The Interaction between Factor H and Von Willebrand Factor

Complement factor H (fH) is a plasma protein that regulates activation of the alternative pathway, and mutations in fH are associated with a rare form of thrombotic microangiopathy (TMA), known as atypical hemolytic uremic syndrome (aHUS). A more common TMA is thrombotic thrombocytopenic purpura, which is caused by the lack of normal ADAMTS-13-mediated cleavage of von Willebrand factor (VWF). We investigated whether fH interacts with VWF and affects cleavage of VWF. We found that factor H binds to VWF in plasma, to plasma-purified VWF, and to recombinant A1 and A2 domains of VWF as detected by co-immunoprecipitation (co-IP) and surface plasmon resonance assays. Factor H enhanced ADAMTS-13-mediated cleavage of recombinant VWF-A2 as determined by quantifying the cleavage products using Western-blotting, enhanced cleavage of a commercially available fragment of VWF-A2 (FRETS-VWF73) as determined by fluorometric assay, and enhanced cleavage of ultralarge (UL) VWF under flow conditions as determined by cleavage of VWF-platelet strings attached to histamine stimulated endothelial cells. Using recombinant full-length and truncated fH molecules, we found that the presence of the C-terminal half of fH molecule is important for binding to VWF-A2 and for enhancing cleavage of the A2 domain by ADAMTS-13. We conclude that factor H binds to VWF and may modulate cleavage of VWF by ADAMTS-13

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

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

The Interaction between Factor H and Von Willebrand Factor

Complement factor H (fH) is a plasma protein that regulates activation of the alternative pathway, and mutations in fH are associated with a rare form of thrombotic microangiopathy (TMA), known as atypical hemolytic uremic syndrome (aHUS). A more common TMA is thrombotic thrombocytopenic purpura, which is caused by the lack of normal ADAMTS-13-mediated cleavage of von Willebrand factor (VWF). We investigated whether fH interacts with VWF and affects cleavage of VWF. We found that factor H binds to VWF in plasma, to plasma-purified VWF, and to recombinant A1 and A2 domains of VWF as detected by co-immunoprecipitation (co-IP) and surface plasmon resonance assays. Factor H enhanced ADAMTS-13-mediated cleavage of recombinant VWF-A2 as determined by quantifying the cleavage products using Western-blotting, enhanced cleavage of a commercially available fragment of VWF-A2 (FRETS-VWF73) as determined by fluorometric assay, and enhanced cleavage of ultralarge (UL) VWF under flow conditions as determined by cleavage of VWF-platelet strings attached to histamine stimulated endothelial cells. Using recombinant full-length and truncated fH molecules, we found that the presence of the C-terminal half of fH molecule is important for binding to VWF-A2 and for enhancing cleavage of the A2 domain by ADAMTS-13. We conclude that factor H binds to VWF and may modulate cleavage of VWF by ADAMTS-13