LPS Mediates the Activation of Platelet by Toll-Like Receptor 4.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3001-3001
Author(s):  
Liping Ma ◽  
Jing Wei ◽  
Jian-Xing Chang ◽  
Cheng Zhang ◽  
Zhi-Xin Pei ◽  
...  
Keyword(s):  
Western Blot ◽  
Platelet Activation ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Platelet Suspension

Abstract Abstract 3001 Poster Board II-978 Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) on various tissues or cells in host. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (CD62P, P-selectin) and CD40L on platelet are the indexes to determine platelet activation. The present experiment was designed to investigate the expression of Toll-like receptor 4 (TLR4) on platelet and to determine whether platelet TLR4 involves in platelet activation induced by Lipopolysaccsharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy people were pretreated with a concentration of 0.2μg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4,CD62P and CD40L on platelets were detected through flow cytometry, and platelet TLR4 was further determined by performing western blot analysis. The results show that the percentage of TLR4-positive platelet induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, P<0.01). TLR4 expression on platelets treated with LPS was remarkedly elevated in the presence or absent of thrombin. However, the expression level was much higher in presence of both than thrombin alone( 39.16%,P<0.01). Moreover, the similar results were found in Western blot analysis. Synchronously, the expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin(42.68% and 14.80%) and LPS respectively, and the increase of CD62P was more significant when stimulated with both of LPS and thrombin(63.03%). Although anti-TLR4 antibody inhibited significantly the increases of TLR4, CD62P and CD40L on platelets induced by LPS, it didn't effect their increases induced by thrombin. The experiment results support the evidence that functional TLR4 can be expressed on human platelet. It may involve in platelet activation as an important mediator of LPS- induced CD62P and CD40L expressions on platelets. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bone Components Downregulate Expression of Toll-Like Receptor 4 on the Surface of Human Monocytic U937 Cells: A Cell Model for Postfracture Immune Dysfunction

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Jui-An Lin ◽  
Feng-Yen Lin ◽  
Ta-Liang Chen
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Model ◽  
Immune Dysfunction ◽  
Endotoxin Tolerance ◽  
Surface Expression ◽  
U937 Cells ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4

To mimic the immune status of monocyte in the localized fracture region, toll-like receptor 4 (TLR4) surface expression in human monocytic U937 cells was used as the main target to assess immune dysfunction following bone component exposure. We first identified the effects of bone components (including the marrow content) on TLR4 surface expression and then examined the mechanisms underlying the changes. The level of microRNA-146a expression, an indicator of endotoxin tolerance, was also assayed. Bone component exposure downregulated TLR4 surface expression at 24 h by flow cytometry analysis, compatible with the result obtained from the membranous portion of TLR4 by western blot analysis. The cytoplasmic portion of TLR4 paradoxically increased after bone component exposure. Impaired TLR4 trafficking from the cytoplasm to the membrane was related to gp96 downregulation, as observed by western blot analysis, and this was further evidenced by gp96-TLR4 colocalization under confocal microscopy. TaqMan analysis revealed that the expression of microRNA-146a was also upregulated. This cell model demonstrated that bone component exposure downregulated TLR4 surface expression in a gp96-related manner in human monocytic U937 cells, an indicator of immunosuppression at 24 h. Immune dysfunction was further evidenced by upregulation of microRNA-146a expression at the same time point.

Suppression of Interferon Regulatory Factor 8 Modulates Toll Like Receptor 2 and Toll Like Receptor 7/8 Induced Dendritic Cell Activation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2573-2573
Author(s):  
Daniela Werth ◽  
Anita Bringmann ◽  
Katharina Brauer ◽  
Karin von Schwarzenberg ◽  
Stefanie Held ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Interferon Regulatory Factor ◽  
Toll Like Receptor ◽  
Regulatory Factor ◽  
Dendritic Cell Activation

Abstract Interferon regulatory factor 8 (IRF-8) is a member of the IRF family of transcription factors, which are stimulated through interferon mediated pathways. In mice, IRF-8 seems to play an essential role in the development and maturation of dendritic cells (DCs). However, very limited knowledge is available about the potential role of IRF-8 in the human system. To bridge this gap we analyzed function and activation of human monocyte-derived dendritic cells (mDCs) lacking IRF-8 expression. To knockdown IRF-8 protein levels, we electroporated mDCs with different siRNAs against IRF-8. Additionally, we stimulated the electroporated mDCs with the Toll like receptor (TLR) 2 ligand Pam3Cys or the TLR 7/8 ligand R848. IRF-8 knockdown in mDCs was verified constantly by Western Blot analysis using an anti-IRF-8 antibody. We found that IRF-8 knockdown clearly diminished the expression of the human lymphocyte antigen molecules HLA-ABC and HLA-DR in Pam3Cys and R848 stimulated mDCs. To gain functional data, we performed ELISAs to determine cytokine and chemokine secretion. The electroporation of mDCs with IRF-8 specific siRNA resulted in profound inhibition of secretion of the cytokines IL-6, IL-12 and TNF-a as well as the chemokines MIP-1a (CCL3), MCP-1 (CCL2) and RANTES (CCL5). To get additional information on IRF-8 function in human mDCs, the regulation of signal transduction pathways was determined by Western Blot analysis. The suppression of IRF-8 diminished the nuclear translocation of the NF-kB family member’s c-Rel and RelB as well as PU.1 and IRF-3 in activated mDCs. In addition, we showed that the suppression of IRF-8 caused a reduced phosphorylation of ERK and JNK, but had no effect on the expression of STAT3. In summary, the knockdown of IRF-8 reduced the capability of mDCs to develop appropriate phenotype and functions in response to activating stimuli. Our results indicate that these effects are mediated via the ERK, NF-kB and PU.1 signalling pathways. IRF-8 plays an important role in the activation and function of human mDCs.

Rictor Overexpression and mTORC2 Signaling in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3884-3884 ◽  
Author(s):  
Gwen Jordaan ◽  
Wei Liao ◽  
Joe Gera ◽  
Sanjai Sharma
Keyword(s):  
Western Blot ◽  
Mantle Cell Lymphoma ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Mantle Cell ◽  
Bcr Signaling ◽  
Mtorc2 Complex

Abstract Abstract 3884 Signaling via the B-cell receptor (BCR) stimulates growth and survival of CLL leukemic cells and inhibits apoptosis by phosphorylating immunoreceptor tyrosine based activation motifs. This signaling subsequently activates PI3 Kinase/AKT, mTOR, ERK and other pathways. Activation of Akt in turn requires phosphorylation by mTOR kinase, which assembles in two complexes mTORC1 and mTORC2 and it is the mTORC2 complex that phosphorylates and activates Akt. This phosphorylation of Akt in CLL specimen's upregulates anti-apoptotic proteins such as Mcl-1, Bcl-xl and XIAP. We have identified that Rictor, a component of mTORC2 complex is over-expressed in CLL specimens as compared to normal peripheral mononuclear B cells. This over-expression was noted by real time PCR that showed 1.5 to 4 fold upregulation (n=12). Western blot analysis also showed Rictor overexpression in all the twelve CLL specimens tested. Rictor overexpression was also seen in Mantle cell lymphoma cell lines and to study its role in BCR signaling, stable Mantle cell lymphoma lines with SiRNA mediated Rictor knockdown were established. Rictor knockdown resulted in a significant decrease in Akt activation as phosphorylation (phospho S473) of Akt both in unstimulated cells and when the cells were stimulated with BCR crosslinking was decreased. To determine the effect of Rictor and mTORC2 inhibition on CLL specimens, we tested the activity of three compounds isolated via yeast two hybrid drug screen designed to identify molecules that inhibit Rictor/mTOR interaction. When tested on CLL specimens in the presence of BCR crosslinking, these mTORC2 inhibitor compounds inhibited the downstream phosphorylation of Akt S473. Functionally the inhibitors also induced apoptosis in CLL cells with 40–60% of CLL cells undergoing apoptosis (1.0mM, cells treated for 48 hours). In comparison, rapamycin (mTORC1 inhibitor) and ppp242 (mTORC1, 2 inhibitor) were comparatively less active in CLL specimens as they were less effective in the induction of apoptosis. Western blot analysis of mTORC2 inhibitor treated cells also showed PARP cleavage and an increase in the pro-apoptotic protein BAD. Our data indicates that Rictor overexpression in CLL specimens is required for Akt phosphorylation activation and downstream BCR signaling. Inhibition of this pathway by mTORC2 inhibitors in CLL will be an effective therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of PAK6-Mediated Survival and Cell Cycle Controls Selectively Targets Therapy-Resistant CML Stem/Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-6
Author(s):  
Andrew Wu ◽  
Min Chen ◽  
Ryan Yen ◽  
Xiaoyan Jiang
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Progenitor Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Biological Effects ◽  
Negative Regulator ◽  
Resistant Cells

The resistance of chronic myeloid leukemia (CML) leukemic stem cells (LSC) to ABL tyrosine kinase inhibitor (TKI) monotherapy remains a challenge in curing CML. We have recently identified miR-185 as a useful biomarker to predict therapy response in treatment-naïve CML stem/progenitor cells. We also demonstrated that restored miR-185 expression in LSCs impaired survival, sensitizing them to TKIs in vitro and in preclinical patient-derived xenotransplantation models, indicating that miR-185 is a critical regulator mediating TKI response/resistance of CML stem/progenitor cells. PAK6, a serine/threonine-protein kinase, was uncovered as a target gene of miR-185 by RNA-seq and was found to be upregulated in CD34+ TKI-nonresponder cells vs. TKI-responders, but its biological functions in CML are largely unknown. To investigate the biological effects of inhibiting PAK6 activity in TKI-resistant cells, we tested a pre-clinically validated pan-PAK inhibitor (PF-3758309) in vitro. PF-3758309 significantly reduced the growth of IM-resistant cell lines, including K562-resistant cells, BV173 blast cells (IC50 25-70 nM) and CD34+ TKI-nonresponder cells, as assessed by viability and CFC assays, and increased their apoptosis; these effects were significantly enhanced by TKIs (~2-fold, P&lt;0.05). PF-3758309 alone, or in combination with a TKI, did not have obvious inhibitory effects on CD34+ normal bone marrow. These results were further confirmed in IM-resistant cells using a lentiviral knockdown system that specifically inhibits PAK6. Interestingly, PF-3758309 alone, or in combination with a TKI, greatly reduced mitochondrial activity in CD34+ TKI-nonresponder cells, as shown in functional assessments with MitoTracker, a dye that accumulates in active mitochondria, the site of OXPHOS; this was not seen with TKI alone (P&lt;0.002). Similarly, CellROX analysis confirmed a reduction in ROS levels upon PF-3758309 treatment, or a combination of PF-3758309 with TKI, in these cells. In addtion, MDM2, a critical negative regulator of the p53 tumor suppressor, was identified as one of substrates of PAK6, by PhosphoSitePlus analysis. Its expression was found to be correlated with PF-3758309 treatment in CML cells based on CellMinerDB univariate analyses using gene-small-molecule association data from the CTRPv2 database. Indeed, Western blot analysis showed that PAK6 knockdown in K562 and IM-resistant cells led to a reduction in MDM2 protein levels. Furthermore, MDM2 downregulation by PAK6 inhibition corresponded to an increase in p21 levels, suggesting a mechanism of MDM2-mediated p21 regulation independent of p53, as these cells are p53-null. Most interestingly, PAK6 knockdown in IM-resistant cells leads to G2/M phase accumulation and increased senescence levels (2-fold, P&lt;0.05), detected by senescence-associated β-galactosidase staining. PAK6 knockdown induced senescence was further supported by observations of enlarged cell size (p&lt;0.05) and increased granulation, as well as changes in senescence-associated protein markers, including p21, p27, MMP-3 and the DNA damage marker pH2Ax, by Western blot analysis. Hence, our findings indicate that dual targeting of miR-185-PAK6-mediated survival, cell cycle and metabolic pathways, along with BCR-ABL, selectively eradicates drug-resistant CML stem/progenitors. Specifically, PAK6 plays roles in MDM2/p21-mediated apoptosis, senescence and cell cycle controls, offering a valuable therapeutic strategy for improved treatment and care. Disclosures No relevant conflicts of interest to declare.

scholarly journals MiR-221/222 Promote Dexamethasone Resistance of Multiple Myeloma through Inhibition of Autophagy By Targeting ATG12

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4469-4469
Author(s):  
Jian Xu ◽  
Yan Su ◽  
Aoshuang Xu ◽  
Fengjuan Fan ◽  
Haifan Huang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Combined Treatment ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Expression Levels

Abstract Dexamethasone (Dex) is the most widely used chemotherapeutic drug in the treatment of multiple myeloma (MM). Inherent or acquired resistance to Dex is broadly associated with poor prognosis in MM. Many microRNAs are aberrantly expressed in MM, including miR-221/222, which have been reported to act as oncogenes in many cancer types. Recently, accumulating evidence has shown that miR-221/222 are involved in the development of chemoresistance in a variety of cancers. However, there is still a lack of valuable data regarding the role of miR-221/222 in the chemoresistance of MM. Here, we first evaluated the expression levels of miR-221/222 in plasma cells (PCs) from MM patients by qRT-PCR analysis. The results showed that miR-221/222 were markedly upregulated in PCs from newly diagnosed MM patients compared to healthy donors, and they were further upregulated in PCs from patients with relapsed MM. In addition, we found that the expression levels of miR-221/222 were inversely correlated with Dex-sensitivity of human MM cell lines (HMCLs). Importantly, enforced expression of miR-221/222 dramatically reduced the sensitivity of Dex-sensitive HMCLs to Dex, while inhibition of miR-221/222 re-sensitized Dex-resistant HMCLs to Dex. Previous studies have shown that Dex-induced cell death in lymphoid leukemia is mediated through initiation of autophagy. To study whether autophagy was involved in Dex-induced cell death in MM cells, HMCLs were exposed to Dex, and then autophagy in these cells was evaluated by the transmission electron microscopy and western blot analysis. The results showed that Dex induced the occurrence of autophagy in Dex-sensitive HMCLs, but not in Dex-resistant HMCLs. Moreover, pharmacological inhibitors of autophagy could significantly reduce Dex-induced cell death in Dex-sensitive HMCLs. These results reveal that autophagy is critical for the induction of cell death following Dex treatment in MM. MicroRNAs have been reported to play an important role in regulating autophagy. We therefore examined whether miR-221/222 can regulate autophagy in MM cells. Low miR-221/222 expressing MM.1S (Dex-sensitive) or high miR-221/222 expressing MM.1R (Dex-resistant) cells were transfected with agomir-221/222 or antagomir-221/222, respectively, and then the level of autophagy was evaluated. The results showed that overexpression of miR-221/222 reduced the level of autophagy in MM.1S cells, while inhibition of miR-221/222 elevated the level of autophagy in MM.1R cells. Using microRNA target prediction bioinformatics tools and dual-luciferase reporter assay, we confirmed that autophagy-related gene 12 (ATG12) was a novel target gene of miR-221/222. Indeed, miR-221/222 could negatively regulate the expression of ATG12 at both the mRNA and protein levels in MM cells. In addition, knockdown of ATG12 by siRNA markedly reduced the autophagy-inducing and Dex-sensitizing activity of miR-221/222 antagomirs in MM.1R cells. Of note, in MM.1S cells, Dex treatment could further decreased the expression of miR-221/222, accompanied by upregulated expression of ATG12, whereas silencing the expression of ATG12 could significantly inhibited Dex-induced autophagy and cell death. Thus, these results suggest that ATG12 is a key player in miR-221/222-mediated autophagy inhibition and Dex-resistance. Next, we evaluated whether miR-221/222 could regulate autophagy and Dex-sensitivity of MM cells invivo. NOD/SCID mice were subcutaneously injected with MM.1R cells to establish Dex-resistant MM xenografts. Combined treatment with antagomir-221/222 plus Dex showed a remarkable reduction of tumor size compared to antagomir-221/222 or Dex alone (397.6±55.08 mm3 VS 895.8±72.44 mm3 VS 987.3±68.49 mm3). Immunohistochemistry and western blot analysis of the retrieved xenografted tumors showed that combination treatment with antagomir-221/222 plus Dex induced upregulation of ATG12, as well as extended autophagy with increased p62 degradation and Beclin-1 expression. In conclusion, our data reveal that upregulation of miR-221/222 promotes Dex resistance of MM cells through inhibition of autophagy by targeting ATG12. Therefore, miR-221/222-ATG12 autophagy-regulatory axis may potentially be applied in glucocorticoid resistance prediction and treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals ARHGEF12 Is Essential for Human Megakaryocyte Differentiation and Plays Critical Roles in Platelet Function

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 341-341
Author(s):  
Siying Zou ◽  
Alexandra M Teixeira ◽  
Chad D Sanada ◽  
Ping-xia Zhang ◽  
Diane Krause
Keyword(s):  
Platelet Aggregation ◽  
Mouse Model ◽  
Western Blot ◽  
Platelet Activation ◽  
Platelet Function ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Thrombin Receptors ◽  
Platelet Functions

Abstract Megakaryocytopoiesis, the process by which hematopoietic stem cells develop into mature megakaryocytes (MK), and thrombopoiesis, platelet production/release, are critical for blood homeostasis. We tested the hypothesis that the Rho guanine exchange factor, ARHGEF12 (also known as LARG), is critical for MK differentiation and platelet functions based on the following: 1) ARHGEF12 is part of a recurrent translocation with MLL in acute myeloid leukemia. 2) Both published microarray datasets and deep-sequencing data from our lab on primary human CD34+ cells differentiating into MKs show that ARHGEF12 expression goes up dramatically during MK differentiation. 3) ARHGEF12 is one of the most highly expressed guanine exchange factors in platelets. 4) ARHGEF12 forms a complex with G proteins and stimulates Rho-dependent signals. It is known that platelet activation can be initiated by extracellular stimuli working through G protein-coupled receptors and Rho signaling, suggesting that ARHGEF12 may function in platelet activation. 5) Mice with KO of RhoA (a known ARHGEF12 substrate) in the MK-lineage have macrothrombocytopenia and defective platelet activation. To test this hypothesis, we used ARHGEF12 shRNA mediated KD and an ARHGEF12 specific pharmacological inhibitor (Y16) in both murine and human primary cells, and characterized a LARG KO mouse model for MK and platelet phenotypes, and found: ARHGEF12 is differentially upregulated during MK differentiation and is enriched in platelets Using quantitative RT-PCR and western blot analysis at different timepoints of primary FACSorted Mk progenitors induced to differentiate into mature MK in vitro, ARHGEF12 RNA and protein expression increases during MK differentiation in both the murine and human systems. Also western blot analysis of murine platelet rich plasma shows that ARHGEF12 protein is highly expressed in platelets. ARHGEF12 is essential for human MK differentiation To test the function of ARHGEF12 in Mk differentiation, we used lentiviral shRNA to knockdown ARHGEF12 in FACSorted primary human Mk progenitors from mobilized peripheral blood differentiated in vitro to MK. The results show that ARHGEF12 knockdown blocks MK polyploidization (not shown) and maturation (Fig. A). This was confirmed using a published ARHGEF12 inhibitor (Y16) in the differentiation culture of human MK progenitors, in which there was a dose-dependent block in MK differentiation (Fig. B). These data suggested that ARHGEF12 is essential for human MK differentiation. We researched the function of ARHGEF12 in the murine system using a constitutive ARHGEF12 knockout mouse model. The mice have enlarged platelets (p=0.07) and a decreased platelet count (p=0.01). However, the knockout mice have normal BM cellularity with no change in megakaryocyte number or ploidy, suggesting that ARHGEF12 is dispensable for murine MK differentiation in vivo. ARHGEF12 is essential for platelet function in both the murine and human systems: To test whether ARGEF12 functions in platelet activation, we compared WT versus KO platelet activation in vitro. We tested activation in response to ADP, U46619 (Thromboxane), ADP+U46619, and Thrombin. KO plateelts have significantly reduced activation in response to U46619 and thrombin, with no effects on ADP-induced activation. Analogous studies using the ARHGEF12 inhibitor (Y16) on WT platelets revealed supportive evidence. Lastly, we tested ARHGEF12 function in human platelet aggregation using the Y16 compound. Consistent with the murine data, Y16 blocked platelet aggregation in response to both U46619 and Thrombin. Taken together, these data strongly suggest that ARHGEF12 is essential for platelet function and acts downstream of the Thromboxane and Thrombin receptors. In summary, we found that ARHGEF12 is differentially up-regulated in MK differentiation both in human and in mouse system,. It plays a critical role in human Mk differentiation but is dispensable in murine MK differentiation, and ARHGEF12 is critical for platelet functions in both human and mouse systems, potentially acting downstream of Thromboxane and Thrombin receptors. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals FAS-Mediated Apoptosis Assay in Patients with ALPS-like Phenotype Carrying CASP10 Mutations

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4960-4960
Author(s):  
Maurizio Miano ◽  
Enrico Cappelli ◽  
Agnese Pezzulla ◽  
Roberta Venè ◽  
Elena Palmisani ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Clinical Symptoms ◽  
Conflicts Of Interest ◽  
Severe Disease ◽  
Pathogenic Mutation ◽  
Incomplete Penetrance ◽  
Epigenetic Factors ◽  
Apoptosis Pathway

Abstract INTRODUCTION: Autoimmune lymphoproliferative syndrome (ALPS) is a rare congenital disorder characterized by an impaired FAS-mediated apoptosis that leads to chronic benign lymphoproliferation and autoimmunity. In most cases mutation on FAS gene are responsible of the disease and the phenotype of individuals carrying the same variants can vary from asymptomatic/mild forms to severe disease, due to incomplete penetrance of pathogenic mutations. More rarely, other genes involved in this pathway, such as CASP10, are mutated. Few clinical and molecular data have been reported on small numbers of patients carrying CASP10 mutation showing that different genetic variations can produce contrasting phenotypic effects. So far, 2 mutations have been recognized as pathogenic (I406L and L258F) and other have been reported with controversial result on their pathogenicity (V410l, Y446C) or are known to be polymorphic variants (L522l) AIMS: The aim of this study is to evaluate the FAS-mediated apoptosis function in patients with ALPS or ALPS-like symptoms carrying mutations on CASP10 gene. METHODS: We evaluated FAS-mediated apoptosis pathway in all patients presenting with an ALPS/ALPS-like phenotype that were found to carry a CASP10 mutation in our Institution. Molecular findings were obtained by NGS analysis of a panel of genes involved in the most common immune-dysregulation syndromes and immune-deficiencies and were confirmed by Sanger sequencing. Functional studies were performed by Western blot analysis of CASP10, CASP8, and PARP proteins after TRIAL-induced apoptosis stimulation. Healthy individuals were used as control. RESULTS: We identified 6 patients with ALPS (2) or ALPS-like (4) phenotype, carrying the following heterozygous CASP10 mutations: I406L (1), V410l (2), Y446C (1) L522l (2). Western blot analysis showed an impaired activation of CASP10, CASP8, and PARP proteins in all cases compared to healthy control (Fig. 1) CONCLUSIONS: In our symptomatic patients, the CASP10 polymorphic variant L522l and other mutations whose pathogenicity is controversial (V410l, Y446C) were associated with impaired CASP10, CASP8, PARP activity -and therefore with apoptosis dysfunction- as in the case of I406L pathogenic mutation. We can speculate that, in addition to the functional impairment of apoptosis, other genetic and epigenetic factors might be crucial for the development of clinical symptoms in CASP10 mutated patients, as already described in FAS mutations, suggesting that the search of other mutations in patients with ALPS/ALPS-like phenotype should be encouraged. Nonetheless, further studies on epigenetic factors potentially implicated in the expression of symptoms are needed to fully understand the heterogeneity of clinical phenotype of this disorder. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

The Function of Toll-Like Receptor 4 Expressions on Human Platelet in Platelet Activation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5361-5361
Author(s):  
Liping Ma ◽  
Da-Nian Nie ◽  
Xiu-Ju Wang ◽  
Shuang-Fen Xie ◽  
Yi-Qing Li ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Platelet ◽  
Gram Negative Bacteria ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Through Flow ◽  
Membrane Adhesion ◽  
Host Infection ◽  
Pai 1

Abstract Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) in host. Infection increases risk for hemostasis, thrombosis, DIC, and tissue repair. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (P-selectin) is one of the indexes to determine platelet activation. Experiment was designed to study whether TLR4 is expressed on human platelet, and what is the function of TLR4 in platelet activation induced with LPS. Platelet suspensions from 10 heath people were treated with LPS of different concentrations for 1 hour, which were 0mg/L(control),0.1mg/L,0.5mg/L,1mg/L and 5mg/L, respectively. The expressions of TLR4, P-select on platelets and PAI-1 in platelets were detected through flow cytometry (FCM) and western blot(WB) methods. ADP-induced platelet aggregation was measured by LG-PABER aggregometer. Compared with control, the expressions of TLR4,P-selectin on platelets and PAI-1 in platelets after treated with LPS of 0.5mg/L,1mg/L and 5mg/L were increased (P&lt;0.05). positive correlation was observed between TLR4 and P-selectin on platelets, but between TLR4 and PAI-1 in platelets. LPS of all concentrations did not affected ADP-induced platelet aggregation. Therefore, it is evident that functional TLR4 is expressed on human platelet. TLR4 on platelet might be one of the important intermedia between platelet activation and LPS or bacteria, and contribute to the inflammatory process in host. It is also worthy to study whether LPS affect platelet aggregation induced by other inductors.

scholarly journals Exosomes derived from platelet-rich plasma present a novel potential in alleviating knee osteoarthritis by promoting proliferation and inhibiting apoptosis of chondrocyte via Wnt/β-catenin signaling pathway

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Xuchang Liu ◽  
Lubo Wang ◽  
Chengshan Ma ◽  
Guozong Wang ◽  
Yuanji Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Platelet Rich Plasma ◽  
Therapeutic Effects ◽  
Significant Difference ◽  
Better Than

Abstract Background Platelet-rich plasma (PRP) provides a nonsurgical approach for treating osteoarthritis (OA). Exosomes that play vital roles in intercellular communication have been studied extensively. Here, we investigated the therapeutic potential and molecular mechanism of exosomes derived from PRP (PRP-Exos) in alleviating OA. Methods Exosomes derived from PRP(PRP-Exos) were isolated and purified using the exoEasy Maxi Kit and then identified and analyzed. Primary rabbit chondrocytes were isolated and treated with interleukin 1 beta (IL-1β) to establish the OA model in vitro. Proliferation, migration, and apoptosis assays were measured and compared between PRP-Exos and activated PRP (PRP-As) to evaluate the therapeutic effects on OA. The mechanism involving the Wnt/β-catenin signaling pathway was investigated by Western blot analysis. In vivo, we established animal knee OA model by surgery to compare the therapeutic effect of PRP-Exos and PRP-As. Results We successfully isolated and purified exosomes from PRP using the exoEasy Maxi Kit. We also isolated and identified chondrocytes from the New Zealand white rabbit and established the IL-1β-induced OA model; meanwhile, PRP-Exos and PRP-As both inhibited the release of tumor necrosis factor-α(TNF-α) and there was no statistically significant difference between the two. In proliferation, migration, scratch assay, the promoting effect of PRP-Exos was significantly more better than PRP-As. Furthermore, PRP-Exos could significantly decreased apoptotic rate of OA chondrocyte compared with PRP-As. In Western blot analysis, the expression of β-catenin, and RUNX2, Wnt5a were increased in IL-1β-treated chondrocytes, but PRP-Exos and PRP-As could both reverse these changes, and the reversal effect of the former was better than the latter. In vivo, we found that both PRP-Exos and PRP-As displayed the progression of OA, and the effect of PRP-Exos was obviously better than PRP-As by chondrocyte count and Osteoarthritis Research Society International (OARSI) scoring system. Conclusion The therapeutic effects of PRP-Exos on OA were similar or better compared with those of PRP-As in vitro or in vivo. PRP-Exos acting as carriers containing growth factors derived from PRP present a novel therapy for OA by activating the Wnt/β-catenin signaling pathway.

Inhibitory Effect of Staphylokinase on Platelet Aggregation

1993 ◽  
Vol 70 (05) ◽  
pp. 834-837 ◽  
Author(s):  
Akira Suehiro ◽  
Yoshio Oura ◽  
Motoo Ueda ◽  
Eizo Kakishita
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Degradation Products ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Effective Concentration ◽  
Inhibit Platelet Aggregation ◽  
Platelet Suspension ◽  
Washed Platelet ◽  
Human Platelet Aggregation

SummaryWe investigated the effect of staphylokinase (SAK), which has specific thrombolytic properties, on human platelet aggregation. Platelet aggregation induced with collagen was observed following preincubation of platelets in platelet-rich plasma (PRP) or washed platelet suspension (WP) with SAK at 37° C for 30 min. SAK inhibited platelet aggregation in PRP only at the highest examined concentration (1 x 10-4 g/ml). Although SAK did not inhibit platelet aggregation in WP which contained fibrinogen, it did when the platelets had been preincubated with SAK and plasminogen. The most effective concentration in WP was 1 x 10-6 g/ml. The effect could be inhibited by adding aprotinin or α2-antiplasmin. The highest generation of plasmin in the same preincubation fluid was detected at 1 x 10-6 g/ml SAK. We concluded that SAK can inhibit platelet aggregation in WP by generating plasmin and/or fibrinogen degradation products, but is only partially effective in PRP because of the existence of α2-antiplasmin.

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