David Ginsburg

Albert Einstein College of Medicine, New York, New York, United States

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Publications (120)

  • Rami Khoriaty · Lesley Everett · Jennifer Chase · [...] · David Ginsburg
    [Show abstract] [Hide abstract] ABSTRACT: In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes.
    Article · Jun 2016 · Scientific Reports
  • [Show abstract] [Hide abstract] ABSTRACT: Background: Previous studies identified common variants at the ABO and VWF loci and unknown variants in a chromosome 2q12 linkage interval that contributed to the variation of plasma von Willebrand factor levels (VWF). While the association with ABO haplotypes can be explained by differential VWF clearance, little is known about the mechanisms underlying the association with VWF SNPs or with variants in the chromosome 2 linkage interval. VWF propeptide (VWFpp) and mature VWF are synthesized from the VWF gene and secreted at the same rate but have different plasma half-lives. Therefore, comparison of VWFpp and VWF association signals can be used to assess if the variants are primarily affecting synthesis/secretion or clearance. Methods: We measured plasma VWFpp levels and performed genome-wide linkage and association studies in 3,238 young and healthy individuals for whom VWF levels have been analyzed previously. Results and conclusions: Common variants in an intergenic region on 7q11 were associated with VWFpp. We demonstrate ABO serotype specific SNPs were associated with VWFpp levels in the same direction as VWF but with a much lower effect size. Neither the association at VWF nor the linkage on chromosome 2 previously reported for VWF was observed for VWFpp. Taken together, these results suggest that the major genetic factors affecting plasma VWF levels: variants at ABO, VWF and a locus on chromosome 2, operate primarily through their effects on VWF clearance. This article is protected by copyright. All rights reserved.
    Article · Jun 2016 · Journal of Thrombosis and Haemostasis
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    [Show abstract] [Hide abstract] ABSTRACT: During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp) deletion causing a frameshift in exon 27 of the Nbeal2 gene was identified. Though initially considered as a plausible thrombosis modifier, this Nbeal2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules. Mice homozygous for the Nbeal2 8 bp deletion (Nbeal2gps/gps) exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10-7). Nbeal2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted Nbeal2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains.
    Full-text Article · Mar 2016 · PLoS ONE
  • David Ginsburg
    Article · Aug 2015 · The Journal of clinical investigation
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    Colin A Kretz · Manhong Dai · Onuralp Soylemez · [...] · David Ginsburg
    [Show abstract] [Hide abstract] ABSTRACT: Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.
    Full-text Article · Jul 2015 · Proceedings of the National Academy of Sciences
  • [Show abstract] [Hide abstract] ABSTRACT: Binding to the von Willebrand factor (VWF) D'D3 domains protects factor VIII (FVIII) from rapid clearance. We applied single-particle electron microscopy (EM) analysis of negatively stained specimens to examine the architecture of D'D3 alone and in complex with FVIII. The D'D3 dimer ([D'D3]2) is comprised of two antiparallel D3 monomers with flexibly attached protrusions of D'. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D' domain while weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. Modeling the FVIII structure into the three-dimensional EM reconstructions of [D'D3]2-FVIII ternary and quaternary complexes indicates conformational rearrangements of the FVIII C domains compared to their disposition in the unbound bound state. These results illustrate the cooperative plasticity between VWF and FVIII that coordinate their high affinity interaction. Copyright © 2015 American Society of Hematology.
    Article · Jun 2015 · Blood
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    Karl C. Desch · Colin Kretz · Andrew Yee · [...] · David Ginsburg
    [Show abstract] [Hide abstract] ABSTRACT: Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2' and P11', for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13-VWF exosite interactions outside of VWF73.
    Full-text Article · Apr 2015 · PLoS ONE
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    Full-text Dataset · Mar 2015
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    [Show abstract] [Hide abstract] ABSTRACT: Analyses of genome-wide association study (GWAS) data have revealed that detectable genetic mosaicism involving large (>2 Mb) structural autosomal alterations occurs in a fraction of individuals. We present results for a set of 24,849 genotyped individuals (total GWAS set II [TGSII]) in whom 341 large autosomal abnormalities were observed in 168 (0.68%) individuals. Merging data from the new TGSII set with data from two prior reports (the Gene-Environment Association Studies and the total GWAS set I) generated a large dataset of 127,179 individuals; we then conducted a meta-analysis to investigate the patterns of detectable autosomal mosaicism (n = 1,315 events in 925 [0.73%] individuals). Restricting to events >2 Mb in size, we observed an increase in event frequency as event size decreased. The combined results underscore that the rate of detectable mosaicism increases with age (p value = 5.5 × 10(-31)) and is higher in men (p value = 0.002) but lower in participants of African ancestry (p value = 0.003). In a subset of 47 individuals from whom serial samples were collected up to 6 years apart, complex changes were noted over time and showed an overall increase in the proportion of mosaic cells as age increased. Our large combined sample allowed for a unique ability to characterize detectable genetic mosaicism involving large structural events and strengthens the emerging evidence of non-random erosion of the genome in the aging population. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
    Full-text Article · Mar 2015 · The American Journal of Human Genetics
  • Karl C. Desch · Qianyi Ma · Ayse Bilge Ozel · [...] · David Ginsburg
    Conference Paper · Dec 2014
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    [Show abstract] [Hide abstract] ABSTRACT: Plasminogen is the precursor of the serine protease plasmin, a central enzyme of the fibrinolytic system. Plasma levels of plasminogen vary by almost two-fold among healthy individuals, yet little is known about its heritability or genetic determinants in the general population. In order to identify genetic factors affecting the natural variation of plasminogen levels, we performed a genome-wide association study and linkage analysis in a sample of 3,456 young healthy individuals who participated in the Genes and Blood Clotting Study (GABC) or the Trinity Student Study (TSS). Heritability of plasminogen levels was 48.1% - 60.0%. Tobacco smoking and female sex were associated with higher levels of plasminogen. In the meta-analysis, eleven SNPs in two regions reached genome-wide significance (P < 5.0E-8). Of these, nine SNPs were near the PLG or LPA genes on Chr6q26, while two were on Chr19q13 and 5'-upstream of SIGLEC14. These 11 SNPs represented 4 independent signals and collectively explained 6.8% of plasminogen level variation in the study populations. The strongest association was observed for a non-synonymous SNP in the PLG gene (R523W). Individuals bearing an additional copy of this allele had an average of 13.4% decrease in plasma plasminogen level.
    Full-text Article · Sep 2014 · Blood
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    Rami Khoriaty · Matthew P Vasievich · Morgan Jones · [...] · David Ginsburg
    [Show abstract] [Hide abstract] ABSTRACT: Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease of ineffective erythropoiesis characterized by increased bi/multinucleated erythroid precursors in the bone marrow. CDAII results from mutations in SEC23B. The SEC23 protein is a core component of coat protein complex II-coated vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Though the genetic defect underlying CDAII has been identified, the pathophysiology of this disease remains unknown. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration, with this early mortality limiting evaluation of the adult hematopoietic compartment. We now report that mice with SEC23B deficiency restricted to the hematopoietic compartment survive normally and do not exhibit anemia or other CDAII characteristics. We also demonstrate that SEC23B-deficient hematopoietic stem cells (HSC) do not exhibit a disadvantage at reconstituting hematopoiesis when compared directly to wild-type HSC in a competitive repopulation assay. Secondary bone marrow transplants demonstrated continued equivalence of SEC23B-deficient and WT HSC in their hematopoietic reconstitution potential. The surprising discordance in phenotypes between SEC23B-deficient mice and humans may reflect an evolutionary shift in SEC23 paralog function and/or expression, or a change in a specific COPII cargo critical for erythropoiesis.
    Full-text Article · Jul 2014 · Molecular and Cellular Biology
  • Elizabeth J Adams · Xiao-Wei Chen · K Sue O'Shea · David Ginsburg
    [Show abstract] [Hide abstract] ABSTRACT: COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution.
    Article · May 2014 · Journal of Biological Chemistry
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    Andrew Yee · Robert D Gildersleeve · Shufang Gu · [...] · David Ginsburg
    [Show abstract] [Hide abstract] ABSTRACT: Plasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D'D3 domains (S764-P1247), either as a monomer or dimer, is sufficient to raise FVIII levels in Vwf-/- mice from a baseline of ~5-10% to ~50-100%. These results demonstrate that a fragment containing only ~20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D'D3 fragment fused at its C-terminus to the Fc segment of IgG1 results in markedly enhanced survival in the circulation (t1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf-/- mice. Although the VWF D'D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the co-transfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D'D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D'D3 and VWF D'D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D'D3/D'D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding.
    Full-text Article · May 2014 · Blood
  • [Show abstract] [Hide abstract] ABSTRACT: The primary cellular source of FVIII biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. LMAN1 is a cargo receptor in the early secretory pathway that is responsible for the efficient secretion of FV and FVIII to the plasma. Lman1 mutations result in combined deficiency of FV and FVIII, with levels of both factors reduced to ~10-15% of normal in human patients. We generated Lman1 conditional knockout mice to characterize the FVIII secretion profiles of endothelial cells and hepatocytes. We demonstrate that endothelial cells are the primary biosynthetic source of murine FVIII, and that hepatocytes make no significant contribution to the plasma FVIII pool. Utilizing RiboTag mice and polyribosome immunoprecipitation, we performed endothelial cell-specific mRNA isolation and qPCR analyses to confirm that endothelial cells highly express F8, and to explore the heterogeneity of F8 expression in different vascular beds. We demonstrate that endothelial cells from multiple, but not all, tissues contribute to the plasma FVIII pool in the mouse.
    Article · Apr 2014 · Blood
  • D. Motto · G. Levy · B. McGee · [...] · D. Ginsburg
    Article · Mar 2014 · Blood
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    [Show abstract] [Hide abstract] ABSTRACT: We previously reported the identification and development of novel inhibitors of streptokinase (SK) expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic screen. Although phenotypic screening is well-suited to identifying compounds that exert desired biological effects in potentially novel ways, it requires follow-up experiments to determine the macromolecular target(s) of active compounds. We therefore designed and synthesized several classes of chemical probes for target identification studies, guided by previously established structure-activity relationships. The probes were designed to first irreversibly photolabel target proteins in the intact bacteria, followed by cell lysis and click ligation with fluorescent tags to allow for visualization on SDS-PAGE gels. This stepwise, 'tag-free' approach allows for a significant reduction in molecular weight and polar surface area compared to full-length fluorescent or biotinylated probes, potentially enhancing membrane permeability and the maintenance of activity. Of the seven probes produced, the three most biologically active were employed in preliminary target identification trials. Despite the potent activity of these probes, specific labeling events were not conclusively observed due to a considerable degree of nonspecific protein binding. Nevertheless, the successful synthesis of potent biologically active probe molecules will serve as a starting point for initiating more sensitive methods of probe-based target identification.
    Full-text Article · Feb 2014 · Bioorganic & medicinal chemistry letters
  • Jordan A Shavit · David Ginsburg
    [Show abstract] [Hide abstract] ABSTRACT: This chapter covers hemophilias and other disordes of hemostasis, focusing on genetic factors and mutations associated with these disorders.
    Article · Dec 2013
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    [Show abstract] [Hide abstract] ABSTRACT: The interaction of protein C (PC) with the endothelial PC receptor (EPCR) enhances activated PC generation. We performed targeted gene sequencing of the PC gene (PROC) and EPCR genes (PROCR) in patients with unprovoked venous thromboembolism (VTE) to determine whether mutations that impair PC-EPCR interactions are associated with an increased risk of VTE. We sequenced exon 3 of PROC and exons 2 and 3 of PROCR (the exons that encode the protein-protein binding domains of PC and EPCR) in 653 patients with unprovoked VTE and in 627 healthy controls. Five single nucleotide variants, each in individual patients, were identified that result in abnormal PC (Arg9Cys, Val34Met, and Arg-1Cys) or abnormal EPCR proteins (Arg96Cys and Val170Leu). None of these single nucleotide variants were found in the controls. When the PC variants were expressed in human embryonic kidney 293 cells, all exhibited decreased synthesis, and 2 of the variants had reduced capacity for activated PC generation. When expressed on the surface of human embryonic kidney 293 cells, the EPCR variants showed reduced affinity for fluorescently labeled PC. In addition, the previously reported EPCR A3 haplotype, which promotes cellular shedding of EPCR, is over-represented in the patient group (P=0.001). This is the first targeted DNA sequencing analysis of PROC and PROCR in a large group of patients with unprovoked VTE. Our data suggest that mutations that impair PC-EPCR interactions may be associated with an increased risk of VTE.
    Full-text Article · Sep 2013 · Arteriosclerosis Thrombosis and Vascular Biology
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    Andrew Yee · Fen-Lai Tan · David Ginsburg
    [Show abstract] [Hide abstract] ABSTRACT: von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.
    Full-text Article · Sep 2013 · PLoS ONE

Publication Stats

6k Citations

Institutions

  • 2014
    • Albert Einstein College of Medicine
      New York, New York, United States
  • 2006
    • Harvard University
      Cambridge, Massachusetts, United States
  • 2003-2005
    • Howard Hughes Medical Institute
      Ашбърн, Virginia, United States
  • 1993
    • Concordia University–Ann Arbor
      Ann Arbor, Michigan, United States