Polo-Like Kinase 3, a Novel Regulator of Thrombosis: Positive Regulation of TxA2 Generation, Granular Secretion and Clot Retraction, but Negative Regulation of Integrin Activation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 379-379
Author(s):  
Meghna Ulhas Naik ◽  
Brendan Bachman ◽  
John C Kostyak ◽  
Wei Dai ◽  
Ulhas P Naik
Keyword(s):  
Platelet Function ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Fibrin Clot ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Mitotic Kinase

Abstract Abstract 379 Polo-like kinase (Plk) family members are serine/threonine kinases involved in cell cycle regulation. Their expression and function in platelets are not known. We identified the presence of Plk3, a member of this family, in human and mouse platelets. We found that Plk3 is localized to the filopodia of activated platelets. Furthermore, it co-immunoprecipitates with integrin aIIbb3 in an aggregation-dependent manner. To understand the physiological function of Plk3 in thrombosis, we obtained Plk3−/− mice in C57Bl/6 background, examined the tail bleeding time of Plk3−/− mice, and compared it to the Plk3+/+ (WT) mice of the same genetic background. We found that the average tail bleeding time for WT mice was about 130 s, consistent with the values reported in the literature. Interestingly, the Plk3−/− mice had a significantly (P<0.05) delayed average tail bleeding time (325 s), suggesting that Plk3 deficiency results in a bleeding phenotype. These results suggest that there may be defects in the thrombotic process in these mice. To evaluate the in vivo thrombotic phenotype, we performed a 10% FeCl3-induced carotid artery injury and observed any differences in time of occlusion or unstable occlusions in Plk3−/− mice compared to WT mice. Consistent with our finding of extended tail bleeding time in Plk3−/− mice, our results showed that the WT mouse vessel occluded within 7–9 min, whereas Plk3−/− mouse took nearly twice that time (∼14 min) to initiate vessel occlusion (P<0.001). We also performed a collagen/epinephrine-induced pulmonary thromboembolism assay to investigate the role of platelet Plk3 in thrombosis. Our result suggests a marked protection from thromboembolism in Plk3−/− mice, since significantly more (P<0.0004) survived compared to WT mice. By assessing the ability of Evans blue dye to pass through the pulmonary circulation, we determined that this better survival rate in Plk3−/− mice is due to the failure of occlusion of pulmonary vessels in these mice. This was further supported by the histological examination of the lungs of these mice, which showed decreased size of the emboli and the reduced extent of arterial occlusion compared to WT. Ex vivo platelet functional studies suggested that thrombin-induced generation of TxA2, a potent activator of platelet function, was significantly attenuated (P<0.03) in Plk3−/− mice compared to WT. When tested for activation of cPLA2, a key enzyme in TxA2 generation, we found that the phosphorylation of cPLA2 is significantly attenuated (P<0.05) in Plk3 null platelets. Furthermore, thrombin-induced secretion of both a- and d- granules was significantly reduced (P<0.007) in Plk3−/− mouse platelets compared to WT, consistent with the observed anti-thrombotic phenotype in vivo. Surprisingly, however, platelet aggregation by low dose of thrombin or PAR4 peptide was significantly augmented (P<0.02) in Plk3 null platelets compared to WT. This was further supported by the significantly increased (P<0.05) fibrinogen receptor exposure on platelets. To determine the molecular mechanism of the observed hyperaggregation, we analyzed signaling events such as ERK1/2 and Akt, an upstream regulator of integrin aIIbb3 activation. Interestingly, we found that agonist-induced activation of ERK2 and Akt (both T308 and S473 phosphorylation) is significantly enhanced in the absence of Plk3. Furthermore, we found that levels of PTEN, a negative regulator of PI3-K/Akt pathway is reduced in the absence of Plk3. The severity of the anti-thrombotic phenotype in Plk3−/− mice may have been dampened due to the opposing role of Plk3. We next asked if the integrin outside-in signaling is also enhanced in these mice. As expected, platelet adhesion to immobilized fibrinogen was significantly increased (P<0.05) in the absence of Plk3. Surprisingly, when analyzed for fibrin clot retraction, we found that Plk3 null platelets failed to retract fibrin clot. These results suggest that Plk3, a mitotic kinase, plays a significant role in regulation of platelet function such as TxA2 generation, granular secretion, and clot retraction, thus affecting the process of thrombosis. Disclosures: No relevant conflicts of interest to declare.

Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ahmed Alarabi ◽  
Zubair Karim ◽  
Victoria Hinojos ◽  
Patricia A Lozano ◽  
Keziah Hernandez ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Clot Retraction ◽  
Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

Feedback Activation of Factor XI by Thrombin Is Essential for Hemostasis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2127-2127
Author(s):  
Henri M. H. Spronk ◽  
Sabine Wilhelm ◽  
Rene Van Oerle ◽  
Menno L. Knetsch ◽  
David Gailani ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Fibrin Clot ◽  
Intrauterine Death ◽  
Factor Xii ◽  
Dependent Manner ◽  
Factor Xi ◽  
Feedback Activation

Abstract Abstract 2127 Poster Board II-102 Background: The revised model of coagulation proposes that factor XI (FXI) can be activated by thrombin, which is generated upon activation of the tissue factor (TF) pathway. This concept, however, has not been tested in vivo. A recent study questioned the existence of this feedback loop and suggested that factor XII (FXII) is the sole activator of FXI. Here, we analyze the feedback activation of FXI in plasma and in genetically altered mice. Methods and results: Fluorescence-based assays indicated that particle-bound thrombin caused thrombin generation in plasma both in the absence of TF and in the presence of active site inhibited factor VIIa. Thrombin failed to activate FXII and thrombin generation was almost completely abolished by an anti-FXIa antibody and in FXI-deficient plasma. Surface bound thrombin induced complex formation of FXI, with its major inhibitor C1 inhibitor, even in FXII-deficient plasma in a time and dose dependent manner. To determine if thrombin-driven FXI activation is important for hemostasis in vivo we used TF deficient mice (low TF), which have severely reduced thrombin formation. Low TF mice were crossed with mice deficient in one of the intrinsic pathway proteases FXII, FXI, or FIX. Double deficiency in TF and either FIX or FXI resulted in the intrauterine death of embryos due to hemorrhage. In contrast low TF/FXII-null mice were viable and the bleeding phenotype was unchanged from low TF animals. Conclusions: Surface-bound thrombin, a model for fibrin clot-protected thrombin, generates thrombin in a FXI dependent manner, independently from FXII. In addition to corroborating an amplifying role of FXI in thrombin generation, we provide the first evidence that at low TF levels FXI is essential in generating a sufficient ambient level of thrombin to permit embryonic development. Disclosures: No relevant conflicts of interest to declare.

Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 588-588
Author(s):  
Karrune Woan ◽  
Fengdong Cheng ◽  
Hongwei Wang ◽  
Jennifer Rock-Klotz ◽  
Zi Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Egfp Expression ◽  
In Vivo Models

Abstract Abstract 588 We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models. 1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100. 2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3183-3191 ◽  
Author(s):  
Catherine Léon ◽  
Anita Eckly ◽  
Béatrice Hechler ◽  
Boris Aleil ◽  
Monique Freund ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Strong Increase ◽  
Clot Retraction ◽  
Gene Encoding ◽  
Normal Platelet ◽  
Coated Surface ◽  
Platelet Functions

Abstract Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9Δ mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction. However, platelet aggregation and secretion in response to any agonist were near normal despite absence of initial platelet contraction. By contrast, integrin outside-in signaling was impaired, as observed by a decrease in integrin β3 phosphorylation and PtdIns(3,4)P2 accumulation following stimulation. Upon adhesion on a fibrinogen-coated surface, MYH9Δ platelets were still able to extend lamellipodia but without stress fiber–like formation. As a consequence, thrombus growth and organization, investigated under flow by perfusing whole blood over collagen, were strongly impaired. Thrombus stability was also decreased in vivo in a model of FeCl3-induced injury of carotid arteries. Overall, these results demonstrate that while myosin seems dispensable for aggregation and secretion in suspension, it plays a key role in platelet contractile phenomena and outside-in signaling. These roles of myosin in platelet functions, in addition to thrombocytopenia, account for the strong hemostatic defects observed in MYH9Δ mice.

scholarly journals TULA-2 Inhibits the Platelet FcγRIIA-Syk Pathway and Heparin-Induced Thrombocytopenia in Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 411-411 ◽  
Author(s):  
Yuhang Zhou ◽  
Shaji Abraham ◽  
Leonard C. Edelstein ◽  
Carol Dangelmaier ◽  
Alexander Tsygankov ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Platelet Reactivity ◽  
Negative Regulator ◽  
Severe Thrombocytopenia ◽  
Clot Retraction ◽  
Relative Level ◽  
Heparin Induced Thrombocytopenia ◽  
Hel Cells ◽  
Platelet Spreading

Abstract Heparin-induced thrombocytopenia (HIT) is a life-threatening disease in which IgG antibodies against the heparin-PF4 complex activate platelets via FcγRIIA. We previously reported that TULA-2 serves as a negative regulator of FcγRIIA pathway by dephosphorylating Syk in HEL cells. To further investigate the effect of TULA-2 on the FcγRIIA pathway and HIT, we crossed TULA-2-/- with FcγRIIA+/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of Syk, LAT, and PLCγ2 in platelets after FcγRIIA activation. Integrin activation, calcium mobilization, and P-selectin exposure were also enhanced in TULA-2-/- murine platelets compared to TULA-2+/+. Further, anti-GPIX antibody-induced HIT-like thrombocytopenia and thrombin generation were also augmented in TULA-2-/- mice (Figure A). We also found that decreased TULA-2 level shortened tail-bleeding time in mice (Figure B), suggesting a role of TULA-2 in physiological hemostasis. Additionally, TULA-2 KO and WT platelets did not show significant differences in platelet spreading and clot retraction, indicating that outside-in signaling is not affected by the absence of TULA-2. At the protein level, TULA-2 heterozygous knockout (TULA-2+/-) platelets express 50% as much protein as their wildtype counterparts. Interestingly, TULA-2+/- mice showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. Together the data indicate that not only the absence of TULA-2, but also the relative level of TULA-2 expression modulate FcγRIIA-mediated platelet reactivity, HIT pathogenesis, and hemostasis. Considering that TULA-2 is also a key regulator in platelet GPVI pathway, measuring TULA-2 expression may be a valuable predictor for HIT susceptibility and platelet reactivity in general. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2333-2333
Author(s):  
Brian D. Adams ◽  
Shangqin Guo ◽  
Haitao Bai ◽  
Changchun Xiao ◽  
E. Premkumar Reddy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3513-3513
Author(s):  
Wenxiu Yi ◽  
Wei Li ◽  
Lijie Ren ◽  
Xinliang Mao ◽  
Li Zhu
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Venous Blood ◽  
Pi3k Pathway ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Occlusion Time

Abstract The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

Apoptosis Signal-Regulating Kinase 1 Regulates Platelet Function through Integrin aIIbb3 Outside-In Signaling

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 328-328
Author(s):  
Meghna Ulhas Naik ◽  
Hidinori Ichijo ◽  
Ulhas P Naik
Keyword(s):  
Map Kinase ◽  
Platelet Function ◽  
Stress Responses ◽  
Intracellular Signaling ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Phosphorylation Site ◽  
Mapk Cascades ◽  
Induced Aggregation

Abstract Abstract 328 Atherosclerosis tends to develop in areas of the circulation where the blood flow is either low or turbulent, such as regions where the arteries bifurcate. Studies have shown that shear stress stimulates endothelial cells and modulates platelet function. However, the intracellular signaling events that coordinate these effects are not completely understood. The mitogen-activated protein kinase (MAPK) cascades are crucial in regulating cellular stress responses. It is known that a number of diseases, including cardiovascular diseases, are intimately related to stress related mechanisms mediated by MAPK cascades. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family which responses to diverse array of stresses. Here, we show for the first time that ASK1, a 155 kDa protein, is present in human and murine platelets and its role as a novel regulator of platelet function. In human platelets, when stimulated with thrombin, ASK1 is rapidly activated within 30 sec, as indicated by phosphorylation of Thr 845 on its activation loop. Interestingly, we found that ASK1 is rapidly inactivated after 3 min by dephosphorylation of phospho-Thr 845. Concomitantly, ASK1 is phosphorylated on Ser 967, a known inhibitory phosphorylation site. The activation of ASK1 coincided well with its downstream effector, p38 Map kinase. These results suggested that ASK1 may play a role in platelet function. We therefore tested the hemostatic functions in congenic Ask-1 knockout mice and compared it to C57/BL6 wild type (Wt) mice. We found a significant delay in tail bleeding time (P=0.2×10−9) in Ask1 null mice. While all Wt mice stopped bleeding with an average bleeding time of 100 sec, the Ask1 null mice had an average bleeding time of 576 sec, with 5 out of 9 mice that did not stop bleeding. A 10% FeCl3-induced carotid artery injury, a well-established in vivo thrombosis model, showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine. This finding was further supported by the histological examination of the lung tissue of mice surviving after 3 min of injection, which showed very few thrombi in Ask1 null mice compared to Wt mice. We next asked if the platelet function is affected in Ask1 null mice. We found that low dose of PAR-4 peptide-induced aggregation is significantly less in Ask1 null mice compared to Wt mice. When analyzed for the ability of Ask1 null platelets to retract clots, we found that while clot retraction was completed within 2h in Wt platelets, Ask1 null platelets failed to retract clots even after 18h. When assessed for phosphorylation of the integrin b3 subunit as an indicator of outside-in signaling, we found that Wt platelet exposure to immobilized fibrinogen showed a robust phosphorylation of b3 subunit. Interestingly, Ask1 null platelets failed to show any induction of b3 phosphorylation. These in vitro and in vivo results strongly suggest that ASK1 plays a significant role in the regulation of platelet function and hemostasis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IGFBP7 As a Potential Therapeutic Target for AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5134-5134
Author(s):  
Yina Niu ◽  
Shuiyan Wu ◽  
Shaoyan Hu
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Dependent Manner ◽  
Mice Model ◽  
Cell Counts ◽  
And Migration ◽  
Proliferation And Migration

Abstract Insulin-like growth factor binding proteins (IGFBPs) are secretory factors that play essential roles in regulation of insulin-like growth factors (IGFs) in tissue as well as in modulating IGF binding to its receptors. IGFBP7, known as IGFBP-related protein 1 (IGFBP-rP1), mac25/angiomodulin, function as a potential tumor suppressor in various human solid cancers, including breast, prostate, gastric and liver cancer. We have reported the overexpression of IGFBP7 in the context of acute myeloid leukemia (AML), showing that IGFBP7 expression level in AML patients is significantly increased compared with controls (P<0.001). IGFBP7 expression was obviously decreased in AML patients achieving complete remission (P<0.01), and was significantly increased in relapsed AML patients (P<0.01). In addition, AML patients with high expression of IGFBP7 had shorter overall survival. Here, we investigate the role and mechanism of IGFBP7 in the development and progression of AML. In order to study the role of IGFBP7 in AML, stable cell lines expressing IGFBP7 and control in AML cells were constructed using lentiviral packaging system. Expression microarray assay was carried out to analyze the global gene level changes driven by IGFBP7. MTT and transwell assays were performed to evaluate the effect of IGFBP7 on cell proliferation and migration. Bioinformatics results found that IGFBP7 appeared to utilize multiple cellular processes for its oncogenic roles, including adhesion, migration, and proliferation. Experimental data showed overexpression of IGFBP7 in K562 cells resulted in a 2-3 fold increase in migration in contrast to control cells. Moreover, enforced expression of IGFBP7 also led to phosphorylation of Akt and Erk, whose activities inactivation by pharmacologically inhibitors resulted in the loss of ability to migrating. Finally, knockdown of IGFBP7 in cells with high IGFBP7 level, their migration abilities were significantly decreased. To assess the role of IGFBP7 in leukemogenesis in vivo, the same numbers of K562/IGFBP7 and K562-Vector cells, U937-shIGFBP7 and U937-shNEG cells were injected into NOD-SCID mice by tail vein injection, respectively. About two weeks later, it was showed that mice of K562/IGFBP7 and U937 groups displayed higher white blood cell counts compared with mice of K562-Vector and U937-shIGFBP7 groups, respectively. Mice of K562/IGFBP7 and U937 groups had more severe splenomegaly and hepatomagaly compared with its corresponding control groups. We further characterized the molecular mechanism underlying leukemogenesis driven by IGFBP7 in AML cell lines. The global expression profiling and molecular biological experiments showed PI3K/AKT signaling was activated by overexpression of IGFBP7, and knockdown of IGFBP7 in AML cells led to a decrease of PI3K/AKT activity in PTEN-dependent manner. IGFBP7 promotes proliferation and migration of AML cells, the promotion could be suppressed by both RNA interference and pharmacological inhibition of PI3K/AKT pathway. Immuno-precipitation assay showed that IGFBP7 associated with AXAN2 and induced PTEN degradation. The expression of ANXA2 was significantly positive correlated with the expression ANXA2 in AML patients. The expression of IGFBP7 in AML, overexpression as well as knockdown of IGFBP7 in leukemia cells and in mice model, all suggest that IGFBP7 is a potential proto-oncogene. Collectively this work suggests that targeting IGFBP7 activity may be an effective therapeutic strategy for AML. Disclosures No relevant conflicts of interest to declare.

Role of Thiol Isomerase ERp5 in Thrombus Formation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 370-370
Author(s):  
Freda H. Passam ◽  
Lin Lin ◽  
Mingdong Huang ◽  
Jonathan M. Gibbins ◽  
Bruce Furie ◽  
...  
Keyword(s):  
Cho Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Wild Type ◽  
In Vivo Models ◽  
Platelet Accumulation

Abstract Abstract 370 Protein disulfide isomerase is required for thrombus formation in various in vivo models of thrombosis. Another member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5), is released from activated platelets and co-immunoprecipitates with beta 3 integrin (Jordan et al, 2005). We further investigated the association of ERp5 with the platelet fibrinogen receptor alpha IIb beta 3 and the significance of ERp5 release in thrombus formation in vivo. Recombinant purified ERp5 was labeled with Alexa 488 and used in direct binding assays to CHO cells expressing wild type (WT) alpha IIb beta 3, CHO cells expressing mutant alpha IIb beta 3 (containing an Asp119Tyr substitution in the beta 3 subunit) and to control CHO cells. The mutant alpha IIb beta 3 does not bind fibrinogen. ERp5 bound to CHO cells expressing wild type (WT) alpha IIb beta 3 in a dose-dependent manner but did not bind to CHO cells expressing mutant alpha IIb beta 3 or to control CHO cells. The relative increase in the geomean of Alexa 488-labeled ERp5 binding to 0.5 ×106 WT alpha IIb beta 3 CHO cells over that bound to control CHO cells was 20, 45 and 85% for ERp5 concentrations of 80, 160 and 400 nM respectively. Binding of ERp5 (160 nM) to WT alpha IIb beta 3 expressing CHO cells was further increased by 75% when the integrin was activated with 2 mM Mn2+ compared to non-activated WT alpha IIb beta 3 CHO cells. A role for ERp5 in thrombus formation was studied in the laser injury model of thrombosis in mouse cremaster arterioles using a rabbit polyclonal anti-ERp5 antibody, immunoaffinity purified against recombinant ERp5. This antibody detected ERp5 in the releasate of thrombin-activated mouse platelets in vitro by Western blot and on the surface of thrombin-activated mouse platelets by flow cytometry. Dylight 649-labeled anti-CD42b was infused into the mouse circulation to detect platelet accumulation and Alexa 488-labeled anti-ERp5 antibody at 0.05 ug/g, a dose that does not inhibit thrombus formation, was infused to detect ERp5. The fluorescent anti-ERp5 signal detected at the thrombus site was compared to the signal produced by a non-specific IgG labeled with Alexa 488 infused into a control mouse. Anti-ERp5 fluorescence was detected in the thrombus with kinetics that followed platelet accumulation whereas there was minimal signal from the control IgG. We examined whether higher doses of anti-ERp5 affect thrombus formation. Platelet and fibrin accumulation were detected using fluorescently labeled anti-CD42b antibody and monoclonal anti-fibrin-specific antibody respectively before or after the injection of unlabeled anti-ERp5 antibody or pre-immune IgG at 2.5 ug/g. Platelet and fibrin accumulation, expressed as area under the curve of the median integrated fluorescence over time, was obtained from 14 thrombi in 6 mice formed before infusion of antibody, 18 thrombi in 2 mice formed after infusion of control IgG and 29 thrombi in 3 mice formed after infusion of anti-ERp5. Anti-ERp5 infusion caused a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (p<0.01). There was no difference in platelet and fibrin accumulation before infusion of antibody and after infusion of control antibody. These results provide evidence for a role of a second thiol isomerase, ERp5, in thrombus formation, a function which may be mediated through its association with alpha IIb beta 3. Disclosures: No relevant conflicts of interest to declare.

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