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.

scholarly journals Homozygous STXBP1 variant causes encephalopathy and gain-of-function in synaptic transmission

Brain ◽  
2019 ◽  
Vol 143 (2) ◽  
pp. 441-451 ◽  
Author(s):  
Hanna C A Lammertse ◽  
Annemiek A van Berkel ◽  
Michele Iacomino ◽  
Ruud F Toonen ◽  
Pasquale Striano ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Synaptic Transmission ◽  
Protein Stability ◽  
Developmental Delay ◽  
Fold Increase ◽  
Null Mouse ◽  
Gain Of Function ◽  
Gene Encoding ◽  
Cellular Effects ◽  
Protein Levels

Abstract Heterozygous mutations in the STXBP1 gene encoding the presynaptic protein MUNC18-1 cause STXBP1 encephalopathy, characterized by developmental delay, intellectual disability and epilepsy. Impaired mutant protein stability leading to reduced synaptic transmission is considered the main underlying pathogenetic mechanism. Here, we report the first two cases carrying a homozygous STXBP1 mutation, where their heterozygous siblings and mother are asymptomatic. Both cases were diagnosed with Lennox-Gastaut syndrome. In Munc18-1 null mouse neurons, protein stability of the disease variant (L446F) is less dramatically affected than previously observed for heterozygous disease mutants. Neurons expressing Munc18L446F showed minor changes in morphology and synapse density. However, patch clamp recordings demonstrated that L446F causes a 2-fold increase in evoked synaptic transmission. Conversely, paired pulse plasticity was reduced and recovery after stimulus trains also. Spontaneous release frequency and amplitude, the readily releasable vesicle pool and the kinetics of short-term plasticity were all normal. Hence, the homozygous L446F mutation causes a gain-of-function phenotype regarding release probability and synaptic transmission while having less impact on protein levels than previously reported (heterozygous) mutations. These data show that STXBP1 mutations produce divergent cellular effects, resulting in different clinical features, while sharing the overarching encephalopathic phenotype (developmental delay, intellectual disability and epilepsy).

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.

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.

Surface expression of glycoprotein Ibα is dependent on glycoprotein Ibβ: evidence from a novel mutation causing Bernard-Soulier syndrome

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 532-539 ◽  
Author(s):  
Niamh Moran ◽  
Patricia A. Morateck ◽  
Adele Deering ◽  
Michelle Ryan ◽  
Robert R. Montgomery ◽  
...  
Keyword(s):  
Novel Mutation ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Confocal Imaging ◽  
Stable Cell Line ◽  
Platelet Glycoprotein ◽  
Gene Encoding ◽  
Platelet Surface ◽  
Platelet Receptor ◽  
Bernard Soulier Syndrome

Abstract Bernard-Soulier syndrome is a rare bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP) Ib-IX-V complex. The complex, which serves as a platelet receptor for von Willebrand factor, is composed of 4 subunits: GPIb, GPIbβ, GPIX, and GPV. We here describe the molecular basis of a novel form of Bernard-Soulier syndrome in a patient in whom the components of the GPIb-IX-V complex were undetectable on the platelet surface. Although confocal imaging confirmed that GPIb was not present on the platelet surface, GPIb was readily detectable in the patient's platelets. Moreover, immunoprecipitation of plasma with specific monoclonal antibodies identified circulating, soluble GPIb. DNA-sequence analysis revealed normal sequences for GPIb and GPIX. There was a G to A substitution at position 159 of the gene encoding GPIbβ, resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIbβ, together with wild-type GPIb and GPIX, demonstrated a failure of GPIX expression on the surface of HEK 293T cells. Similar results were obtained with Chinese hamster ovary  IX cells, a stable cell line expressing GPIb that retains the capacity to re-express GPIX. Thus, we found that GPIbβ affects the surface expression of the GPIb-IX complex by failing to support the insertion of GPIb and GPIX into the platelet membrane.

scholarly journals Phenotypic and Genotypic Characterization of Avian Escherichia coli O86:K61 Isolates Possessing a Gamma-Like Intimin

2002 ◽  
Vol 68 (10) ◽  
pp. 4932-4942 ◽  
Author(s):  
R. M. La Ragione ◽  
I. M. McLaren ◽  
G. Foster ◽  
W. A. Cooley ◽  
M. J. Woodward
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Wild Birds ◽  
Gene Encoding ◽  
Infantile Diarrhea ◽  
E Coli ◽  
Gene Coding ◽  
Transmission Electron ◽  
Specific Pathogen

ABSTRACT Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli O86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.

Bernard-Soulier Syndrome with Severe Bleeding: Absent Platelet Glycoprotein lb alpha Due to a Homozygous One-base Deletion

1996 ◽  
Vol 76 (05) ◽  
pp. 670-674 ◽  
Author(s):  
Chaoyang Li ◽  
Dominick N Pasquale ◽  
Gerald J Roth
Keyword(s):  
Stop Codon ◽  
Surface Membrane ◽  
Genetic Defect ◽  
Premature Stop Codon ◽  
Platelet Glycoprotein ◽  
Severe Bleeding ◽  
Giant Platelets ◽  
Platelet Disorder ◽  
Oligonucleotide Hybridization ◽  
Bernard Soulier Syndrome

SummaryBernard-Soulier syndrome is a rare congenital platelet disorder that affects a surface membrane adhesion receptor, glycoprotein (GP) Ib-V-IX. Both the genetic defects and the bleeding diatheses associated with the syndrome are heterogeneous due, in part, to the complexity of the involved receptor which consists of four different members, GPs: Ibα-Mr 143 K (contains the von Willebrand factor-binding site), Ibβ-Mr 22 K, V-Mr 83 K and IX-Mr 20 K. We studied a kindred that includes a 40 year-old man with severe Bernard-Soulier syndrome: life-threatening gastrointestinal bleeding, thrombocytopenia, giant platelets and absent ristocetin-dependent platelet aggregation. By Southern blotting, PCR amplification/sequencing, hetero-duplex analysis, and allele-specific oligonucleotide hybridization, the Ib-V-IX genes were analyzed, and the molecular genetic defect was defined as a one-base deletion in the GPIbα gene, involving an adenine of codon 19. The mutation, K19R, homozygous in the propositus and heterozygous in the available unaffected relatives, leads to a frame shift in codons 19-21 and a premature stop codon after codon 21. No functional GPIbα can be produced from the mutant allele, implying that the platelets of the affected patient lack all GPIbα. Within the spectrum of Bernard-Soulier syndrome, this patient’s disorder exemplifies a severe or “classic” extreme; an “experiment of Nature” that illustrates the effect of a complete deficiency of the ligand-binding chain (GPIbα) of the GPIb-V-IX receptor.

Sign in / Sign up

Export Citation Format

Share Document