Abstract 598: A Platelet Glycoprotein Ib-IX-specific 14-3-3/Rac1/LIMK1 Signaling Pathway Promotes Thrombin-Induced Platelet Activation

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Brian Estevez ◽  
Keegan Delaney ◽  
Aleksandra Stojanovic-Terpo ◽  
Xiaoping Du
Keyword(s):  
Binding Site ◽  
Signaling Pathway ◽  
Platelet Activation ◽  
Intracellular Signaling ◽  
Low Dose ◽  
Cell Model ◽  
Chinese Hamster ◽  
Platelet Glycoprotein ◽  
Protease Activated Receptors

Rationale: Thrombin-induced platelet activation requires protease-activated receptors. However, the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds to thrombin and is important for low dose thrombin-induced platelet activation. It is unclear how GPIb-IX promotes thrombin-induced platelet activation. Objective: To clearly elucidate the mechanism by which GPIb-IX promotes thrombin-induced platelet activation. Methods and Results: We reconstituted GPIb-IX (GPIb) /Protease-activated receptors (PARs) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression. This effect of GPIb appears to require GPIb signaling, as mutation of a cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-IX-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown by pretreating human platelets with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of GPIbα, which inhibited thrombin-induced platelet activation, but did not affect thrombin binding to platelets. Furthermore, 14-3-3 binding site deletion in GPIbα or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1. To determine the role of the Rac1/LIMK1 signaling pathway in mediating thrombin-induced GPIb signaling and platelet activation, we examined the effects of Rac1 knockout, LIMK1 knockout and Rac1-inhibitor on low dose thrombin-induced calcium response and platelet activation. Rac1 inhibitor, NSC23766, abolished the GPIb-dependent cell response in a reconstituted CHO cell model. Rac1 knockout platelets showed diminished platelet response to thrombin and were not different from wild type platelets in the presence of MPαC. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation. Conclusions: The stimulatory role of GPIb in thrombin-induced platelet activation requires a thrombin-induced GPIb-specific 14-3-3/Rac1/LIMK1 signaling pathway.

scholarly journals A Signaling Mechanism By Which Platelet Glycoprotein Ib-IX Promotes Thrombin-Induced Platelet Activation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2759-2759
Author(s):  
Brian Estevez ◽  
Michael Keegan Delaney ◽  
Aleksandra Stojanovic-Terpo ◽  
Xiaoping Du
Keyword(s):  
Binding Site ◽  
Platelet Activation ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Inhibitory Peptide ◽  
Protease Activated Receptors ◽  
Signaling Mechanism

Abstract Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. We reconstituted GPIb-IX (GPIb) /Protease-activated receptor (PAR) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression, and this effect of GPIb appears to require GPIb signaling, as deletion of the cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown in human platelets, in which pretreatment with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of the GPIbα, inhibited thrombin-induced platelet activation. Furthermore, 14-3-3 binding site deletion in GPIba or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1, both of which have been shown to mediate von Willebrand factor-induced GPIb signaling, and the role of GPIb in promoting thrombin signaling was abolished with a Rac-inhibitor, NSC23766 or in Rac1-/- platelets. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation. Disclosures No relevant conflicts of interest to declare.

Abstract 11947: Unraveling the Mechanism for the Stimulatory Role of Platelet GPIb-IX-V in Thrombin-Induced Platelet Activation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Brian Estevez ◽  
Michael K Delaney ◽  
Aleksandra Stojanovic-Terpo ◽  
Xiaoping Du
Keyword(s):  
Platelet Activation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Inhibitory Peptide ◽  
Protease Activated Receptors ◽  
Signaling Mechanism ◽  
Ovary Cells

Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. Pretreatment of human platelets with MPalphaC, an inhibitory peptide based on a critical 14-3-3 signaling protein binding site on the cytoplasmic domain of the GPIb alpha chain, inhibited thrombin-induced platelet activation. MPalphaC-treatment inhibited thrombin-induced activation of Rac1 and LIMK1, both of which are known to play essential roles in GPIb signaling. To more specifically determine the role of GPIb-IX, we reconstituted GPIb-IX-facilitated thrombin signaling in Chinese Hamster Ovary cells expressing PAR1. Thrombin induced signaling was significantly enhanced by GPIb-expression, and deletion of the cytoplasmic 14-3-3-binding domain of GPIb alpha abolished the stimulatory effect of GPIb on thrombin signaling. Furthermore, the role of GPIb-IX in promoting thrombin signaling requires Rac1, and GPIb-IX-dependent Rac1 activation and LIMK phosphorylation are abolished in delta 605 cells expressing a 14-3-3-binding defective mutant GPIb alpha. Taken together, these data suggest that the stimulatory role of GPIb in thrombin signaling requires a C-terminal 14-3-3-binding region which mediates activation of a Rac1/LIMK1 pathway that promotes thrombin signaling leading to platelet activation.

THE ROLE OF AGONISTS AND ANTAGONISTS OF THE WNT SIGNALING PATHWAY IN PATIENTS WITH ANDROGENIC ALOPECIA AND ALOPECIA AREATA

2021 ◽  
pp. 87-95
Author(s):  
Samoylova A.V. ◽  
Snimshchikova I.A. ◽  
Plotnikova M.O. ◽  
Yakushkina N.Y.
Keyword(s):  
Wnt Signaling ◽  
Hair Follicle ◽  
Signaling Pathway ◽  
Alopecia Areata ◽  
Intracellular Signaling ◽  
Wnt Signaling Pathway ◽  
Follicle Cells ◽  
Androgenic Alopecia ◽  
Active Population

Alopecia is a common pathology among the active population, which leads not only to cosmetic defects, but also to the development of somatic diseases against the background of traumatic effects and chronic stress. The pathogenetic mechanisms of hair follicle formation are complex and diverse, since numerous factors, including the components of the Wnt signaling pathway, have an effect on its morphogenesis, the study of which is the subject of this study. The search for possible early markers of the development of alopecia led to interest in the study of the main morphogenic proteins of WNT - the signaling pathway (one of the intracellular signaling pathways, which control the development of blood vessels, as well as the growth and division of hair follicle cells) sclerostin and β-catenin among patients with androgenic and alopecia areata. The article presents data on the quantitative content of β-catenin and sclerostin in the blood serum in patients with androgenic and alopecia areata. Their possible pathways of complex interaction and influence on the morphogenesis of the hair follicle and the activity of the Wnt-signaling pathway have been analyzed, and the relationship between changes in the level of morphogenic proteins of the WNT-signaling pathway with sex and the course of the disease has been described. Establishment of the prognostic role of morphogenic proteins of the WNT signaling pathway in androgenic and alopecia areata will allow not only identify the personal risk of disease progression and to determine approaches to targeted therapy, but to develop and introduce updated diagnostic screening into dermatological practice.

scholarly journals Lipotoxicity-Induced mtDNA Release Promotes Diabetic Cardiomyopathy by Activating the cGAS-STING Pathway in Obesity Related Diabetes

2021 ◽  
Author(s):  
Xiu Mei Ma ◽  
Kang Geng ◽  
Betty Yuen-Kwan Law ◽  
Peng Wang ◽  
Yue Li Pu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Signaling Pathway ◽  
Diabetic Cardiomyopathy ◽  
Cell Model ◽  
Critical Role ◽  
Kidney Tissue ◽  
H9c2 Cells ◽  
Downstream Targets ◽  
Sting Pathway

Abstract Diabetic cardiomyopathy (DCM) is characterized by lipid accumulation, mitochondrial dysfunction, and aseptic inflammatory activation. Mitochondria-derived cytosolic DNA has been reported to induce inflammation by activating cyclic GMP-AMP synthase (cGAS)/the stimulator of interferon genes (STING) pathway in the adipose, liver, and kidney tissue. However, the role of cytosolic mtDNA in the progression of DCM is unclear. In this study, with an obesity-related DCM mouse model established by feeding db/db mice with a high-fat diet (HFD), we observed increased mtDNA in the cytosol and activated cGAS-STING signaling pathway during DCM, as well as the downstream targets, IRF3, NF-κB, IL-18, and IL-1β. In further study with a palmitic acid (PA)-induced lipotoxic cell model established in H9C2 cells, we revealed that the cytosolic mtDNA was resulted from PA-induced overproduction of mitochondrial ROS, which also led to the activation of the cGAS/STING system and its downstream targets. Notably, treatment of extracted mtDNA alone was sufficient to activate the cGAS-STING signaling pathway in cultured H9C2 cells. Besides, both knockdown of STING in PA-induced H9C2 cells and inhibition of STING by C-176 injection in the DCM mouse model could remarkably block the inflammation and apoptosis of cardiomyocytes. In conclusion, our study elucidated the critical role of cytosolic mtDNA-induced cGAS-STING activation in the pathogenesis of obesity-related DCM and provided preclinical validation for using a STING inhibitor as a new potential therapeutic strategy for the treatment of DCM.

The Role of miRNAs in PI3K Signaling Pathway in Blood Malignancies: A Review Article

2021 ◽  
Author(s):  
Haniyeh Gaffari-Nazari ◽  
Samira Karami ◽  
Leila Noorazar ◽  
Sayeh Parkhideh ◽  
Elham Roshandel ◽  
...  
Keyword(s):  
Treatment Failure ◽  
Signaling Pathway ◽  
Intracellular Signaling ◽  
Pi3k Pathway ◽  
Pi3k Signaling ◽  
Laboratory Findings ◽  
A Cell ◽  
Pi3k Signaling Pathway ◽  
Relationship Of

Background: The PI3K/Akt/mTOR signaling pathway is one of the most important intracellular signaling pathways by regulating the cell cycle process. The direct relationship of this pathway with important mechanisms such as cell quiescence, longevity, and proliferation has been established. The overactive PI3K pathway with decreased and increased apoptosis and cell proliferation respectively is involved in pathogenesis of many cancers, including blood malignancies such as leukemia. Methods: Laboratory findings have shown that different factors, such as miRNAs, play a role in regulating PI3K signaling pathway. These molecules can alter the fate of a cell by interfering in suppression/overexpression of mRNA, transcription factors or stimulating the transcription of some genes. In this article, we reviewed the role of miRNAs in regulating the PI3K/Akt/mTOR pathway and its effect on leukemic progression and treatment failure. Conclusion: At present, miRNAs are known to be one of the causes of treatment failure and relapse in cancers.

scholarly journals BK polyomavirus infection promotes growth and aggressiveness in bladder cancer

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yigang Zeng ◽  
Jiajia Sun ◽  
Juan Bao ◽  
Tongyu Zhu
Keyword(s):  
Bladder Cancer ◽  
Signaling Pathway ◽  
Epithelial Mesenchymal Transition ◽  
Cell Model ◽  
Mesenchymal Transition ◽  
Invasion And Migration ◽  
Urothelial Carcinomas ◽  
Bk Polyomavirus ◽  
And Migration

Abstract Background Recent studies have confirmed the integration of the BK polyomavirus (BKPyV) gene into the cellular genome of urothelial carcinomas in transplant recipients, further confirming the correlation between BKPyV and urothelial carcinomas after transplantation. However, the role BKPyV infections play in the biological function of bladder cancer remains unclear. Methods We developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections. Results Our research proves that BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells, while the activation of β-catenin signaling pathway is one of its mediation mechanisms. Conclusions We first described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer. We verified the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyV-infected bladder cancer. These results provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.

scholarly journals LncRNA MEG3 influences the proliferation and apoptosis of psoriasis epidermal cells by targeting miR-21/caspase-8

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Hai-Yan Jia ◽  
Kai Zhang ◽  
Wen-Jing Lu ◽  
Gui-Wen Xu ◽  
Jian-Fen Zhang ◽  
...  
Keyword(s):  
Binding Site ◽  
Cell Model ◽  
Mrna Level ◽  
Epidermal Cells ◽  
Luciferase Reporter ◽  
Psoriatic Skin ◽  
Caspase 8 ◽  
Proliferation And Apoptosis

Abstract Background It was reported that microRNA-21(miR-21) was differentially expressed in the keratinocytes of psoriasis patients, and it may influence the apoptosis and proliferation of cells. The role of lncRNA maternally expressed gene3 (MEG3), a competing endogenous RNAs of miR-21, in the progression of psoriasis remains unclear. We aimed to unfold the influence of MEG3 and miR-21 on the proliferation and apoptosis of psoriasis epidermal cells. Methods 50μg/L TNF-α was used to treat HaCaTs and NHEKs cells for 24 h, and then different experiments were conducted. qRT-PCR were applied for measuring the mRNA level of MEG3, miR-2, and caspase-8, and the protein expression of caspase-8 was measured with western blotting. Flow cytometry was used for assessing apoptosis. Cell proliferation was detected using MTT and colony formation assays. Dual luciferase reporter assay was applied for confirming the binding site between MEG3 and miR-21, miR-21 and Caspase-8. Results A cell model for in vitro studying the role of MEG3 in psoriasis pathophysiology was established using HaCaT and HHEKs. MEG3 was significantly down-regulated in HaCaT, HHEKs, and psoriatic skin samples. MEG3 inhibits proliferation and promotes apoptosis of Activated-HaCaT (Act-HaCaT) and Activated-HHEKs (Act- HHEK) by regulating miR-21, and the binding site between MEG3 and miR-21 was identified. We also found that miR-21 could inhibit the level of caspase-8 and identified the binding site between caspase-8 and miR-21. Some down-stream proteins of caspase-8, Cleaved caspase-8, cytc, and apaf-1 were regulated by miR-21 and MEG3. Conclusion MEG3/miR-21 axis may regulate the expression of caspase-8, and further influence the proliferation and apoptosis of psoriasis keratinocyte, Act-HaCaT and Act- HHEK. Therefore, our findings may provide a new thought for the study of pathogenesis and treatment of psoriasis.

scholarly journals Role of a JAK3-dependent Biochemical Signaling Pathway in Platelet Activation and Aggregation

2001 ◽  
Vol 276 (21) ◽  
pp. 17815-17822 ◽  
Author(s):  
Heather E. Tibbles ◽  
Alexei Vassilev ◽  
Heather Wendorf ◽  
Dawn Schonhoff ◽  
Dan Zhu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Platelet Activation

Identification of a novel 14-3-3ζ binding site within the cytoplasmic tail of platelet glycoprotein Ibα

Blood ◽  
2004 ◽  
Vol 104 (2) ◽  
pp. 420-427 ◽  
Author(s):  
Pierre Mangin ◽  
Tovo David ◽  
Vincent Lavaud ◽  
Susan L. Cranmer ◽  
Inna Pikovski ◽  
...  
Keyword(s):  
Binding Site ◽  
Critical Role ◽  
Chinese Hamster ◽  
Adaptor Protein ◽  
Cytoplasmic Tail ◽  
Cell Spreading ◽  
Platelet Glycoprotein ◽  
Platelet Spreading ◽  
Mutant Forms ◽  
Von Willebrand

Abstract The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin αIIbβ3-dependent stable adhesion and spreading. Interaction of the 14-3-3ζ adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIbα subunit has been implicated in the control of αIIbβ3 activation and cell spreading. In this study, we have examined potentially novel 14-3-3ζ binding sites by expressing mutant forms of GPIbα in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIbα-14-3-3ζ interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3ζ from GPIbα. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIbα-14-3-3ζ binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3ζ binding site within the cytoplasmic tail of GPIbα that has an important functional role in regulating integrin-dependent cell spreading. (Blood. 2004;104:420-427)

Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression

2005 ◽  
Vol 98 (4) ◽  
pp. 1414-1419 ◽  
Author(s):  
Antonino Coppola ◽  
Ludovico Coppola ◽  
Liliana dalla Mora ◽  
Francesco M. Limongelli ◽  
Antonio Grassia ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
B Lymphocytes ◽  
Platelet Activation ◽  
Critical Role ◽  
Strenuous Exercise ◽  
Platelet Glycoprotein ◽  
Vigorous Exercise ◽  
Platelet Aggregates

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the “in vitro” ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

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