1. The dominant-negative von Willebrand factor gene deletion p
The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation
by Caterina Casari, Mirko Pinotti, Stefano Lancellotti, Elena Adinolfi, Alessandra Casonato, Raimondo De Cristofaro, and Francesco Bernardi
Blood
Volume 116(24):
December 9, 2010
©2010 by American Society of Hematology

2. Expression plasmids for VWF variants and breakpoint-specific siRNAs.
Expression plasmids for VWF variants and breakpoint-specific siRNAs. Schematic representation of the expression vectors for the wild-type (pWT) and in-frame deleted (pDEL) VWF variants. The amino, carboxyl-terminus, and deleted (r del, p.1127_C1948delinsR) VWF domains are reported as white, light gray, and dark gray rectangles, respectively. Accordingly, PCR primers (VWF-25F, VWF-25R. VWF-35F, and VWF-35R) used to create the deletion are indicated by short white or light gray bars. The black arrow in the pDEL vector indicates the breakpoint. Position and numbering of sequences targeted by siRNA molecules are indicated by small flags. Square brackets indicate the localization of VWF epitopes recognized by antibody pools (M13, M31) used for Western blotting. The filled triangle represents the position (134 bases downstream of the stop codon) of the suppressed SacI restriction site. (Inset) Capillary electrophoresis of wild-type and deleted mRNA reverse-transcribed PCR products from cells transfected with equimolar amounts of the indicated vectors, and on treatment with 40nM si3681 (right).
Caterina Casari et al. Blood 2010;116:
©2010 by American Society of Hematology

3. Wild-type and mutant VWF expression in cellular models.
Wild-type and mutant VWF expression in cellular models. (A) Western blot analysis of wild-type and mutant VWF in conditioned media (top panel) and in cell lysates (bottom panels) with the monoclonal antibody pool M31,22,23 recognizing domains downstream of the breakpoint junction. Migration of the mature wild-type (matureWT-VWF) and deleted (matureDEL-VWF) proteins, and of forms containing the propeptide (proWT-VWF), is indicated. For comparison, see the Western blots in supplemental Figure 4. (B) VWF:Ag (white bars) and VWF:CB (gray bars) levels in conditioned media. The relative molar amount of pWT, pDEL, and vectors and the addition of siRNAs (40nM) are indicated in the table. 100% VWF:Ag corresponds to ± 54.5 ng/mL and 100% of VWF:CB to 108.0% ± 14.7% of normal standard. Results from at least 3 independent experiments are reported as mean ± SEM. Statistical significance was evaluated by one-way analysis of variance with Bonferroni posttest: *P < .001; ○P < .01. nd indicates not detectable.
Caterina Casari et al. Blood 2010;116:
©2010 by American Society of Hematology

4. Multimer analysis of VWF in conditioned media.
Multimer analysis of VWF in conditioned media. (A) High-resolution multimer analysis (3% agarose gels) and (C) schematic representation of multimer composition. Dimers (D) and tetramers (T) are indicated together with sub-band composition in accordance with the schematic representation.30,31 DI-III and TI-V (subscript Roman numerals) indicate polymers containing combinations of wild-type VWF (pro- or mature VWF). D1-3 and T1-5 (Italics, subscript Arabic numerals) indicate polymers containing deleted VWF (mature). A dotted line flanks the fast-migrating and smeared bands containing heterotetramers. The similar extent of the deleted and propeptide region causes comigration of bands with different subunit composition. Comigrating proteins are indicated in the same line (scheme), and comigrating bands (DI + D3, TIII + T5) are indicated by the sum of specific dimers or tetramers. A total of 1 ng (left and center panels) and 0.25 ng (right panel) of protein were loaded in each lane, based on VWF:Ag concentration in media. Accordingly, the gel in the right panel was overexposed. (B) Multimer analysis in 1.8% agarose gels. Equal volumes of media (15 μL) were loaded in each lane to highlight both quantitative and qualitative differences among samples. The relative molar amounts of each vector and siRNA are reported in the tables. N (normal plasma from an healthy person with normal values of VWF) was diluted 1/200.
Caterina Casari et al. Blood 2010;116:
©2010 by American Society of Hematology

5. Intracellular distribution of recombinant VWF in late endosomal compartment.
Intracellular distribution of recombinant VWF in late endosomal compartment. Cells transfected with vectors and siRNA indicated above the images (A-H) were coimmunostained for VWF (tetramethylrhodamine isothiocyanate, red) and mannose 6-phosphate receptor (fluorescein isothiocyanate, green) as late endosome marker. Images were taken with Carl Zeiss LSM 510 equipped with a Fluar 40×/1.3 oil immersion objective at room temperature on fixed cells mounting in glycerol/1,4 diazabicyclo[2.2.2]octane/4,6-diamidino-2-phenylindole medium. Briefly, 488- and 543-nm excitation wavelengths were provided, respectively, by Argon/2 and HeNE laser sources at a 5% intensity. Superimposition of red and green fluorescence, not specifically caused by colocalization, was excluded by applying to the green channel a beam path admitting the acquisition of fluorescence composed among 505 and 550 nm and to the red channel a light path excluding all fluorescence less than 560 nm. Pinhole values ranged from 40 to 80 μm. Images in the top panels were digitally zoomed in twice the original size with LSM examiner software Version 3.0 (Carl Zeiss).
Caterina Casari et al. Blood 2010;116:
©2010 by American Society of Hematology