5 research outputs found
Binding of recombinant full-length and truncated fH molecules to VWF and VWF-A2.
<p>(<b>A</b>) Recombinant GST VWF-A2 protein (100 nM) was mixed with recombinant full-length or truncated fH (30 nM) and precipitated using glutathione agarose beads. The precipitated proteins were immunoblotted (IB) with anti-fH antibody for examining the association between VWF-A2 and fH molecules. Immunoblotting with anti-VWF antibody served as loading control. A representative blot is shown (n=3). (<b>B</b>) We repeated the above experiment but used a monoclonal anti-fH antibody (clone 90X) recognizing SCR1 to immunoblot fH fragments precipitated with GST-VWF-A2. (<b>C</b>) Recombinant VWF-A1 protein (100 nM) was mixed with recombinant full-length or truncated fH (30 nM) and precipitated using polyclonal anti-VWF antibody. The precipitated proteins were immunoblotted with monoclonal anti-fH antibody (clone 90X) for examining the association between VWF-A1 and fH molecules. (<b>D</b>) SPR sensograms were generated by injecting recombinant fH proteins (F1, F2, F3, or F4) at a concentration of 10 µg/ml over immobilized VWF-A2 or VWF for 60s followed by dissociation for 2 min. (<b>E</b>) SPR sensograms generated by injecting fH proteins (F1, F2, F3, and F4) over immobilized ovalbumin (OVA), served as a negative control.</p
Recombinant full-length and truncated Factor H.
<p>(<b>A</b>) Schematic representation of fH structure that includes 20 SCR domains and design of recombinant fH molecules used in our experiments. Amino acid lengths and SCR domains of each recombinant fragment are shown. (<b>B</b>) Immunoblotting (IB) of purified recombinant full-length and truncated fH molecules using polyclonal anti-fH antibody. (<b>C</b>) Purified recombinant full-length and truncated fH molecules were detected after PAGE (10%) and Coomassie blue staining. (<b>D</b>) C3b was incubated with plasma purified and recombinant fH molecules and immunoprecipitated using polyclonal anti-C3 antibody. Precipitated proteins were immunoblotted using anti-fH antibody. (<b>E</b>) Cofactor activity of plasma purified and recombinant fH molecules was investigated by incubating them with purified C3b and factor I. Factor I-mediated cleavage of C3b generated two cleavage products (arrowheads) originated form C3b α chain.</p
The effect of recombinant full-length and truncated fH on ADAMTS-13-mediated cleavage of VWF-A2.
<p>(<b>A</b>) Cleavage of recombinant VWF-A2 (100 nM) after 30 minutes of incubation with ADAMTS-13 (10 nM) in the presence or absence of full-length or truncated fH (100 nM) was studied by Western-blotting using anti-GST antibody. A representative blot is shown (n=7). (<b>B</b>) The band intensity of the VWF-A2 cleavage products was compared between samples with full-length and truncated fH (n=7, t-test).</p
Factor H enhances ADAMTS-13-mediated cleavage of recombinant VWF-A2.
<p>(<b>A</b>) Cleavage of VWF-A2 (100 nM) by ADAMTS-13 (10 nM) after 1 hr incubation in the presence or absence of fH (0.6 µM) was detected by immunoblotting (IB) with polyclonal anti-VWF antibody. A representative blot is shown (n=5). (<b>B</b>) The results of three separate experiments with ADAMTS-13-mediated VWF-A2 cleavage were summarized as bar graphs. The VWF-A2 cleavage (%) was calculated by measuring the ratio of band density of cleaved to uncleaved + cleaved VWF-A2. (<b>C</b>) Recombinant GST VWF-A2 (100 nM) was incubated with ADAMTS-13 (10 nM), in the presence or absence of fH (0.6 µM) for different time intervals. The cleavage products were detected by immunoblotting with anti-GST antibody. A representative blot is shown (n=3). (<b>D</b>) The half maximal cleavage time of GST VWF-A2 by ADAMTS-13 was calculated using the data obtained from different incubation intervals (20 minutes to 2 hours), in the presence or absence of fH. The results of these experiments were summarized as a linear graph (n=3, t-test, ** p<0.01, * p<0.05).</p
Factor H binds to VWF-A1 and –A2 domains.
<p>(<b>A</b>) Binding of recombinant fragments of VWF (A1, A2, A3, 200 nM each) to immobilized fH (30 nM) was studied using ELISA. The results are shown as mean with standard deviation (n=3, t-test). (<b>B</b>) Recombinant His-tagged VWF-A1, –A2, or –A3 domain (100 nM) was incubated with purified fH (30 nM) and immunoprecipitated (IP) by anti-fH antibody or IgG. Co-precipitated proteins were immunoblotted (IB) with anti-VWF antibody. A representative blot is shown (n=3). (<b>C</b>) Recombinant His-tagged VWF-A2 (100 nM) and Myc-tagged VWF-BC domains (100 nM), both purified from transfected HEK 293 cells, were incubated with purified fH (30 nM), precipitated with anti-fH antibody or IgG, separated by SDS- PAGE, and transferred to immobilon-P membranes. The same membrane was blotted with anti-VWF (upper panel) or anti-fH antibody (lower panel). (<b>D</b>) In a Biacore assay, two-fold increasing concentration (40 to 640 nM) of fH were injected over immobilized VWF-A1, VWF-A2, or VWF-A3 (200 nM each) at a flow rate of 20 µL/min. Baseline corrected SPR response curves are shown in black, with lower curve corresponding to lower concentration of fH injected. The sensorgrams for fH binding to VWF-A1 were fitted to the predefined two-state model (fitted curves are shown in red). For fH to VWF-A2 binding interaction, the responses at equilibrium of the SPR curves were fitted to a one-site binding isotherm (inset) to obtain the equilibrium dissociation constant (<i>K</i><sub>D</sub>). Binding of fH to VWF-A3 was negligible.</p