Shear-Stress-Induced Conformational Changes of von Willebrand Factor in a Water–Glycerol Mixture Observed with Single Molecule Microscopy

The von Willebrand factor (VWF) is a human plasma protein that plays a key role in the initiation of the formation of thrombi under high shear stress in both normal and pathological situations. It is believed that VWF undergoes a conformational transition from a compacted, globular to an extended form at high shear stress. In this paper, we develop and employ an approach to visualize the large-scale conformation of VWF in a (pressure-driven) Poiseuille flow of water–glycerol buffers with wide-field single molecule fluorescence microscopy as a function of shear stress. Comparison of the imaging results for VWF with the results of a control with λ-phage double-stranded DNA shows that the detection of individual VWF multimers in flow is feasible. A small fraction of VWF multimers are observed as visibly extended along one axis up to lengths of 2.0 μm at high applied shear stresses. The size of this fraction of molecules seems to exhibit an apparent dependency on shear stress. We further demonstrate that the obtained results are independent of the charge of the fluorophore used to label VWF. The obtained results support the hypothesis of the conformational extension of VWF in shear flow

Effets de l'ordonnance d'expropriation sur les baux ruraux

Important cellular events such as division require drastic changes in the shape of the membrane. These remodeling processes can be triggered by the binding of specific proteins or by changes in membrane composition and are linked to phospholipid metabolism for which dedicated enzymes, named phospholipases, are responsible. Here wide-field fluorescence microscopy is used to visualize shape changes induced by the action of phospholipase A1 on dye-labeled supported membranes of POPC (1-palmitoyl-2-oleoly-<i>sn</i>-glycero-3-phosphocholine). Time-lapse imaging demonstrates that layers either shrink and disappear or fold and collapse into vesicles. These vesicles can undergo further transformations such as budding, tubulation, and pearling within 5 min of formation. Using dye-labeled phospholipases, we can monitor the presence of the enzyme at specific positions on the membrane as the shape transformations occur. Furthermore, incorporating the products of hydrolysis into POPC membranes is shown to induce transformations similar to those observed for enzyme action. The results suggest that phospholipase-mediated hydrolysis plays an important role in membrane transformations by altering the membrane composition, and a model is proposed for membrane curvature based on the presence and shape of hydrolysis products

miRNA-mediated knockdown of GPIbα by transfection of CHO GPIb-IX cells.

<p>Representative flow cytometry histograms from CHO GPIb-IX control cells (white) and cells transfected with (A) pCMV-mi<i>GPIBA</i>-2-eGFP (green), (B) pCMV-mi<i>GPIBA</i>-3-eGFP (red), (C) pCMV-mi<i>GPIBA</i>-2+2-eGFP (orange), or (D) pCMV-mi<i>GPIBA</i>-2+3-eGFP (purple) expressing GPIbα 48h post transfection. Negative control in which no anti-GPIbα moAb 6B4 was added is depicted in each histogram in grey. Percentages of cells expressing GPIbα are indicated next to each histogram. (F) Flow cytometry analysis representing mean fluorescence intensities of each population of CHO GPIb-IX cells expressing GPIbα ± SEM (n>3). Statistical analysis was performed using Anova followed by Dunnett’s post-test (** p<0.01).</p

Morphological changes in differentiating megakaryoblastic DAMI cells.

<p>(A-C) Representative confocal images of differentiating mock transfected DAMI cells (A), DAMI cells transfected with pCMV-eGFP (note extensive eGFP fluorescence) (B) and DAMI cells transfected with pCMV-mi<i>GPIBA</i>-2+3-eGFP (C-D). Cells were stained for GPIbα (green), the actin cytoskeleton (red) and the nucleus (blue/purple). Scale bar is 25 μm. The width (W) and length (L) of the cells along two perpendicular axes used to calculate cell aspect ratios are indicated in (C). (D) GPIbα expression in untransfected (white) or pCMV-eGFP (black) or pCMV-mi<i>GPIBA</i>-2+3-eGFP (purple) transfected DAMI cells was determined by flow cytometry. DAMI transfected cells were stimulated with PMA for 48h as indicated. Data represent mean fluorescence intensities of GPIbα expression ± SEM (n>3). (E) Quantitative analysis showing cell aspect ratios (W/L) represent mean ± SEM (n >3; 20 cells analyzed per condition). Statistical analysis was performed using the unpaired Student t test (* p<0.05; *** p<0.01).</p

Reduced ADAMTS13 levels in patients with acute and chronic cerebrovascular disease

<div><p>Von Willebrand Factor (VWF) plays a major role in thrombosis and hemostasis and its thrombogenicity is controlled by ADAMTS13. Whereas increasing evidence shows a clear association between VWF levels and acute ischemic stroke, little is known about a correlation with ADAMTS13. Therefore, the aim of this study was to compare plasma levels of ADAMTS13 between 85 healthy volunteers (HV), 104 patients with acute ischemic stroke and 112 patients with a chronic cerebrovascular disease (CCD). In this case-control study, plasma ADAMTS13 antigen levels were measured by ELISA and plasma VWF levels, measured previously, were next used to calculate VWF:ADAMTS13 ratios. ADAMTS13 levels and VWF:ADAMTS13 ratios were subsequently correlated with key demographic and clinical parameters. ADAMTS13 levels were significantly lower in acute ischemic stroke patients (82.6 ± 21.0%) compared with HV (110.6 ± 26.9%). Also, CCD patients (99.6 ± 24.5%) had significantly lower ADAMTS13 levels compared with HV however these were still higher than in acute stroke patients. Furthermore, when assessing the VWF:ADAMTS13 ratios, an even greater difference was revealed between stroke patients (2.7 ± 1.9), HV (1.1 ± 0.5) and CCD patients (1.7 ± 0.7). The VWF:ADAMTS13 ratio was significantly associated with stroke severity and modality. In conclusion, both in acute and chronic cerebrovascular disease patients, ADAMTS13 levels were significantly decreased, with the lowest ADAMTS13 levels found in acute stroke patients. This difference was even more distinct when the ratio of VWF:ADAMTS13 was considered. These results demonstrate the potentially important involvement of the VWF/ADAMTS13 axis in ischemic stroke.</p></div

Baseline characteristics of the acute stroke patients (acute ischemic stroke (AIS) and transient ischemic attack (TIA)).

<p>Data are represented as mean ± standard deviation (SD). (NIHSS: National Institutes of Health stroke scale; TOAST: Trial of Org 10172 in Acute Stroke Treatment).</p

Patients with acute or chronic cerebrovascular disease have lower ADAMTS13 levels and a higher VWF:ADAMTS13 ratio compared to healthy volunteers.

<p>ADAMTS13 antigen levels from 104 patients with acute ischemic stroke, 112 patients with a chronic cerebrovascular disease (CCD) and 85 healthy volunteers, were measured. Levels of ADAMTS13 were plotted as a percentage of the level in a normal human plasma pool (NHP), consisting of pooled plasma from 20 healthy donors. VWF levels were measured previously and were used to calculate the VWF:ADAMTS13 ratios. Both ADAMTS13 levels <b>[A]</b> and VWF:ADAMTS13 ratios <b>[B]</b> are depicted in box-and-whisker plots indicating the first and third quartiles as well as the interquartile range (IQR, Tukey plot). Outliers outside the 1.5 IQR are visualized by single dots. ADAMTS13 levels and the VWF:ADAMTS13 ratios showed significant differences over the three groups. Data were analyzed using a Kruskal-Wallis test with a Dunn's multiple comparison test. *: p < 0.05; ****: p < 0.0001.</p

Knockdown of GPIb-IX reduces ristocetin induced VWF-dependent CHO GPIb-IX cell aggregation.

<p>CHO GPIb-IX cells were incubated with ristocetin without (A) or with (B-E) VWF on a rotary shaker to induce aggregate formation. Representative overlay bright field and fluorescent (GFP) images from (A-B) mock, (C) pCMV-eGFP and (D) pCMV-mi<i>GPIBA</i>-2+3-eGFP transfected cells are shown. Quantitative analysis was performed by measuring (E) the number of aggregates and (F) the aggregate size (a.u.: arbitrary units). Data represent mean ± SEM (n = 4). Statistical analysis was performed using the unpaired Student t test (* p<0.05).</p

Knockdown of GPIbα expression following transfection of CHO GPIb-IX cells with siRNA.

<p>Representative flow cytometry histograms from mock transfected cells (white) and cells transfected with (A) si<i>GPIBA</i>-1 (blue), (B) si<i>GPIBA</i>-2 (green), (C) si<i>GPIBA</i>-3 (red), (D) si<i>GPIBA</i>-2+3 (purple) and (E) si<i>GPIBA</i>-1+2+3 (black) expressing GPIbα 48h post transfection. Negative control in which no anti-GPIbα moAb 6B4 was added is depicted in each histogram in grey. Percentages of cells expressing GPIbα are indicated next to each histogram. (F) Flow cytometry analysis representing mean fluorescence intensities of each population of CHO GPIb-IX cells expressing GPIbα ± SEM (n>3). Statistical analysis was performed using Anova followed by Dunnett’s post-test (* p<0.05; ** p<0.01).</p

Multivariate analysis of ADAMTS13 levels and the VWF:ADAMTS13 ratios with demographic and clinical parameters.

<p>Multivariate analysis of ADAMTS13 levels and the VWF:ADAMTS13 ratios with demographic and clinical parameters.</p