In Vitro and In Vivo Mouse Models of the Type 1 Von Willebrand Disease Mutations Y1584C and R1205H.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 26-26
Author(s):  
Cynthia M. Pruss ◽  
Kate Sponagle ◽  
Kimberly Laverty ◽  
Carol A. Hegadorn ◽  
Yvette Chirinian ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Transient Transfection ◽  
Von Willebrand Disease ◽  
Wild Type ◽  
Time Points ◽  
High Molecular Weight Material ◽  
Von Willebrand

Abstract Abstract 26 Introduction: Type 1 von Willebrand disease (VWD) is caused by mutations that result in moderate decreases in VWF (5-50% of normal levels) and a mild bleeding phenotype. The VWF missense mutation Y1584C is associated with mildly decreased VWF:Ag levels, increased ADAMTS13 cleavage, as well as a possible increase in clearance. The Vicenza mutation, R1205H, exhibits a more severe phenotype (VWF:Ag ∼10%), as well as accelerated clearance. However, extensive controlled in vitro and in vivo investigation of these mutations has yet to be described. In this study, we examine both Y1584C and R1205H in comparison to wild type VWF using in vitro and in vivo strategies employing both human and mouse VWF. Methods: Recombinant murine and human VWF and ADAMTS13 were produced via transient transfection in HEK293T cells in serum free OPTIMEM for 72 hours. Full length ADAMTS13 digests were performed in a Tris-Urea system and analyzed via multimer pattern. VWF115 digests were performed in an ELISA based assay. Hydrodynamic injections were performed using 100 μg wild type (WT) or mutant ET-mVWF plasmid DNA in Ringer's solution in 7-9 week old C57Bl6 VWF knockout mice. Mice were sampled at days 2, 5, 8, and then weekly. Mouse plasma was analyzed for CBCs, VWF:Ag, and VWF multimer structure. Results: In the HEK293T transient transfection system, secreted mutant protein was similar to that of wild type recombinant protein, with high molecular weight material present. ADAMTS13 digestion of full length recombinant Y1584C versus wild type showed no statistical difference: 50% cleavage for hVWF WT 1.78U hADAMTS13, hVWF Y1584C 1.67 U, P=0.58; mVWF WT 0.32 U mADAMTS13; Y1584C 0.42 U, P=0.11. In contrast, the Y1584C substitution in the hVWF115 construct required 40% less hADAMTS13 to effect equivalent cleavage (WT 0.087±0.014, 4; Y1584C 0.052±0.005, 8, mean U/ml ADAMTS13±SEM, N, P=0.013). Mouse ADAMTS13 cleavage of mVWF115 was also increased 20% for Y1584C (WT: 0.20±0.06, 4; Y1584C: 0.16±0.01, 4, P=0.014). Hydrodynamic injection caused no adverse events in any animals. CBC values were not statistically significantly different between wild type and mutants. Initial high VWF:Ag values were similar for wild type VWF (25.6±2.9, 15, mean U/ml±SEM, n) and Y1584C (27.2±5.0, 11), but R1205H levels were 36% lower (16.3±2.1, 10). At 14 days, WT VWF:Ag was 5.33±1.13, 15, with R1205H (1.73±0.44, 12) and Y1584C (1.84±0.59, 11) VWF:Ag levels being 68% and 65% lower, respectively. R1205H continued to remain approximately 40% of WT values for the next three weeks, while Y1584C continued to decrease, dropping to 15% on day 21 and 8% on day 28, compared to the wild type values at these time points. The R1205H mutation showed no significant difference in multimer structure defined by observed number of bands (days 2 to 42, mean difference -0.49, P>0.05) to wild type VWF. In contrast, Y1584C had a significant decrease in band number (3.38, P<0.001). Conclusions: This study demonstrates that these two type 1 VWD mutations have a strong observable effect in the VWF knockout mouse model. R1205H exhibits a large decrease in VWF:Ag levels at all measured time points, but no alteration in multimer structure. Y1584C, in contrast, shows a loss of high molecular weight material, and an initially high VWF:Ag level that rapidly decays from day 14 onward, suggesting increased ADAMTS13 cleavage and increased clearance. In addition, in vitro ADAMTS13 testing shows that Y1584C responds differently in the two assay systems with only the VWF115 assay showing significant increases in ADAMTS13-mediated cleavage. Disclosures: No relevant conflicts of interest to declare.

Partial in Vivo FVIII Stabilization by VWF Fragments

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 15-15
Author(s):  
Andrew Yee ◽  
Austin N Oleskie ◽  
Robert D Gildersleeve ◽  
Colin A Kretz ◽  
Min Su ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Von Willebrand Disease ◽  
Post Injection ◽  
Time Points ◽  
Platelet Poor Plasma ◽  
Plasma Factor ◽  
Recombinant Fviii ◽  
Von Willebrand

Abstract Abstract 15 Plasma factor VIII (fVIII) circulates in complex with von Willebrand factor (VWF) and is rapidly cleared in the absence of VWF. Previous in vitro studies have 1) localized the fVIII-binding region of VWF to the N-terminus, comprised of the contiguous D' and D3 domains and 2) observed reduced affinity for fVIII upon alterations to the tertiary structure of VWF. To gain insight into the structure-function of VWF for fVIII stabilization, we tested VWF fragments for in vivo fVIII stabilization and investigated the architecture of a VWF fragment in complex with fVIII. For the in vivo study, fragments of murine Vwf cDNA were cloned into the hepatic-specific expression vector, pLIVE, and modified to fuse tandem E and FLAG tags to the C-terminus. The following VWF fragments were expressed in vivo by hydrodynamic tail vein injection into Vwf−/− mice: 1) a monomer of the VWF D'D3 domains (monoD'D3; M1-C22, S764-P1274); 2) a truncation of monomeric D'D3 (truncD'D3; M1-C22, S764-R1035); 3) dimers of D'D3 (diD'D3; M1-P1274); 4) multimers of D'D3 (multiD'D3; M1-P1274, G2713-K2813); 5) dimers of mature VWF subunits (DPro, M1-C22, S764-K2813); or 6) full length, multimeric VWF (wtVWF, M1-K2813). Expression of all VWF fragments persisted throughout the period of observation (4 weeks) with peak antigenic levels at 1 or 3 days post-injection. Prolonged elevation of plasma fVIII activity (fVIIIa) from ∼10% to ∼50–200% were observed (100% defined as the fVIIIa level of pooled platelet poor plasma from 10 wild type C57BL/6 mice) for all but the truncated monomer of D'D3 (Figure 1). Significantly increased fVIIIa levels (p<0.05, relative to pre-injection) were first observed at 1 day, peaked at 3 days, and persisted for the duration of observation. A minimal VWF fragment (S764-R1035, truncD'D3) reported to bind fVIII in vitro significantly increased plasma fVIIIa to 34% only at 3 days post-injection. Clearance of VWF fragments from circulation were determined from injections of pooled platelet poor plasma containing recombinant VWF fragments derived from hydrodynamically injected mice into naïve Vwf−/− mice. Nonlinear regression estimated the half-life for monoD'D3 (3.4hr), diD'D3 (2.1hr), multiD'D3 (2.3hr), DPro (2.8hr), and wtVWF (3.5hr). To examine how dimers of D'D3 bind fVIII, diD'D3 from HepG2 conditioned media was purified either alone or with recombinant fVIII, and negative stained samples were visualized by electron microscopy (EM). Single-particle EM analysis revealed that each subunit of the dimer binds 1 fVIII molecule. 3D EM reconstructions indicate that the light chain of fVIII directly interacts with, and potentially induces torsion in the flexible D'D3 domains of VWF. Together, these results emphasize the importance of VWF's tertiary structure in fVIII stabilization and that the N-terminal D'D3 alone is sufficient to support fVIII survival in vivo. These findings could lead to improved methods of recombinant fVIII production and the development of novel approaches to treatment for hemophilia and von Willebrand disease. Figure 1. fVIIIa of hydrodynamically injected mice at indicated time points. Figure 1. fVIIIa of hydrodynamically injected mice at indicated time points. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

scholarly journals Characterization of Type 2N Von Willebrand Disease Mutations Using Ιn Vitro and Ιn Vivo Mouse Models

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 471-471
Author(s):  
Laura L Swystun ◽  
Ilinca Georgescu ◽  
Meghan Deforest ◽  
Mia Golder ◽  
Kate Sponagle ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Compound Heterozygous ◽  
Wild Type ◽  
Binding Ability ◽  
Von Willebrand ◽  
Deficient Mice

Abstract Introduction: Von Willebrand factor (VWF) is a multimeric glycoprotein that serves as the carrier for the essential coagulation cofactor, factor VIII (FVIII). Both plasma levels of VWF and its FVIII-binding ability can influence plasma levels of FVIII. Type 2N von Willebrand disease (VWD) is associated with a reduced binding affinity of VWF for FVIII, resulting in accelerated proteolysis and clearance of FVIII (plasma levels 5 – 30% of normal). Type 2N VWD is a recessive trait and patients are either homozygous or compound heterozygous for 2N alleles. We hypothesize that type 2N VWD mutations can alter the expression and FVIII-binding ability of VWF. In these studies, we characterize three type 2N VWD mutations in vitro and in a murine model. R854Q (20-30% FVIII) is the most common 2N allele and is associated with a mild phenotype, while R816W (<10% FVIII) is associated with a severe phenotype. The R763A mutation inhibits propeptide cleavage that likely sterically interferes with the FVIII-binding ability of VWF. Methods: Type 2N VWD mutations were generated in the murine VWF cDNA. Heterologous VWF synthesis/secretion was characterized in vitro using HEK 293T cells and in vivo using hydrodynamic gene transfer of the murine VWF cDNA into VWF deficient mice. Binding of FVIII to type 2N variants was assessed in vitro using a solid phase binding assay and in vivo in VWF deficient mice by a FVIII chromogenic activity assay. Results: In HEK 293 T cells, biosynthesis of type 2N VWD variants was not significantly different from wild type VWF while secretion of all type 2N VWD variants was decreased relative to wild type: R763A (66%, p=0.0043), R816W (53%, p=0.0004), R854Q (4%, p<0.0001). Immunofluorescent staining of transfected HEK 293 cells demonstrated impaired pseudo-Weibel Palade body formation for the R854Q variant. Western blot analysis under denaturing conditions demonstrated that approximately 50% of the secreted R763A protein remained attached to the propeptide. Multimeric profiles of plasma-derived type 2N VWD mutants were normal. In vitro binding of plasma-derived murine type 2N VWD mutants to recombinant human FVIII was reduced relative to wild type VWF: R763A (56%, p=0.0009), R816W (10%, p<0.0001), R854Q (46%, p=0.0002). Type 2N VWD mutants were expressed alone or in a compound heterozygous state (R816W/R854Q) in VWF deficient mice. A trend of lower VWF:Ag levels were observed for type 2N VWD mutants relative to wild type (average 4.8 U/mL) after 14 days: R763A (35.7%), R816W (53.1%), R854Q (21.3%), except for compound heterozygous condition R816W/R854Q (103%). Plasma levels of FVIII:C are significantly reduced in VWF deficient mice (15-20% of normal). We measured the ability of hydrodynamically expressed type 2N VWD mutants to stabilize endogenous FVIII:C in VWF deficient mice. Hepatic expression of wild type VWF stabilized endogenous plasma FVIII:C, resulting in a significant increase in FVIII:C after 14 days (7.7-fold increase above baseline, p=0.0002). For the type 2N VWD mutants, variable partial stabilization of endogenous FVIII:C was observed relative to baseline: R763A (4.7-fold increase, p=0.01), R816W (1.2-fold decrease, p=0.04), R816W/R854Q (4.8-fold increase, p<0.0001), R854Q (2.1-fold increase, p=0.06). The correlation coefficient between VWF:Ag and FVIII:C was assessed for samples with VWF:Ag between 0.5-10 U/mL. Correlation between wild type VWF expression and FVIII:C was highly positive (r2=0.85, slope=189.5 ± 15.7, p<0.0001). Correlation between VWF:Ag and FVIII:C for mice expressing type 2N VWD mutants was variable: R763A (r2=0.89, slope=235.3 ± 18.15, p<0.0001), R816W (r2=0.591, slope=0.96 ± 2.8, p=0.7433), R816W/854Q (r2=0.72, slope=91.32 ± 10.64, p<0.0001) and R854Q (r2=0.705, slope=156.7 ± 24.4, p=0.0002). The slopes for R816W (p<0.0001) and R816W/R854Q (p=0.009) mutants were significantly different from wild type, suggesting impaired FVIII-stabilization in vivo. Conclusion: Expression of the type 2N VWD severe mutant R816W or the compound heterozygous R816W/R854Q mutant can recapitulate type 2N VWD in a murine model. Type 2N VWD mutations are associated with impaired secretion of VWF and/or decreased binding and stabilization of endogenous FVIII. Disclosures No relevant conflicts of interest to declare.

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2339-2345 ◽  
Author(s):  
Wolf Achim Hassenpflug ◽  
Ulrich Budde ◽  
Tobias Obser ◽  
Dorothea Angerhaus ◽  
Elke Drewke ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Von Willebrand ◽  
A2 Domain ◽  
The Impact ◽  
Willebrand Factor ◽  
High Molecular Weight Multimers

Abstract Classical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of high-molecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.

Human von Willebrand Disease Mutations in Mouse von Willebrand Factor Alter Its Cleavage by ADAMTS13.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2708-2708
Author(s):  
Cynthia M. Pruss ◽  
Carol A. Hegadorn ◽  
Colleen R.P. Notley ◽  
Rouzbeh Chegeni ◽  
Aly S. Dhala ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Disease ◽  
Full Length ◽  
P Value ◽  
Wild Type ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers in plasma is regulated by the specific VWF cleaving protease, ADAMTS13, that cleaves VWF at the Y1605-M1606 bond in the VWF A2 domain. The adhesive properties of VWF is directly related to the multimer size, with loss of high molecular weight VWF leading to the bleeding phenotype in Type 2A von Willebrand disease (VWD) and ultra high molecular weight VWF multimers observed in the prothrombotic state of thrombotic thrombocytopenic purpura (TTP). VWF mutations leading to multimer changes have not been examined in an animal model. Although the human and mouse VWF genes are highly conserved, mutations in the two genes have not been compared experimentally. To that end, we have made mutations in the mouse VWF (mVWF) cDNA to compare the ADAMTS13 cleavage patterns seen with human VWF mutations in two in vitro assays. Recombinant multimerized full-length mVWF was digested with different concentrations of recombinant mouse ADAMTS13 (mADAMTS13) and 1.5M urea, and analyzed via multimer migration distance. The GST and histidine-tagged G1554-T1668 A2 domain region of mVWF (mVWF115) was assayed via ELISA as follows: the mVWF115 was bound to anti-GST coated plates, digested with mADAMTS13, and intact mVWF115 detected via HRP-labeled anti-histidine tag antibody. We examined R1597W (VWD Type 2A), R1306W, (VWD Type 2B), Y1584C (VWD Type 1), as well as two previously described changes that drastically lower VWF cleavage, Y1605A/M1606A, and D1614A/E1615A/K1617A. Y1584C showed a statistically insignificant 41% decrease in cleavage in the full-length assay, but a 20% increase in the mVWF115 assay. Conversely, R1597W had a 64% decrease in the full-length assay, but a 28% increase in the mVWF115 assay. Y1605A/M1606A showed a dramatic loss of cleavage, with no observable loss of high molecular weight multimers at 32 units/ml of mADAMTS13, and a 126-fold higher mADAMTS13 concentration than wild type in the mVWF115 assay. This contrasted with the 8-fold increase observed with D1614A/E1615A/K1617A. The R1306W A1 domain mutation showed a significant increase in cleavage, with 63% less ADAMTS13 necessary in the full-length assay. The triple mutant R1306W/Y1605A/M1606A did not cleave at the highest concentration, 32 U/ml, similar to that of Y1605A/M1606A, showing that the R1306W mutation was not able to increase cleavage of the Y1605A/M1606A change. These results show a strong correlation between the mouse mutations described here and previously reported human VWF mutations in their susceptibility to ADAMTS13 cleavage. These results provide a rationale for the development of mouse models of type 1, 2A, and 2B VWD, as well as mVWF mutations that might lead to a prothrombotic state similar to TTP in humans. mADAMTS13 Concentration for 50% mVWF Cleavage ADAMTS13 Concentration (Units/ml) Relative mADAMTS13 Concentration (to Wild Type) mVWF Multimer (n=1) mVWF115 (n=4) mVWF Multimer (n=1) mVWF115 (n=4) *: P value <0.05, **: P Value < 0.001 in 2 tailed T test. Wild Type 0.301 0.197 1 1 Y1584C 0.423 0.157* 1.41 0.80* R1597W 0.109** 0.253* 0.36** 1.28* Y1605A/M1606A >32** 24.8** >100** 126** D1614A/E1615A/K1617A (−) 1.58** (−) 8.0** R1306W 0.112** (−) 0.37** (−) R1306W/Y1605A/M1606A >32** (−) >100** (−)

A randomised pilot trial of the anti-von Willebrand factor aptamer ARC1779 in patients with type 2b von Willebrand disease

2010 ◽  
Vol 104 (09) ◽  
pp. 563-570 ◽  
Author(s):  
Petra Paulinska ◽  
Petra Jilma-Stohlawetz ◽  
James Gilbert ◽  
Renta Hutabarat ◽  
Paul Knöbl ◽  
...  
Keyword(s):  
Pilot Trial ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
Double Blind ◽  
Clinical Trial Registration ◽  
Maximal Increase ◽  
Double Blind Design ◽  
Von Willebrand

SummaryDesmopressin aggravates thrombocytopenia in type 2B von Willebrand disease (VWF type 2B) by release of large and hyper-adhesive von Wille-brand Factor (VWF) multimers. This pilot study investigated whether the anti-VWF aptamer ARC1779 can prevent desmopressin-induced thrombocytopenia and interferes with the excessive VWF turnover in patients with VWF type 2B. Concentration effect curves of ARC1779 were established for five patients in vitro and two patients with VWF type 2B were treated by infusion of ARC1779, desmopressin, or their combination in a randomised, controlled, double-blind design. ARC1779 concentrations in the range of 1–3 μg/ml blocked free A1 domain binding sites by 90% in vitro. In vivo, desmopressin alone induced a profound (-90%) drop in platelet counts in one of the patients. ARC1779 (4–5 μg/ml) completely inhibited VWF A1 domains and prevented this desmopress-in-induced platelet drop. Desmopressin alone increased VWF antigen two- to three-fold, accompanied by concordant changes in VWF Ristocetin cofactor activity (RCo) and coagulation factor VIII activity. ARC1779 substantially enhanced the desmopressin-induced maximal increase in these parameters, and improved multimer patterns. No treatment related adverse events were observed and no bleeding occurred despite marked thrombocytopenia. These data provide first proof of concept in humans and evidence that ARC1779 is a potent inhibitor of VWF. ARC1779 prevented the rapid consumption of VWF multimers together with agglutinated platelets that occurred in response to desmopressin challenge in patients with VWD type 2B.Clinical Trial registration number: NCT00632242.

scholarly journals Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo

2003 ◽  
Vol 77 (2) ◽  
pp. 1382-1391 ◽  
Author(s):  
Michiko Tanaka ◽  
Hiroyuki Kagawa ◽  
Yuji Yamanashi ◽  
Tetsutaro Sata ◽  
Yasushi Kawaguchi
Keyword(s):  
Vero Cells ◽  
Infectious Molecular Clone ◽  
Wild Type ◽  
Molecular Clone ◽  
Viral Genes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT In recent years, several laboratories have reported on the cloning of herpes simplex virus type 1 (HSV-1) genomes as bacterial artificial chromosomes (BACs) in Escherichia coli and on procedures to manipulate these genomes by using the bacterial recombination machinery. However, the HSV-BACs reported so far are either replication incompetent or infectious, with a deletion of one or more viral genes due to the BAC vector insertion. For use as a multipurpose clone in research on HSV-1, we attempted to generate infectious HSV-BACs containing the full genome of HSV-1 without any loss of viral genes. Our results were as follows. (i) E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAC flanked by loxP sites was inserted into the intergenic region between UL3 and UL4 was constructed. (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii) The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by coinfection of Vero cells with YK304 and a recombinant adenovirus, AxCANCre, expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus, the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulence in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics, and a recombinant virus carrying amino acid substitutions in both copies of the α0 gene was generated. pYEbac102 will have multiple applications to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.

A novel von Willebrand disease–causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 560-568 ◽  
Author(s):  
Simon Allen ◽  
Adel M. Abuzenadah ◽  
Joanna Hinks ◽  
Joanna L. Blagg ◽  
Turkiz Gursel ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Family Members ◽  
Amino Acid Position ◽  
Von Willebrand Disease ◽  
Molecular Defect ◽  
Wild Type ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.

Abstract 165: The 18 kDa FGF-2 Prevents the Doxorubicin-induced Cardiac Damage and Amp-activated Kinase Activation, in vitro and in vivo

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Navid Koleini ◽  
Jon Jon Santiago ◽  
Barbara E Nickel ◽  
Robert Fandrich ◽  
Davinder S Jassal ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cell Death ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Wild Type ◽  
Rat Cardiomyocytes ◽  
Ventricular Ejection Fraction ◽  
Compound C

Introduction: Protection of the heart from chemotherapeutic (Doxorubicin, DOX) drug-induced toxicity is a desirable goal, to limit side effects of cancer treatments. DOX toxicity has been linked to the activation (phosphorylation) of the AMP-activated kinase, AMPK. The 18 kDa low molecular weight isoform of fibroblast growth factor 2 (Lo-FGF-2) is a known cardioprotective and cytoprotective agent. In this study we have tested the ability of Lo-FGF-2 to protect from DOX-induced damage in rat cardiomyocytes in vitro, and in transgenic mouse models in vivo, in relation to AMPK activation. Methods: Rat neonatal cardiomyocytes in culture were exposed to DOX (0.5 μM) in the presence or absence of pre-treatment Lo-FGF-2 (10 ng/ml). Compound C was used to block phosphorylation (activity) of AMPK. Levels of cell viability/death (using Calcein-AM/Propidium iodide assay), phospho -and total AMPK, and apoptotic markers such as active caspase 3 were analyzed. In addition, transgenic mice expressing only Lo-FGF2, and wild type mice, expressing both high molecular weight (Hi-FGF2) as well as Lo-FGF2 were subjected to DOX injection (20 mg/kg, intraperitoneal); echocardiography was used to examine cardiac function at baseline and at 10 days post-DOX. Results: DOX-induced cell death of cardiomyocytes in culture was maximal at 24 hours post-DOX coinciding with significantly increased in activated (phosphorylated) AMPK. Compound C attenuated DOX-induced cardiomyocyte loss. Pre-incubation with Lo-FGF-2 decreased DOX induced cell death, and also attenuated the phosphorylation of AMPK post-DOX. Relative levels of phospho-AMPK were lower in the hearts of Lo-FGF2-expressing male mice compared to wild type. DOX-induced loss of contractile function (left ventricular ejection fraction and endocardial velocity) was negligible in Lo-FGF2-expressing mice but significant in wild type mice. Conclusion: Lo-FGF-2 protects the heart from DOX-induced damage in vitro and in vivo, by a mechanism likely involving an attenuation of AMPK activity.

Reduced Von Willebrand Factor Secretion Is Associated with Loss of Weibel-Palade Formation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 541-541
Author(s):  
Giancarlo Castaman ◽  
Sofia Helene Giacomelli ◽  
Paula M. Jacobi ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Negative Impact ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Wild Type ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 541 Background. Von Willebrand Disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes patients with quantitative plasma VWF deficiency with normal VWF structure and function. Aim of the study. We report three different novel type 1 VWF mutations (A1716P, C2190Y and R2663C) which although located in different VWF domains are associated with reduced secretion and lack of formation of Weibel-Palade body-like granules. Methods. Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding, and GpIb binding assessed in comparison to wild-type VWF. Furthermore, expression was also examined in HEK293 cells that form Weibel-Palade body (WPB)-like granules when transfected with wt VWF. Results. The multimer analysis of plasma VWF was compatible with type 1 VWD. The results of 3 different expression experiments showed a slightly reduced VWF synthesis and drastically impaired secretion into the medium with homozygous expression. In HEK293 cells, homozygous A1716P and C2190Y VWF variants failed to form WPB-like granules, while R2663C was capable of forming granules, but had fewer cells with granules and more with ER-localized VWF. Heterozygous expression of A1716P and C2160Y VWF variants had a negative impact on wild-type VWF and WPB-like granules were observed in transfected cells. Conclusions. Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations can be associated with inability to form endothelial Weibel-Palade-like granules and mutations in different VWF domains can affect the formation of these organelles. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regions of the Herpes Simplex Virus Type 1 Latency-Associated Transcript That Protect Cells from Apoptosis In Vitro and Protect Neuronal Cells In Vivo

2002 ◽  
Vol 76 (2) ◽  
pp. 717-729 ◽  
Author(s):  
Maryam Ahmed ◽  
Martin Lock ◽  
Cathie G. Miller ◽  
Nigel W. Fraser
Keyword(s):  
Herpes Simplex ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Exon 1 ◽  
Induced Apoptosis ◽  
Simplex Virus ◽  
In Cells

ABSTRACT Recent studies have suggested that the latency-associated transcript (LAT) region of herpes simplex virus type 1 (HSV-1) is effective at blocking virus-induced apoptosis both in vitro and in the trigeminal ganglia of acutely infected rabbits (Inman et al., J. Virol. 75:3636–3646, 2001; Perng et al., Science 287:1500–1503, 2000). By transfecting cells with a construct expressing the Pst-Mlu segment of the LAT, encompassing the LAT exon 1, the stable 2.0-kb intron, and 5′ part of exon 2, we confirmed that this region was able to diminish the onset of programmed cell death initiated by anti-Fas and camptothecin treatment. In addition, caspase 8-induced apoptosis was specifically inhibited in cells expressing the Pst-Mlu LAT fragment. To further delineate the minimal region of LAT that is necessary for this antiapoptotic function, LAT mutants were used in our cotransfection assays. In HeLa cells, the plasmids lacking exon sequences were the least effective at blocking apoptosis. However, similar to previous work (Inman et al., op. cit.), our data also indicated that the 5′ end of the stable 2.0-kb LAT intron appeared to contribute to the promotion of cell survival. Furthermore, cells productively infected with the 17N/H LAT mutant virus, a virus deleted in the LAT promoter, exon 1, and about half of the intron, exhibited a greater degree of DNA fragmentation than cells infected with wild-type HSV-1. These data support the finding that the exon 1 and 2.0-kb intron region of the LAT transcription unit display an antiapoptotic function both in transfected cells and in the context of the virus infection in vitro. In trigeminal ganglia of mice acutely infected with the wild-type virus, 17, and 17ΔSty, a virus lacking most of exon 1, apoptosis was not detected in cells that were positive for virus particles. However, dual staining was observed in cells from mice infected with 17N/H virus, indicating that the LAT antiapoptotic function demonstrated in cells transfected by LAT-expressing constructs may also play a role in protecting cells from virus-induced apoptosis during acute viral infection in vivo.

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