Molecular characterization, recombinant protein expression, and mRNA analysis of type 3 von Willebrand disease: Studies of an Italian cohort of 10 patients

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

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Evaluation of the Abnormal Platelet Function in von Willebrand Disease by the Blood Filtration Test

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650600 ◽

1996 ◽

Vol 76 (03) ◽

pp. 460-468 ◽

Cited By ~ 7

Author(s):

Francesco I Pareti ◽

Marco Cattaneo ◽

Luca Carpinelli ◽

Maddalena L Zighetti ◽

Caterina Bressi ◽

...

Keyword(s):

Platelet Function ◽

Relevant Information ◽

Von Willebrand Disease ◽

Type I ◽

Cumulative Number ◽

Primary Hemostasis ◽

Normal Platelet ◽

Filter Test ◽

Type 3 ◽

Von Willebrand

SummaryWe have evaluated platelet function in different subtypes of von Willebrand disease (vWD) by pushing blood through the capillarysized channels of a glass filter. Patients, including those with type IIB vWD, showed lower than normal platelet retention and increased cumulative number of blood drops passing through the filter as a function of time. In contrast, shear-induced platelet aggregation, measured in the cone-and-plate viscometer, was paradoxically increased in type IIB patients. Treatment with l-desamino-8-D-arginine vasopressin (DDAVP) tended to normalize the filter test in patients with type I-platelet normal and type I-platelet low vWD, but infusion of a factor VUI/von Willebrand factor (vWF) concentrate lacking the largest vWF multimers was without effect in type 3 patients. Experiments with specific monoclonal antibodies demonstrated that the A1 and A3 domains of vWF, as well as the glycoproteins Ibα and Ilb-IIIa on platelets, are required for platelet retention in the filter. Thus, the test may reflect vWF function with regard to both platelet adhesion and aggregation under high shear stress, and provide relevant information on mechanisms involved in primary hemostasis.

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Toward Personalized Treatment for Patients with Low von Willebrand Factor and Quantitative von Willebrand Disease

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0041-1722864 ◽

2021 ◽

Vol 47 (02) ◽

pp. 192-200

Author(s):

James S. O'Donnell

Keyword(s):

Von Willebrand Factor ◽

Bleeding Risk ◽

Von Willebrand Disease ◽

Personalized Treatment ◽

Treatment Plans ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

AbstractThe biological mechanisms involved in the pathogenesis of type 2 and type 3 von Willebrand disease (VWD) have been studied extensively. In contrast, although accounting for the majority of VWD cases, the pathobiology underlying partial quantitative VWD has remained somewhat elusive. However, important insights have been attained following several recent cohort studies that have investigated mechanisms in patients with type 1 VWD and low von Willebrand factor (VWF), respectively. These studies have demonstrated that reduced plasma VWF levels may result from either (1) decreased VWF biosynthesis and/or secretion in endothelial cells and (2) pathological increased VWF clearance. In addition, it has become clear that some patients with only mild to moderate reductions in plasma VWF levels in the 30 to 50 IU/dL range may have significant bleeding phenotypes. Importantly in these low VWF patients, bleeding risk fails to correlate with plasma VWF levels and inheritance is typically independent of the VWF gene. Although plasma VWF levels may increase to > 50 IU/dL with progressive aging or pregnancy in these subjects, emerging data suggest that this apparent normalization in VWF levels does not necessarily equate to a complete correction in bleeding phenotype in patients with partial quantitative VWD. In this review, these recent advances in our understanding of quantitative VWD pathogenesis are discussed. Furthermore, the translational implications of these emerging findings are considered, particularly with respect to designing personalized treatment plans for VWD patients undergoing elective procedures.

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Choice of selectable marker affects recombinant protein expression in cells and exosomes

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.100838 ◽

2021 ◽

pp. 100838

Author(s):

Chenxu Guo ◽

Francis K. Fordjour ◽

Shang Jui Tsai ◽

James C. Morrell ◽

Stephen J. Gould

Keyword(s):

Recombinant Protein ◽

Protein Expression ◽

Selectable Marker ◽

Recombinant Protein Expression ◽

In Cells

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BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells

Methods in Molecular Biology - Stem Cell Nanotechnology ◽

10.1007/7651_2019_249 ◽

2019 ◽

pp. 205-208 ◽

Cited By ~ 1

Author(s):

Deepak B. Thimiri Govinda Raj ◽

Niamat Ali Khan ◽

Srisaran Venkatachalam ◽

Sivakumar Arumugam

Keyword(s):

Recombinant Protein ◽

Protein Expression ◽

Mammalian Cells ◽

Recombinant Protein Expression ◽

Expression In Mammalian Cells

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Evaluation of somatic embryos of alfalfa for recombinant protein expression

Plant Cell Reports ◽

10.1007/s00299-014-1700-x ◽

2014 ◽

Vol 34 (2) ◽

pp. 211-221 ◽

Cited By ~ 4

Author(s):

Guohua Fu ◽

Vojislava Grbic ◽

Shengwu Ma ◽

Lining Tian

Keyword(s):

Recombinant Protein ◽

Protein Expression ◽

Somatic Embryos ◽

Recombinant Protein Expression

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Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication

Journal of Virology ◽

10.1128/jvi.00423-15 ◽

2015 ◽

Vol 89 (13) ◽

pp. 6746-6760 ◽

Cited By ~ 26

Author(s):

Nenavath Gopal Naik ◽

Huey-Nan Wu

Keyword(s):

Life Cycle ◽

Recombinant Protein ◽

Dengue Virus ◽

Protein Expression ◽

Nonstructural Protein ◽

Recombinant Protein Expression ◽

Rna Replication ◽

Viral Production ◽

Glycosylation Sites ◽

Infected Cells

ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

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