients

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 549-554 ◽  
Author(s):  
János Polgár ◽  
Petra Eichler ◽  
Andreas Greinacher ◽  
Kenneth J. Clemetson
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Heparin Therapy ◽  
Dense Granule ◽  
Heparin Induced Thrombocytopenia ◽  
Adp Receptor ◽  
Induced Aggregation ◽  
Granule Release

The molecular basis for heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, is not yet fully understood. We found that pretreatment of platelets with AR-C66096 (formerly FPL 66096), a specific platelet adenosine diphosphate (ADP) receptor antagonist, at a concentration of 100 to 200 nmol/L that blocked ADP-dependent platelet aggregation, resulted in complete loss of platelet aggregation responses to HIT sera. AR-C66096 also totally inhibited HIT serum-induced dense granule release, as judged by measurement of adenosine triphosphate (ATP) release. Apyrase, added to platelets at a concentration that had only minor effects on thrombin- or arachidonic acid-induced aggregation, also blocked completely HIT serum-induced platelet aggregation. Furthermore, AR-C66096 inhibited platelet aggregation and ATP release induced by cross-linking FcγRIIA with specific antibodies. These data show that released ADP and the platelet ADP receptor play a pivotal role in HIT serum-induced platelet activation/aggregation. The thromboxane receptor inhibitor, Daltroban, had no effect on HIT serum-induced platelet activation whereas GPIIb-IIIa antagonists blocked platelet aggregation but had only a moderate effect on HIT serum-induced dense granule release. Pretreatment of platelets with chondroitinases but not with heparinases resulted in concentration dependent inhibition of HIT serum-induced platelet aggregation. These novel data relating to the mechanism of platelet activation induced by HIT sera suggest that the possibility should be examined that ADP receptor antagonists or compounds that inhibit ADP release may be effective as therapeutic agents for the prevention or treatment of complications associated with heparin therapy.

scholarly journals Alterations in Platelet Alpha-Granule Secretion and Adhesion on Collagen under Flow in Mice Lacking the Atypical Rho GTPase RhoBTB3

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 149 ◽  
Author(s):  
Martin Berger ◽  
David Lutz ◽  
Julia Lutz ◽  
Jawad Khalil ◽  
Ahmed Aburima ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Domain Architecture ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Knockout Mouse Model ◽  
Dense Granules ◽  
Granule Secretion ◽  
Static Conditions

Typical Rho GTPases, such as Rac1, Cdc42, and RhoA, act as molecular switches regulating various aspects of platelet cytoskeleton reorganization. The loss of these enzymes results in reduced platelet functionality. Atypical Rho GTPases of the RhoBTB subfamily are characterized by divergent domain architecture. One family member, RhoBTB3, is expressed in platelets, but its function is unclear. In the present study we examined the role of RhoBTB3 in platelet function using a knockout mouse model. We found the platelet count, size, numbers of both alpha and dense granules, and surface receptor profile in these mice were comparable to wild-type mice. Deletion of Rhobtb3 had no effect on aggregation and dense granule secretion in response to a range of agonists including thrombin, collagen, and adenosine diphosphate (ADP). By contrast, alpha-granule secretion increased in mice lacking RhoBTB3 in response to thrombin, collagen related peptide (CRP) and U46619/ADP. Integrin activation and spreading on fibrinogen and collagen under static conditions were also unimpaired; however, we observed reduced platelet accrual on collagen under flow conditions. These defects did not translate into alterations in tail bleeding time. We conclude that genetic deletion of Rhobtb3 leads to subtle alterations in alpha-granule secretion and adhesion to collagen without significant effects on hemostasis in vivo.

scholarly journals Platelet malondialdehyde production and aggregation responses induced by arachidonate, prostaglandin-G2, collagen, and epinephrine in 12 patients with storage pool deficiency

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 27-33 ◽  
Author(s):  
HJ Weiss ◽  
B Lages
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Synthetic Pathway ◽  
Dense Granules ◽  
Storage Pool ◽  
Increased Sensitivity

Abstract We assessed the integrity of the prostaglandin synthetic pathway by measuring malondialdehyde (MDA) production and studied platelet aggregation responses to arachidonic acid and PGG2 in 12 patients with storage pool deficiency (SPD). Eight patients were deficient only in dense granules (delta-SPD) and four were deficient in both dense and alpha-granules (alpha delta-SPD). Production of MDA in response to arachidonic acid (AA), epinephrine, and collagen suggested that the transformation of AA to prostaglandin metabolites was normal in delta- SPD but abnormal in alpha delta-SPD and that the liberation of AA from phospholipids were abnormal in the majority of patients with SPD. Since the content of secretable adenosine diphosphate (ADP) is diminished in SPD platelets, the aggregation responses of these platelets to AA and PGG2 were studied to help answer the question whether these agents aggregate platelets directly or through release of endogenous ADP. Among patients with delta-SPD, aggregation by both AA and PGG2 was decreased in four albinos whose platelets were markedly deficient in ADP. In contrast, normal, or less strikingly abnormal, responses were observed in patients whose platelets either contained higher levels of platelet ADP or showed increased sensitivity to ADP. The more marked impaired responses to AA and PGG2 in patients with alpha delta-SPD suggest that substances derived from alpha-granules may also play a role in platelet aggregation by these agents. The aggregation responses in these patients with various types of SPD is consistent with a theory that granule-derived ADP mediates platelet aggregation by AA and PGG2.

scholarly journals p21ras couples the T cell antigen receptor to extracellular signal-regulated kinase 2 in T lymphocytes.

1993 ◽  
Vol 178 (4) ◽  
pp. 1199-1208 ◽  
Author(s):  
M Izquierdo ◽  
S J Leevers ◽  
C J Marshall ◽  
D Cantrell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Receptor ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Extracellular Signal Regulated Kinase ◽  
Cell Antigen ◽  
Extracellular Signal ◽  
Erk2 Activation ◽  
Constitutively Active

It has previously been shown in T cells that stimulation of protein kinase C (PKC) or the T cell antigen receptor (TCR) induces the rapid accumulation of the active guanosine triphosphate-bound form of p21ras. These stimuli also induce the activation of extracellular signal-regulated kinase 2 (ERK2), a serine/threonine kinase that is rapidly activated via a kinase cascade in response to a variety of growth factors in many cell types. In this study, we show that p21ras is a component of the TCR signaling pathway that controls ERK2 activation. In the human Jurkat T cell line, transient expression of constitutively active p21ras induces ERK2 activation, measured as an increase in the ability of an ERK2-tag reporter protein to phosphorylate myelin basic protein. Thus, constitutively active p21ras bypasses the requirement for PKC activation or TCR triggering to induce ERK2 activation. In addition, activation of PKC or the TCR produces signals that cooperate with activated p21ras to stimulate ERK2. Conversely, expression of a dominant negative mutant of ras, Ha-ras N17, blocks ERK2 activation after TCR stimulation, indicating that endogenous p21ras function is necessary for the TCR-stimulated ERK2 activation. Taken together, these results demonstrate that the activation of p21ras is both necessary and sufficient to induce ERK2 activation in T cells.

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