ATZ11 Recognizes Not Only Z-α₁-Antitrypsin-Polymers and Complexed Forms of Non-Z-α₁-Antitrypsin but Also the von Willebrand Factor

<div><p>Aims</p><p>The ATZ11 antibody has been well established for the identification of α₁-anti-trypsin (AAT) molecule type PiZ (Z-AAT) in blood samples and liver tissue. In this study, we systematically analyzed the antibody for additional binding sites in human tissue.</p><p>Methods and Results</p><p>Ultrastructural ATZ11 binding was investigated immunoelectron microscopically in human umbilical vein endothelial cells (HUVECs) and in platelets of a healthy individual. Human embryonic kidney (HEK293) cells were transiently transfected with Von Willebrand factor (VWF) and analyzed immunocytochemically using confocal microscopy and SDS-PAGE electrophoresis followed by western blotting (WB). Platelets and serum samples of VWF-competent and VWF-deficient patients were investigated using native PAGE and SDS-PAGE electrophoresis followed by WB. The specificity of the ATZ11 reaction was tested immunohistochemically by extensive antibody-mediated blocking of AAT- and VWF-antigens.</p><p>ATZ11-positive epitopes could be detected in Weibel-Palade bodies (WPBs) of HUVECs and α-granules of platelets. ATZ11 stains pseudo-WBP containing recombinant wild-type VWF (rVWF-WT) in HEK293 cells. In SDS-PAGE electrophoresis followed by WB, anti-VWF and ATZ11 both identified rVWF-WT. However, neither rVWF-WT-multimers, human VWF-multimers, nor serum proteins of VWF-deficient patients were detected using ATZ11 by WB, whereas anti-VWF antibody (anti-VWF) detected rVWF-WT-multimers as well as human VWF-multimers. In human tissue specimens, AAT-antigen blockade using anti-AAT antibody abolished ATZ11 staining of Z-AAT in a heterozygous AAT-deficient patient, whereas VWF-antigen blockade using anti-VWF abolished ATZ11 staining of endothelial cells and megakaryocytes.</p><p>Conclusions</p><p>ATZ11 reacts with cellular bound and denatured rVWF-WT and human VWF as shown using immunocytochemistry and subsequent confocal imaging, immunoelectron microscopy, SDS-PAGE and WB, and immunohistology. These immunoreactions are independent of the binding of Z-AAT-molecules and non-Z-AAT complexes.</p></div

Confocal imaging of VWF-transfected HEK293 cells: Pseudo-Weibel-Palade-Body (pseudo-WPB) granules formed after transfection of HEK293 cells using recombinant wild-type VWF (rVWF-WT) constructs: (A, B) Pseudo-WPB granules are shown in green (anti-VWF staining).

<p>(C) The same intracellular structures are stained with ATZ11 (red). (D) Small dot-like signals of less than .25 µm were found in very few HEK293 cells stained with anti-AAT (arrow). (E) Merged images of anti-VWF and ATZ11 stains highlight the co-localization of the antibody-binding sites. At a single cell level, small dot-like positive signals were found in the ATZ11 reaction, which were not co-localized with VWF staining (arrow). (F) Merged images of anti-VWF staining and anti-AAT signals demonstrated that the dot-like anti-AAT positive signals were not associated with pseudo-WPBs (arrow). Scale bar = 10 µm.</p

Recombinant von Willebrand Factor Expression in four Transfection Experiments in HEK293 cell lines.

<p>rVFW – recombinant von Willebrand factor.</p

Comparative immunostaining of temporary artery specimens.

<p>(A) Localization of AAT in the temporal artery. Specimen stained with polyclonal anti-AAT (1∶5000) showed immunoreactivity on the endothelial surface and a gradient of presumably soluble AAT within the vessel wall. (B) ATZ11 (1∶100) showed a distinct staining of the endothelial layer. (C) The endothelial layer is distinctly stained using anti-VFW (1∶500). (D) After saturation with anti-AAT antibody (1∶10), ATZ11 labeled a thin endothelial layer. (E) Blockade with anti-VWF antibody (1∶10) abolished ATZ11 staining (1∶100) of the endothelial layer. (F) Sequential blockade with anti-AAT (1∶10) and anti-VWF (1∶10) completely abolished ATZ11 staining (1∶100).</p

Protein-electrophoretic studies on VWF-transfected HEK293 cells and human serum samples:

<p>(A) <b>SDS-PAGE electrophoresis and subsequent western blotting (WB) and visualization using anti-VWF: (lane 1) cell lysates of recombinant wild-type VWF (rVWF-WT)-transfected HEK293 cells, (lane 2) mock-transfected HEK293 cells.</b> (B) SDS-PAGE electrophoresis and subsequent WB and visualization using ATZ11: (lane 1) of cell lysates of rVWF-WT-transfected HEK293 cells and (lane 2) mock-transfected HEK293 cells. A congruent single band of 225 kDa was detected in the VWF-transfected HEK293 cells using both anti-VWF (A) and ATZ11 (B). (C) SDS-PAGE electrophoresis and subsequent WB of human serum samples of a non-Z healthy individual (lanes 1–2) and of VWF-deficient patients (lanes 3–5) stained with anti-VWF. (D) SDS-PAGE electrophoresis and subsequent WB of serum samples of a non-Z healthy individual (lanes 1–2) and of VWF-deficient patients (lanes 3–5) stained with ATZ11. (E) Native PAGE electrophoresis and subsequent WB of a recombinant VWF (lanes 1–2) and serum samples of a non-Z healthy individual (lanes 3–5) stained with the anti-VWF antibody.</p