DISTINCT ROLES for Rap1b In PLATELET SECRETION and INTEGRIN aIIBb3 OUTSIDE-In SIGNALING

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2200-2200
Author(s):  
Guoying Zhang ◽  
Binggang Xiang ◽  
Ye Shaojing ◽  
Magdalena Chrzanowska-Wodnicka ◽  
Andrew J. Morris ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Critical Role ◽  
Wild Type ◽  
Clot Retraction ◽  
Platelet Factor ◽  
Dense Granules ◽  
Activation Mechanisms ◽  
Platelet Spreading ◽  
Inside Out ◽  
Platelet Secretion

Abstract Abstract 2200 Rap1b is activated by platelet agonists, and plays a critical role in integrin aIIbb3 inside-out signaling and platelet aggregation. In this study, we identify two novel functions of Rap1b in platelets. We show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that aIIbb3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Defect in secretion of Rap1b deficient platelets was not due to reduced granule contents, because the amount of serotonin (5HT) and platelet factor 4 (PF4) in Rap1b deficient platelets is similar to that in wild type platelets. Data from transmission electron microscopy indicate that under resting conditions, wild type and Rap1b deficient platelets had normal discoid shapes with similar numbers of granules. When stimulated with thrombin, wild type platelets showed an irregular appearance with protruding filopodia and lack of granules. In contrast, thrombin-stimulated Rap1b−/– platelets showed that more Rap1b−/– platelets contained visible a granules than wild type platelets. Dense granules were also more obvious in thrombin-stimulated Rap1b−/– platelets than wild type platelets. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TP deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-BAPTA. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b deficient platelets compared with wild type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b deficient platelets were not rescued by addition of MnCl2, which elicits aIIbb3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin aIIbb3 outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of Integrin αIIbβ3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets

2020 ◽  
Vol 21 (18) ◽  
pp. 6563
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Soochong Kim
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Tyrosine Kinase 2 ◽  
Platelet Spreading ◽  
Protein Kinase Cδ

Engagement of integrin αIIbβ3 promotes platelet–platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

Failure of Erythropoiesis and Megakaryocytopoiesis in RASA3 Mutant Scat Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 680-680
Author(s):  
Lionel Blanc ◽  
Babette Gwynn ◽  
Steven L. Ciciotte ◽  
Luanne L. Peters
Keyword(s):  
Bone Marrow ◽  
Positional Cloning ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Inbred Mouse Strain ◽  
Recessive Mutation ◽  
Initial Increase ◽  
Severe Anemia ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 680 Scat (severe combined anemia and thrombocytopenia) is a spontaneous, autosomal recessive mutation coisogenic with the BALB/cBy inbred mouse strain. Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. As its name implies, crisis episodes are characterized by severe anemia and thrombocytopenia, but significant lymphocyte depletion occurs as well. The first crisis episode begins in utero, lasts until postnatal day (P) 9 on average, and is associated with 10–15% mortality. Remarkably, in homozygotes that survive the first crisis, a remission phase occurs wherein the disease phenotype reverts to normal. This remission is transient, however, and is followed by a second crisis episode during which 94% of scat/scat mice die by P30. Previously we showed that the scat phenotype is transferrable via the hematopoietic stem cells and is also recapitulated in scat/scat, Hox11−/− double homozygotes in which a spleen does not develop, indicating that the splenic micro-environment plays little or no role in disease appearance or progression. Positional cloning of scat revealed a missense mutation in Rasa3 encoding a GTPase activating protein (GAP) that negatively regulates Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. We further showed that Rasa3 is a conserved gene critical to vertebrate erythropoiesis via morpholino knockdowns in zebrafish which resulted in profound anemia. Here we report data that shed further light on RASA3 function during hematopoiesis. Overall, the data indicate that defects in RASA3 profoundly and negatively impact erythropoiesis and megakaryocytopoieis through, at least in part, a Ras-mediated mechanism. FACS analyses of scat spleen and bone marrow erythroid populations reveal a severe block in erythropoiesis during crisis periods. In the spleen, despite an initial increase in size due to expansion of Ter-119+ cells, there is ultimately a loss of compensatory erythropoiesis resulting in a return to normal cellularity and a striking loss of hemoglobinized cells as the crisis phenotype deepens. In addition, the bone marrow shows loss of Ter-119+ cells and overall cell depletion during crisis. Megakaryocyte numbers are increased in scat crisis BM and spleen. By transmission electron microscopy, scat crisis megakaryocytes display features characteristic of a significant developmental delay: a disorganized demarcation membrane system with no platelet forming areas and few granules with hypersegmented nuclei and excess rough endoplasmic reticulum. In addition to the severe anemia and thrombocytopenia, a significant lymphopenia occurs in scat crisis mice. However, the scat phenotype is not lymphocyte mediated, as the scat phenotype is completely recapitulated in mice doubly homozygous for scat and the immunodeficient mutations, scid and Rag1tm1Mom, in which B- and T-lymphocytes are completely depleted. Together these results suggest that lymphopenia is a secondary phenomenon in scat, and the severe anemia and thrombocytopenia aspect of the phenotype neither follows from nor is dependent upon loss of lymphocytes. Despite the delay observed in erythroid differentiation, some mature red cells are produced although ∼50% of these are reticulocytes. By confocal microscopy, we show that RASA3 protein localizes to the plasma membrane as well as internal membrane compartments in wild type reticulocytes, where it partially colocalizes with CD71. Western blot analyses of reticulocytes after Percoll gradient purification show that RASA3 is lost during the maturation step, both in vivo and in vitro. Interestingly, in scat, RASA3 is present in reticulocytes, but appears to be mislocalized, the protein being found in the cytosol. Preparation of ghosts from wild type and scat reticulocytes confirms that RASA3 is not attached to the membrane in scat animals during crisis. In pull-down assays active GTP-bound Ras is increased in scat crisis reticulocytes when compared to wild type, suggesting that scat is a RASA3 loss of function mutation due to its mislocalization and demonstrating a critical role for the RASA3-Ras axis during mammalian erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals C-CBL Is a Critical Negative Regulator of Megakaryopoesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1581-1581
Author(s):  
Sebastian J. Saur ◽  
Melanie Märklin ◽  
Alexandra Poljak ◽  
Manuela Ganser ◽  
David E. James ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Platelet Factor ◽  
Physiological Level ◽  
Hematopoietic Stem ◽  
Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors in order to maintain a physiological level of circulating platelets. Thrombopoietin (TPO) is the main regulator of megakaryopoiesis modulating megakaryocyte differentiation, promoting endomitosis and proplatelet formation and as such supports the self-renewal and survival of hematopoietic stem cells. To allow proper proliferation and differentiation of different hematopoetic lineages, TPO signal transduction must be tightly regulated. Several mechanisms negatively modulating hematopoiesis and differentiation of the megakaryocytic lineage have previously been identified. Among those are suppressors cytokine signaling, protein phosphatases as well as a multitude of negative regulatory signaling pathways. However, one of the most effective mechanisms to permanently disable activated signaling proteins is by targeted degradation via lysosomes or proteasomes. In this study, we investigated the mechanisms that regulate TPO-mediated MPL degradation in primary mouse cells. Previous studies have identified CBL as an E3 ligase responsible for the ubiquitination of MPL in cell lines. In order to determine the potential role of c-CBL in murine thrombopoiesis, we used Cre/loxP technology to specifically delete c-CBL in the megakaryocytic lineage. Mice expressing two floxed c-CBL alleles were crossed to mice expressing Cre recombinase under the control of the platelet factor 4 (PF4) promoter. This yielded progeny with the desired genotype of c-CBLfl/fl PF4-Cre (CBL ko) after two generations of breeding. The desired cohort exhibited a quantitative absence of c-CBL in megakaryocytes and platelets as assessed by western blotting compared with wild type C57/BL6 mice. The expression of CBL in other hematopoietic cells such as B cells, T cells, neutrophils, monocytes and dendritic cells remained unaffected in this conditional ko strain. The experimental cohort showed significantly higher numbers of megakaryocytes in the bone marrow and of platelets in the peripheral blood as compared to wild type mice (1.2 mio vs. 1.8 mio cells/µl, p<0.0001). In addition, the platelets from the mutant mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). To evaluate the role of c-CBL in mature megakaryocytes, total bone marrow was collected from 12 wk old CBL ko mice and grown in TPO-containing culture medium for 72 h. Megakaryocytes derived from the bone marrow of wild type mice served as controls. Mature megakaryocytes were eventually isolated on a BSA-density gradient. Subsequent Western Blot analysis revealed a significant reduction of MPL ubiquitination in the CBL ko mice as compared to wild type mice, thereby identifying c-CBL as a critical negative regulator of megakaryopoesis. Taken together, we have successfully ablated c-CBL specifically from the megakaryocyte lineage and could demonstrate that this has profound effects on platelet counts and platelet size. In addition, we were able to show that c-CBL ablation leads to reduced ubiquitination of MPL and a consecutively longer half life of this protein culminating in substantially increased megakaryopoiesis in the c-CBL ko cohort. In summary, these data enhance our understanding of the regulation of TPO signaling and the physiological role of CBL in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

scholarly journals M6PR-Specific Targeting of Lysosomal Heparanase Regulates Platelet Hemostatic Function in Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1059-1059
Author(s):  
Nathan Eaton ◽  
Caleb Drew ◽  
Theresa A. Dlugi ◽  
Karin M. Hoffmeister ◽  
Hervé Falet
Keyword(s):  
Platelet Function ◽  
Conflicts Of Interest ◽  
Blood Platelets ◽  
Critical Role ◽  
Fluorescent Microscopy ◽  
Type I ◽  
Dense Granules ◽  
Specific Targeting

Besides α-granules and dense granules, which play critical roles in and beyond hemostasis, circulating blood platelets and their precursor cells megakaryocytes contain lysosomes, the contents of which are also secreted during platelet activation. In their delivery to the lysosome, acid hydrolases bearing phosphomannosyl residues are trafficked from the trans-Golgi network to the acidic late-endosomal compartment via the mannose 6-phosphate receptor (M6PR) pathway. To determine the role of M6PR-specific targeting of lysosomal enzymes in platelet function, platelet parameters were investigated in M6pr-/- mice lacking the 46-kDa M6PR, the physiological role of which is unclear. M6pr-/- mice had normal platelet count but displayed an increased number of distinct proplatelet-like cells compared to control mice, as determined by immunofluorescent microscopy. Moreover, transmission electron microscopy revealed the presence of abnormal membrane tubulations, elongated and electron-dense granules, and large vacuole-like structures within resting M6pr-/- platelets. M6pr-/- platelets expressed normally major glycoproteins on their surface and von Willebrand factor and fibrinogen in their α-granules. M6pr-/- mice were hyper-thrombotic, as assessed by tail bleeding time, and M6pr-/- platelets adhered to type I collagen with a significantly greater propensity than control platelets under arterial shear in in vitro flow experiments. Heparanase, an endo-β-glucuronidase that cleaves extracellular matrix heparan sulfate proteoglycans, is the most abundant lysosomal enzyme in platelets. Thus, its contribution to the phenotype of M6pr-/- mice was investigated further. Heparanase expression was decreased in the bone marrow megakaryocytes and blood platelets of M6pr-/- mice and increased in M6pr-/- plasma, as evidenced by immunoblot and fluorescent microscopy analysis, consistent with its mistargeting in the absence of M6PR. Interestingly, pharmacological inhibition of heparanase with OGT 2115 normalized the adhesion of M6pr-/- platelets to collagen in vitro, indicating that increased plasma heparanase contributes to the thrombotic phenotype of M6pr-/- mice. Taken together, the data suggest that the M6PR-specific targeting of lysosomal heparanase plays a critical role in platelet function, thereby regulating hemostasis. Disclosures No relevant conflicts of interest to declare.

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.

Influence of Protein Tyrosine Phosphatase Polymorphisms on the Development of Antibodies to Platelet Factor 4 and the Risk of Heparin-Induced Thrombocytopenia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3683-3683
Author(s):  
Jerôme Rollin ◽  
Claire Pouplard ◽  
Dorothee Leroux ◽  
Marc-Antoine May ◽  
Yves Gruel
Keyword(s):  
Immune Response ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Platelet Factor 4 ◽  
Control Group ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Protein Tyrosine ◽  
Significant Difference

Abstract Abstract 3683 Introduction. Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of platelet-activating IgG antibodies that activate platelets via FcgRIIa receptors. The reasons explaining why only a subset of patients treated with heparin develop IgG to PF4/H complexes, and why most patients who synthesize these antibodies do not develop HIT, have not been fully defined. The immune response in HIT involves both B and T cells, and protein tyrosine kinases (PTKs) and phosphatases (PTPs) are crucial for regulating antigen receptor-induced lymphocyte activation. Moreover, some PTPs such as CD148 and low-molecular-weight PTP (LMW-PTP) could also have a critical role in platelet activation. Dysregulation of the equilibrium between PTK and PTP function could therefore have pathologic consequences and influence the pathogenesis of HIT. Aim of the study. To investigate an association between polymorphisms affecting genes encoding 4 different PTPs i.e. CD45 (PTPRC), CD148 (PTPRJ), LYP (PTPN22) and LMW-PTP (ACP1) and the development of heparin-dependent antibodies to PF4 and HIT. Patients and methods. A cohort of 89 patients with definite HIT (positive PF4-specific ELISA and positive serotonin release assay) and two control groups were studied. The first control group (Abneg) consisted of 179 patients who had undergone cardiopulmonary bypass (CBP) with high doses of heparin and who did not develop Abs to PF4 post-operatively. The second control group (Abpos) consisted of 160 patients who had also undergone cardiac surgery with CPB and heparin, who had all developed significant levels of PF4-specific antibodies but without HIT. Genotypes of PTPRC 77C/G (rs17612648), PTPN22 1858C/T (rs2476601), PTPRJ 2965 C/G (rs4752904) and PTPRJ 1176 A/C (rs1566734) were studied by a PCR-HRM method using the LightCycler 480 (Roche). In addition, the ACP1 A, B, C alleles were defined by combining the analysis of T/C transition at codon 43 of exon 3 (rs11553742) and T/C transition at codon 41 of exon 4 (rs11553746). Results. The frequency of PTPRC 77G and PTPN22 1858T alleles was not different in HIT patients and controls, whether they had developed antibodies to PF4 or not. The third PTP gene analyzed was ACP1, in which three alleles (A, B and C) were previously associated with the synthesis of distinct active LMW-PTP isoforms exhibiting different catalytic properties. The percentage of subjects in our study carrying the AC, BB and BC genotypes was significantly higher in the HIT and the Abpos groups than in patients without antibodies to PF4 after CPB (Abneg). In addition, the ACP1 A allele was less frequent in patients with antibodies to PF4, whether they had developed HIT (25%) or not (27.5% in Abpos controls), than in Abneg subjects (37%). The AC, BB and BC genotypes (associated in Caucasians with the highest LMW-PTP enzyme activity) therefore appeared to increase the risk of antibody formation in heparin-treated patients (OR 1.8; 95% CI 1.2–2.6, p=0.004 after comparing Abpos + HIT vs. Abneg). We also evaluated 2 SNPs affecting PTPRJ encoding CD148. No significant difference was found concerning the 2965 C/G polymorphism, but the frequency of PTPRJ 1176 AC and CC genotypes was significantly lower in the HIT (17%) than in the Abneg and Abpos groups (35%, p=0.003 and 29.5%, p=0.041, respectively). The C allele therefore appeared to provide a significant protection from the risk of HIT (OR 0.52; 95%CI 0.29–0.94, p=0.041) in patients with antibodies to PF4. Discussion-Conclusion. Recent studies have demonstrated that CD148 is a positive regulator of platelet activation by maintaining a pool of active SFKs in platelets. This non-synonym PTPRJ 1176 A/C SNP is associated with a Q276P substitution inducing a torsional stress of a fibronectin domain that is critical for the activity of CD148 and may influence the pathogenic effects of HIT Abs. This study supports the hypothesis that PTPs such as LMW-PTP and CD148 influence the immune response to heparin and the risk of HIT in patients with antibodies to PF4. Disclosures: No relevant conflicts of interest to declare.

The Role of Akt3 in Mediating Outside-in Signaling of the Platelet Integrin αIIbβ3

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1134-1134
Author(s):  
Kelly A O'Brien ◽  
Nissim Hay ◽  
Xiaoping Du
Keyword(s):  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Akt Inhibitor ◽  
Integrin Αiibβ3 ◽  
Akt Activation ◽  
Downstream Effector ◽  
Platelet Spreading ◽  
Human And Mouse

Abstract Abstract 1134 Ligand binding to integrins mediates cell adhesion and transmits “outside in” signals that lead to cell spreading, migration, and proliferation. In platelets, the prototype integrin aIIbb3-mediated outside-in signaling is required for platelet spreading and retraction, and greatly amplifies platelet activation. Previous studies suggest that phosphoinositide 3-Kinases (PI3K) are activated upon binding of integrin αIIbβ3 to its ligand fibrinogen, and is important in outside-in signaling leading to platelet spreading. However, the mechanism by which PI3K transmits outside-in signals has been unclear. A major known downstream effector of PI3K is the Akt (protein kinase B) family of serine/threonine kinases, including Akt1, Akt2, and Akt3. We have recently shown that platelets not only express Akt1 and Akt2 as previously reported, but also express a substantial amount of Akt3. To investigate whether Akt3 is a downstream effector mediating PI3K-dependent integrin outside-in signaling, platelets from Akt3 knockout mice were compared with wild type platelets for their spreading on fibrinogen. Platelets from Akt3−/− mice showed partially, but significantly reduced spreading on fibrinogen, indicating that Akt3 is important in integrin-mediated outside-in signaling leading to platelet spreading. Consistent with the results of Akt3 knockout, treatment of platelets with a pan Akt inhibitor also significantly inhibited spreading of human and mouse platelets on fibrinogen. Akt becomes phosphorylated upon platelet spreading on fibrinogen, which is significantly reduced in Akt3 knockout platelets, and is abolished by PI3 Kinase inhibitor, wortmannin, or Src Family Kinase (SFK) Inhibitor, PP2, suggesting that Akt activation is downstream from PI3K, and SFK during integrin outside-in signaling. Thus, our data reveals that Akt3 is an important downstream effector of PI3K-dependent integrin outside-in signaling promoting platelet spreading. Disclosures: No relevant conflicts of interest to declare.

Nfkb1 Plays a Tumor-Suppressing Role in BCR-ABL-Induced Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1666-1666
Author(s):  
Con Sullivan ◽  
Yaoyu Chen ◽  
Zhiru Guo ◽  
Yi Shan ◽  
Cong Peng ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Critical Role ◽  
Philadelphia Chromosome ◽  
K562 Cells ◽  
Wild Type ◽  
Empty Vector ◽  
Donor Cells ◽  
Rescue Experiment

Abstract Abstract 1666 NF-κB activation has been linked to the promotion of an assortment of malignancies. While in vitro studies have supported a tumor-promoting role for NF-κB in leukemias, in vivo evidence has not been collected. NF-κB has also been proposed as a therapeutic target for Philadelphia chromosome-positive leukemias including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). In this study, we investigate the role the Nfkb1 gene plays in CML and B-ALL using mouse models for these leukemias induced by the BCR-ABL oncogene. Contrary to conventional thinking, we find a tumor-suppressing role for Nfkb1 in BCR-ABL leukemias. To induced CML, we transduced bone marrow cells from 5-FU-primed wild type or Nfkb1−/− mice with BCR-ABL-GFP retrovirus, followed by transplantation of the transduced cells into lethally irradiated recipient mice. We show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells developed CML more rapidly than the wild type recipients, correlating to elevated percentages (82.3% vs 38.5%) and total numbers of Gr-1+GFP+ cells in peripheral blood of the mice. We next tested whether the loss of Nfkb1 also accelerated B-ALL development under the B-ALL-inducing conditions, and show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells succumbed to B-ALL more quickly than the wild type recipients. These results suggest that Nfkb1 does not stimulate leukemia growth, and instead, it plays a tumor-suppressing role in CML and B-ALL. Because these findings are opposite from what people believe in the field, we confirmed our findings by conducting a rescue experiment. The Nfkb1 gene encodes the p105 protein; roughly half of p105 product is selectively proteolyzed to yield the p50 transcription factor. In this rescue experiment, we transduced Nfkb1−/− donor cells with BCR-ABL alone or BCR-ABL and p50 of Nfkb1 to induce leukemia, and we show that acceleration of leukemia development caused by the Nfkb1 loss is partially reversed by p50. To study the underlying mechanism, we focused on CARD11, because it plays a critical role in mediating NF-κB signaling in lymphocytes. We find that CARD11 expression is induced in myeloid and increased in lymphoid cell lines expressing BCR-ABL. Furthermore, we induced B-ALL using Card1−/− mice. We find that similar to the loss of Nfkb1, the loss of Card11 expression also accelerates the development of B-ALL. To increase human relevance of our findings, we overexpressed p50 in human K562 cells by retroviral transduction with p50-IRES-GFP or empty vector, and find that FACS-sorted p50/GFP K562 cells grow significantly slower than empty vector-transduced cells. These findings support an idea that Nfkb1 signaling suppresses BCR-ABL-induce leukemia at least partially through a CARD11-dependent mechanism. In summary, our results demonstrate that Nfkb1 plays a tumor-suppressing role in BCR-ABL-induced leukemias, arguing that we should be careful when targeting of Nfkb1 in leukemia treatment. Disclosures: No relevant conflicts of interest to declare.

A Mechanism For Switch Of Integrin Signaling Direction and a New Anti-Thrombotic Strategy Through Selective Outside-In Signaling Inhibition

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2295-2295
Author(s):  
Bo Shen ◽  
Xiaojuan Zhao ◽  
Kelly A O'Brien ◽  
Aleksandra Stojanovic-Terpo ◽  
Michael Keegan Delaney ◽  
...  
Keyword(s):  
Side Effect ◽  
Early Phase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Late Phase ◽  
Integrin Signaling ◽  
Clot Retraction ◽  
Integrin Antagonists ◽  
Inside Out

Abstract Antagonists of platelet integrin alphaIIbbeta3 are potent anti-thrombotics due to critical roles of integrins in thrombosis. However, integrins are also important in hemostasis, and thus integrin antagonists have potentially life-threatening bleeding side effect. It would be ideal if we can develop integrin antagonists without bleeding side effect. Integrins transmit signals bidirectionally. Intracellular signals activate integrin alphaIIbbeta3, leading to talin-dependent integrin ligation, which is required for platelet adhesion and initial hemostatic thrombus formation. Integrin ligation in turn mediates Galpha13/Src-dependent outside-in signaling that stabilizes and amplify thrombi, which is crucial for occlusive arterial thrombosis. Here we show that talin and Galpha13 bind to mutually exclusive but distinct sites in integrin beta3, and their bindings are dynamically regulated during integrin signaling. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for early phase outside-in signaling. Integrin ligation induces talin dissociation and Galpha13 binding, which selectively mediates early phase outside-in signaling. The second talin binding wave is associated with late phase outside-in signaling and clot retraction. Based on these findings, we have designed a selective inhibitor of Galpha13-integrin interaction, which specifically abolished outside-in signaling without affecting inside-out signaling and integrin ligation. Strikingly, this inhibitor potently inhibits occlusive thrombosis in vivo, but has no effect on tail bleeding time, in contrast to the current integrin antagonists. Thus, we have discovered a mechanism for switching the direction of integrin signaling and a new anti-thrombotic that does not cause bleeding. Disclosures: No elevant conflicts of interest to declare.

scholarly journals Critical Role for Mouse Marginal Zone B Cells in PF4/Heparin Antibody Production

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1175-1175
Author(s):  
Yongwei Zheng ◽  
Mei Yu ◽  
Andrew Podd ◽  
Debra K. Newman ◽  
Renren Wen ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Antibody Production ◽  
Marginal Zone ◽  
Critical Role ◽  
Wild Type ◽  
Platelet Factor ◽  
Specific Antibodies ◽  
Deficient Mice

Abstract Abstract 1175 Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here we show that upon challenge with PF4/heparin complexes, anti-PF4/heparin antibody production is severely impaired in B cell-specific Notch2-deficient mice (CD19CreNotch2fl/fl) that specifically lack marginal zone (MZ) B cells, and that antibody production is readily generated in wild-type mice (CD19CreNotch2+/+). As expected, Notch2-deficient mice responded normally to challenge with T cell-dependent antigen NP-CGG but not T cell-independent antigen TNP-Ficoll, in agreement with the lack of MZ B cells in the mutant mice. PF4/heparin-specific antibodies produced by wild-type mice on a C57BL/6 background were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells from wild-type C57BL/6 mice were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular (FO), B cells adoptively transferred into B cell-deficient muMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells play a critical role in production of PF4/heparin-specific antibodies. Disclosures: Arepally: Teva Pharmaceuticals: Research Funding.

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