Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2102-2107 ◽  
Author(s):  
Taisuke Kanaji ◽  
Susan Russell ◽  
Jerry Ware
Keyword(s):  
Mouse Model ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Chinese Hamster ◽  
Interleukin 4 ◽  
Platelet Glycoprotein ◽  
Severe Bleeding ◽  
Chimeric Receptor ◽  
Α Subunit ◽  
Bernard Soulier Syndrome

Abstract An absent platelet glycoprotein (GP) Ib-IX receptor results in the Bernard-Soulier syndrome and is characterized by severe bleeding and the laboratory presentation of macrothrombocytopenia. Although the macrothrombocytopenic phenotype is directly linked to an absent GP Ib-IX complex, the disrupted molecular mechanisms that produce the macrothrombocytopenia are unknown. We have utilized a mouse model of the Bernard-Soulier syndrome to engineer platelets expressing an α-subunit of GP Ib (GP Ibα) in which most of the extracytoplasmic sequence has been replaced by an isolated domain of the α-subunit of the human interleukin-4 receptor (IL-4Rα). The IL-4Rα/GP Ibα fusion is membrane expressed in Chinese hamster ovary (CHO) cells, and its expression is facilitated by the presence of human GP IX and the β-subunit of GP Ib. Transgenic animals expressing a chimeric receptor were generated and bred into the murine Bernard-Soulier syndrome–producing animals devoid of mouse GP Ibα but expressing the IL-4Rα/GP Ibα fusion sequence. The characterization of these mice revealed a 2-fold increase in circulating platelet count and a 50% reduction in platelet size when compared with platelets from the mouse model of the Bernard-Soulier syndrome. Immunoprecipitation confirmed that the IL-4Rα/GP Ibα subunit interacts with filamin-1 and 14-3-3ζ, known binding proteins to the GP Ibα cytoplasmic tail. Mice expressing the chimeric receptor retain a severe bleeding phenotype, confirming a critical role for the GP Ibα extracytoplasmic domain in hemostasis. These results provide in vivo insights into the structural elements of the GP Ibα subunit that contribute to normal megakaryocyte maturation and thrombopoiesis.

scholarly journals Impediment of Gpibα-Mediated Platelet Clearance By an Anti-Gpibβ Antibody Derivative

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1003-1003
Author(s):  
Wenchun Chen ◽  
Moriah Simone Wilson ◽  
Yingchun Wang ◽  
Francois Lanza ◽  
Renhao Li
Keyword(s):  
Life Span ◽  
Cho Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Chinese Hamster ◽  
Coiled Coil ◽  
Wild Type Mouse ◽  
Interleukin 4 ◽  
Wild Type ◽  
Α Subunit

Abstract Background: Glycoprotein (GP)Ib-IX complex plays a critical role in mediating platelet activation and platelet clearance. Recently, we identified the mechanosensory domain (MSD) in the GPIbα subunit, and demonstrated that unfolding of the MSD and subsequent exposure of the Trigger sequence (residues 473-483) therein activates GPIb-IX and induces rapid platelet clearance. This mechanism could explain acute thrombocytopenia induced by activated VWF, anti-GPIbα antibodies, neuraminidase, and ectodomain shedding of GPIbα. Consistently, platelets in IL4R-IbαTg mice, a transgenic strain in which the entire extracellular domain of human GPIbα except the Trigger sequence was replaced with that of the α-subunit of interleukin-4 receptor, exhibit constitutively more filopodia and are cleared much faster than the wild type. Previously, an anti-GPIbβ antibody RAM.1 was developed. RAM.1 significantly inhibits GPIb-IX-mediated filopodia formation and Ca 2+ signaling in platelets. In addition, it could inhibit GPIb-dependent thrombin generation. These results suggest that targeting GPIbβ could inhibit activation of GPIb-IX induced by MSD unfolding. Objectives: To investigate whether targeting GPIbβ with RAM.1 can impede rapid platelet clearance induced by exposed Trigger sequence and ameliorate related thrombocytopenia. Methods: Spontaneous filopodia in platelets and transfected Chinese hamster ovary (CHO) cells were visualized by fluorescence staining of actin and confocal microscopy. Images were quantified by ImageJ. Platelet signaling events, like P-selectin exposure, β-galactose exposure, and Ca 2+ influx, were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody. Results: CHO cells stably expressing the same mutant GPIb-IX complex in IL4R-IbαTg mouse platelets have been successfully obtained. Like IL4R-IbαTg platelets, these IL4R-IbαTg CHO cells exhibited spontaneous filopodia in the absence of any GPIbα ligands. RAM.1 could inhibit spontaneous filopodia formation in these CHO cells and IL4R-IbαTg platelets (Fig. 1, 2). Compared to wild-type mouse platelets, IL4R-IbαTg platelets constitutively exhibited increased P-selectin exposure, increased β-galactose exposure, and elevated intracellular Ca 2+, all of which could be inhibited by treatment of RAM.1 (Fig. 3). Recombinant RAM.1-GCN4 protein (rRAM.1-GCN4), in which the Fc region of RAM.1 heavy chain was replaced with the GCN4 coiled coil dimerizing sequence, has been generated and used as an alternative of the divalent RAM.1-Fab2. It retained the ability of RAM.1 antibody to inhibit GPIb-IX signaling. Injecting rRAM.1-GCN4 into IL4R-IbαTg mice dramatically improved the life span of endogenous IL4R-IbαTg platelets (Fig. 4). Conclusion: These results demonstrate that the exposed Trigger sequence is sufficient to activate GPIb-IX in transfected CHO cells, and that RAM.1 derivatives can impede GPIbα-mediated rapid platelet clearance. Targeting GPIbβ may be a novel approach to treat GPIb-related thrombocytopenia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Platelet glycoprotein IIb-IIIa (alpha IIb beta 3 integrin) confers fibrinogen- and activation-dependent aggregation on heterologous cells

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 369-376 ◽  
Author(s):  
MM Frojmovic ◽  
TE O'Toole ◽  
EF Plow ◽  
JC Loftus ◽  
MH Ginsberg
Keyword(s):  
Platelet Aggregation ◽  
Cho Cells ◽  
Synthetic Peptide ◽  
Molecular Mechanisms ◽  
Divalent Cations ◽  
Single Cells ◽  
Chinese Hamster ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Antibody 2G12

Abstract To analyze molecular mechanisms of platelet aggregation, we have studied the aggregation of Chinese hamster ovary (CHO) cells expressing between 1 and 4 x 10(5) recombinant human glycoprotein (GP) IIb-IIIa molecules per cell (A5 cells). These cells aggregated as measured by the disappearance of single cells during rotary agitation. Aggregation was dependent on the presence of extracellular fibrinogen (approximately 500 nmol/L) and divalent cations, and required prior activation of the GPIIb-IIIa. A synthetic peptide (GRGDSP) and monoclonal anti-GPIIb-IIIa antibody (2G12) that block platelet aggregation also blocked aggregation of these cells. Parent CHO cells or those expressing recombinant GPIIb-IIIa containing a point mutation that causes variant thrombasthenia both failed to aggregate when stimulated in the presence of fibrinogen. These data show that GPIIb- IIIa is the only unique platelet surface component required for aggregation.

Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4529-4538 ◽  
Author(s):  
Steve N. Georas ◽  
John E. Cumberland ◽  
Thomas F. Burke ◽  
Rongbing Chen ◽  
Ulrike Schindler ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Regulatory Elements ◽  
Jurkat Cells ◽  
Proximal Promoter ◽  
Interleukin 4 ◽  
Th2 Cells ◽  
Th Cells

Abstract The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4–secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4–activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.

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

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

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

Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4529-4538 ◽  
Author(s):  
Steve N. Georas ◽  
John E. Cumberland ◽  
Thomas F. Burke ◽  
Rongbing Chen ◽  
Ulrike Schindler ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Regulatory Elements ◽  
Jurkat Cells ◽  
Proximal Promoter ◽  
Interleukin 4 ◽  
Th2 Cells ◽  
Th Cells

The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4–secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4–activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.

scholarly journals RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia

Blood ◽  
2009 ◽  
Vol 114 (13) ◽  
pp. 2744-2752 ◽  
Author(s):  
Holly Edwards ◽  
Chengzhi Xie ◽  
Katherine M. LaFiura ◽  
Alan A. Dombkowski ◽  
Steven A. Buck ◽  
...  
Keyword(s):  
Cytosine Arabinoside ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Pi3 Kinase ◽  
Akt Pathway ◽  
Chemotherapy Response ◽  
Acute Leukemias ◽  
Α Subunit ◽  
Acute Megakaryocytic Leukemia

Abstract RUNX1 (AML1) encodes the core binding factor α subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P < .001) lower than that in non-DS AMkL cases. Short hairpin RNA knockdown of RUNX1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivity to cytosine arabinoside, accompanied by significantly decreased expression of PIK3CD, which encodes the δ catalytic subunit of the survival kinase, phosphoinositide 3 (PI3)–kinase. Transcriptional regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease.

Correction of Murine Bernard Soulier Syndrome by Lentivirus-Mediated Gene Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 552-552
Author(s):  
Sachiko Kanaji ◽  
Erin L. Kuether ◽  
Scot A. Fahs ◽  
Jocelyn A. Schroeder ◽  
Jerry Ware ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Platelet Glycoprotein ◽  
Dna Flow Cytometry ◽  
Severe Bleeding ◽  
Hematopoietic Stem ◽  
Platelet Counts ◽  
Bernard Soulier Syndrome

Abstract Abstract 552 Bernard Soulier Syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib/IX complex. The main treatment for BSS is platelet transfusion but it is often limited to severe bleeding episodes or surgical interventions due to the risk of developing allo-immunization. Ware and colleagues have developed a murine model of BSS by targeting GPIbα (GPIbαnull), and have shown that the BSS phenotype was rescued by transgenic expression of hGPIbα. We have previously reported successful expression of human GPIbα in human megakaryocytes using a lentiviral vector (LV) encoding hGPIbα under the transcriptional control of integrin aIIb promoter (2bIbα). In this study, we examined the efficacy of this strategy for the gene therapy of BSS using GPIbαnull as a murine model of BSS. GPIbαnull hematopoietic stem cells (HSC) transduced with 2bIbα LV were transplanted into lethally irradiated GPIbαnull littermates. After bone marrow (BM) reconstitution, mice were analyzed. The presence of 2bIbα transgene in recipients was confirmed by PCR amplification of white blood cell derived genomic DNA. Flow cytometry demonstrated that 84.5% ± 9.5% (n = 9) of platelets expressed hGPIbα at 6 weeks after transplantation and stable expression was maintained through the entire observation period of 7 months. Immunofluorescent confocal microscopy demonstrated that transgene protein was expressed on the cell surface of transduced platelets. Tail bleeding times were corrected to normal levels in the GPIbαnull recipients who received LV transduced GPIbαnull HSC (2.3 ± 2.9 minutes, n = 9). On the other hand, recipients who received untransduced GPIbαnull HSC exhibited prolonged bleeding times (8.8 ± 2.4 minutes, n = 4) that were similar to GPIbαnull mice. Macrothrombocytopenia improved with significantly increased platelet counts and decreased platelet sizes in LV transduced GPIbαnull HSC recipients compared to untransduced GPIbαnull HSC recipients (platelet counts; 4.9 ± 1.3×105/μ l, n = 9 vs. 1.8 ± 0.1×105/μ l, n = 4, and mean platelet volume; 6.9 ± 0.7 fL, n = 9 vs. 9.3 ± 0.1 fL, n = 4, respectively). As expected, expression levels of hGPIbα correlated with platelet counts and inversely correlated with the platelet size. Immunoprecipitation followed by Western blot analysis showed that hGPIbα expressed on platelets associated with endogenous murine GPIbβ and GPIX. No antibody to hGPIbα was detected in these recipients. Furthermore, BM mononuclear cells from the primary recipients were transplanted into the secondary GPIbαnull recipients and the results showed sustained expression of hGPIbα leading to the correction of bleeding phenotype as well as macrothrombocytopenia. These results demonstrate that lentivirus mediated gene transfer can provide sustained phenotypic correction of murine BSS, indicating that this approach may be a promising strategy for gene therapy of BSS in human. Disclosures: No relevant conflicts of interest to declare.

Quaternary Organization of GPIb-IX Complex and Insights Into Bernard-Soulier Syndrome Revealed by the Crystal Structures of GPIbβ Ectodomain and a GPIbβ/GPIX Chimera

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2195-2195
Author(s):  
Paul A. McEwan ◽  
Wenjun Yang ◽  
Katherine H. Carr ◽  
Xi Mo ◽  
Xiaofeng Zheng ◽  
...  
Keyword(s):  
Structural Model ◽  
Conflicts Of Interest ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Surface ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Ovary Cells ◽  
Bernard Soulier Syndrome

Abstract Abstract 2195 Platelet glycoprotein (GP)Ib-IX receptor complex contains three subunits, GPIbα, GPIbβ and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome (BSS). We have crystallized the GPIbβ ectodomain (GPIbβE) and determined the structure to reveal a single leucine-rich repeat with N- and C-terminal disulfide bonded capping regions. The central region of the structure can be divided into concave parallel β-sheet and convex loops. The crystal structure of a GPIbβE/GPIXE chimera that contains three non-continguous convex loops of GPIX and retains a GPIbβ-binding site of GPIX (Mo et al. J. Thromb. Haemost. 7:1533–40, 2009) was also determined. The chimera, but not GPIbβE, forms a homotetramer in the crystal, revealing a quaternary interface between GPIbβ and GPIX ectodomains. Central to this interface is residue Tyr106 from GPIbβ that inserts into a shallow and largely hydrophobic pocket generated by two convex loops from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIbβ and GPIX in the full-length complex. Eight GPIbβ missense mutations identified from BSS patients were examined in transiently transfected Chinese hamster ovary cells for changes to the GPIb-IX complex surface expression. Six of the eight mutations lead to secretion defect and/or misfolding of GPIbβE. In contrast, the other two mutations, A108P and P74R, were found to maintain normal secretion and folding of GPIbβE but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIbβE interface with GPIX indicates that residues Ala108 and Pro74 in GPIbβ are located at the GPIbβE/GPIXE interfaces. Based on the tetrameric arrangement of the chimera structure, we propose a structural model for the GPIb-IX complex that embodies its organizing principles and helps to provide mechanistic insights on its assembly, function and regulation. Disclosures: No relevant conflicts of interest to declare.

Genetic deletion of mouse platelet glycoprotein Ibβ produces a Bernard-Soulier phenotype with increased α-granule size

Blood ◽  
2004 ◽  
Vol 104 (8) ◽  
pp. 2339-2344 ◽  
Author(s):  
Kazunobu Kato ◽  
Constantino Martinez ◽  
Susan Russell ◽  
Paquita Nurden ◽  
Alan Nurden ◽  
...  
Keyword(s):  
Fold Increase ◽  
Granule Size ◽  
Platelet Glycoprotein ◽  
Severe Bleeding ◽  
Gene Encoding ◽  
Protein Levels ◽  
Transmission Electron ◽  
Presynaptic Protein ◽  
Bernard Soulier Syndrome

Abstract Here we report the characterization of a mouse model of the Bernard-Soulier syndrome generated by a targeted disruption of the gene encoding the glycoprotein (GP) Ibβ subunit of the GP Ib-IX complex. Similar to a Bernard-Soulier model generated by disruption of the mouse GP Ibα subunit, GP IbβNull mice display macrothrombocytopenia and a severe bleeding phenotype. When examined by transmission electron microscopy, the large platelets produced by a GP IbβNull genotype revealed α-granules with increased size as compared with the α-granules from control mouse platelets. Data are presented linking the overexpression of a septin protein, SEPT5, to the presence of larger α-granules in the GP IbβNull platelet. The SEPT5 gene resides approximately 250 nucleotides 5′ to the GP Ibβ gene and has been associated with modulating exocytosis from neurons and platelets as part of a presynaptic protein complex. Fusion mRNA transcripts present in megakaryocytes can contain both the SEPT5 and GP Ibβ coding sequences as a result in an imperfect polyadenylation signal within the 3′ end of both the human and mouse SEPT5 genes. We observed a 2- to 3-fold increase in SEPT5 protein levels in platelets from GP IbβNull mice. These results implicate SEPT5 levels in the maintenance of normal α-granule size and may explain the variant granules associated with human GP Ibβ mutations and the Bernard-Soulier syndrome.

Sign in / Sign up

Export Citation Format

Share Document