21 research outputs found
A Novel Dimeric Inhibitor Targeting Beta2GPI in Beta2GPI/Antibody Complexes Implicated in Antiphospholipid Syndrome
Background: b2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of b2GPI generated by anti-b2GPI antibodies is pathologically important, in contrast to monomeric b2GPI which is abundant in plasma. Principal Findings: We created a dimeric inhibitor, A1-A1, to selectively target b2GPI in b2GPI/antibody complexes. To make this inhibitor, we isolated the first ligand-binding module from ApoER2 (A1) and connected two A1 modules with a flexible linker. A1-A1 interferes with two pathologically important interactions in APS, the binding of b2GPI/antibody complexes with anionic phospholipids and ApoER2. We compared the efficiency of A1-A1 to monomeric A1 for inhibition of the binding of b2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of b2GPI present in human serum, b2GPI purified from human plasma and the individual domain V of b2GPI. We demonstrated that when b2GPI/antibody complexes are formed, A1-A1 is much more effective than A1 in inhibition of the binding of b2GPI to cardiolipin, regardless of the source of b2GPI. Similarly, A1-A1 strongly inhibits the binding of dimerized domain V of b2GPI to cardiolipin compared to the monomeric A1 inhibitor. In the absence of anti-b2GPI antibodies, both A1-A1 and A1 only weakly inhibit the binding of pathologically inactive monomeric b2GPI to cardiolipin. Conclusions: Our results suggest that the approach of using a dimeric inhibitor to block b2GPI in the pathologica
Dimerized Domain V of Beta2-Glycoprotein I Is Sufficient to Upregulate Procoagulant Activity in PMA-Treated U937 Monocytes and Require Intact Residues in Two Phospholipid-Binding Loops
Upregulation of the procoagulant activity of monocytes by antibodies to beta2-glycoprotein I (β2GPI) is one of the mechanisms contributing to thrombosis in antiphospholipid syndrome. Current knowledge about receptors responsible for the upregulation of procoagulant activity by β2GPI/anti-β2GPI complexes and their binding sites on β2GPI is far from complete. We quantified the procoagulant activity expressed by phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells by measuring clotting kinetics in human plasma exposed to stimulated cells. Cells stimulated with anti-β2GPI were compared to cells treated with dimerized domain V of β2GPI (β2GPI-DV) or point mutants of β2GPI-DV. We demonstrated that dimerized β2GPI-DV is sufficient to induce procoagulant activity in monocytes. Using site-directed mutagenesis, we determined that the phospholipid-binding interface on β2GPI is larger than previously thought and includes Lys308 in β2GPI-DV. Intact residues in two phospholipid-binding loops of β2GPI-DV were important for the potentiation of procoagulant activity. We did not detect a correlation between the ability of β2GPI-DV variants to bind ApoER2 and potentiation of the procoagulant activity of cells. The region on β2GPI inducing procoagulant activity in monocytes can now be narrowed down to β2GPI-DV. The ability of β2GPI-DV dimers to come close to cell membrane and attach to it is important for the stimulation of procoagulant activity
Interleukin-8 and Acute Kidney Injury following Cardiopulmonary Bypass: A Prospective Cohort Study
Crystallographic statistics.
a<p>Values in parenthesis correspond to the highest resolution shell.</p
Comparison of the inhibition of β2GPI in serum with the inhibition of the isolated domain V by monomeric A1 in the absence of dimerization antibodies.
<p>Inhibition of the binding of β2GPI-DV by A1 (circles); inhibition of the binding of β2GPI in serum by A1 (triangles).</p
Inhibition of the binding of purified β2GPI to cardiolipin by A1-A1 and A1 in the absence of anti-β2GPI antibodies.
<p>The purified β2GPI bound to cardiolipin in the absence of anti-β2GPI antibody with increasing amounts of A1-A1 (black bars) and A1 (white bars). The OD<sub>405</sub> values were normalized to OD<sub>405</sub> measured in the absence of inhibitor.</p
Stereoview of the crystal structure of the isolated domain V of β2GPI (β2GPI-DV).
<p>Backbone superposition of the structures of β2GPI-DV chain A (green), chain B (blue) and the crystal structure of domain V from the full-length β2GPI (PDB ID 1C1Z, residues from 244 to 326) (red). The N- and C-termini, and a flexible loop in the domain V are labeled. Figure was generated with the program PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015345#pone.0015345-DeLano1" target="_blank">[61]</a>. The two molecules of β2GPI-DV in the asymmetric unit of the crystal, chains A and B, have nearly identical structures with backbone RMSD of 0.23 Å. Backbone superposition of domain V in the full-length β2GPI onto the structure of β2GPI-DV have RMSD of 1.04 Å and 0.97 Å for chains A and B, respectively.</p
HPLC chromatograms of products formed by oxidative refolding of A1 (A) and A1-A1 (B).
<p>The proteins were dialyzed in redox buffer in the presence of calcium or EDTA and eluted with a linear gradient of 0.1% per minute of acetonitrile containing 0.1% TFA starting at 15 minutes from 21% of acetonitrile/TFA (for A1) or 26% of acetonitrile/TFA (for A1-A1).</p