Pegylated versions of PA inhibit complement activity in a hemolytic assay.

<p>(A) Hemolytic assays using factor B-depleted serum were performed with PA dissolved in DMSO and its pegylated derivatives dissolved in water. Factor B-depleted serum was incubated with 0.77mM of each peptide and then added to sensitized sheep erythrocytes. (B) Titration of increasing amounts of PA and PA-dPEG24 in the hemolytic assay. Water and DMSO were used as vehicle controls in the presence of factor B-depleted serum. GVBS⁺⁺ is a buffer- only control. Values are the means of three independent experiments. Error bars represent the SEM.</p

Linear mode MALDI-TOF mass spectrometry analysis of PA-dPEG24 oligomeric state.

<p>PA-dPEG24 in acidified water was analyzed by linear mode mass spectrometry. The spectrum shows two peaks for PA-dPEG24 with a monomer peak at 2778 and a dimer form at 5553.</p

CD and 1D ¹H NMR of PA-dPEG24.

<p>(A) The PA-dPEG24 peptide at 0.2mg/mL in PBS was analyzed on a Jasco J-815 CD spectrometer. (B) The PA-dPEG24 peptide at 1.8mg/mL in PBS with 10% D₂O was analyzed on a Bruker Avance 400 MHz spectrometer at a temperature of 294 K.</p

PA-dPEG24 inhibits complement activation <i>in vivo</i>.

<p>(A) Rats were IV administered PA-dPEG24 at 10 (n = 2) or 20mg/mL (n = 2) in 0.9% NaCl or received a vehicle control (n = 2) or were sham treated (n = 2). B, Rats were IV administered PA-dPEG24 at 10mg/mL (n = 4), 20mg/mL (n = 4) or 30mg/mL (n = 4) in 10mM Na₂HPO₄, 0.9% NaCl or received a vehicle control (n = 3). At the indicated time points post-administration, blood was collected and plasma isolated. Plasma was tested in hemolytic assays with human AB erythrocytes. Error bars represent the SEM.</p

PA-dPEG24 inhibits complement activation in rat serum, mouse plasma and non-human primate serum.

<p>Hemolytic assays using (A) Wistar rat serum, (B) mouse plasma and (C) cynomolgus monkey serum were performed with increasing amounts PA-dPEG24 dissolved in 100mM Na₂HPO₄ with 0.9% NaCl buffer for rat serum, 10mM Na₂HPO₄ with 0.9% NaCl for cynomolgus monkey serum and 0.9% NaCl for mouse plasma. Serum or plasma were incubated with peptide and then added to human AB erythrocytes. Values are the means of three independent experiments. Error bars represent the SEM.</p

PA binds to MBL and ficolins and does not displace C1s from C1q.

<p>(A) The AcPA and CP2 peptides were adsorbed to a microtiter plate and incubated with a constant amount of MBL or K55Q. Bound MBL was detected with a polyclonal goat anti-MBL sera followed by HRP-conjugated anti-goat sera. (B) The various ficolins indicated in the figure were adsorbed to the microtiter plate and incubated with biotinylated PA followed by a neutravidin conjugate. BSA was used as a negative control for binding. (C) Partially purified C1 was mixed with increasing amounts of CP or PA and added to a microtiter plate coated with monoclonal antibody to C1q. After washing, C1s signal present in intact C1 complexes was measured by ELISA using polyclonal antibody to C1s (CP, red line; PA, blue line) or polyclonal antibody to C1q (green line) to confirm C1q was bound to the plate. Data represent the means of three independent experiments. Error bars denote SEM.</p

Model of PA inhibition of C1/MBL function.

<p>Our data favors a model in which PIC1 derivatives functionally disrupt the C1s-C1r-C1r-C1s/MASP interaction with the CLR of C1q/MBL, respectively. By PIC1 binding to the serine protease interaction site on CLR, the serine proteases are hypothesized to be in a suboptimal conformation for autoactivation and initiation of the classical and/or lectin pathways of complement.</p