Fig 1.
Coomassie stain of (A) rhRod and rhVim in reducing (R) and non-reducing (NR) conditions and (B) rhRod and ΔrhRod in non-reducing and reducing conditions. (A) Non-reduced rhVim and rhRod are approximately twice the size of reduced protein, suggesting dimerization. (B) Coomassie stain of rhRod and ΔrhRod are similar in both the non-reduced and reduced states.
Fig 2.
SPOT-peptide membranes of (A) full-length human vimentin and (B) human P-selectin. The dark spots indicate locations in which IR800-labeled proteins bound to 20 amino acid residues from (A) full-length vimentin or (B) P-selectin. (A) IR-labeled P-sel/Fc bound to 20 amino acid spots of full-length vimentin that are located primarily in the rod domain. There is an 18-aa overlap between adjacent spots. (B) IR800-labled rhRod bound to 20 amino acid spots from P-selectin. There is a 17-aa overlap between adjacent spots. Letters and numbers correlate to the 20-aa sequence list in S1 Table (full-length vimentin) & S2 Table (P-selectin).
Fig 3.
Three views of the crystallized lectin/EGF-domain of P-selectin showing the rhRod-binding regions based on the SPOT array.
P-selectin is shown (A) complexed with the binding region of PSGL-1, SGP-3 (yellow; PDB 1G1S) and (B) without SGP-3 (PDB 1G1Q). The rhRod-binding regions are highlighted based on the SPOT array (see S1 Table; cyan: SPOTs #14–16 (aa residues 43–65), blue: SPOTs #27–33 (aa residues 79–116), and magenta: SPOTs #50–51 (aa residues 148–170)). The rhRod binding regions overlap with where PSGL-1/SGP-3 interacts with P-selectin (ribbon regions highlighted in red). (C) Two views of the top in silico prediction model of rhRod (green) binding to P-selectin in the region correlating to aa residues 107–129 (Psel SPOT #31–42).
Fig 4.
rhRod binds to P-selectin and blocks PSGL-1-P-selectin interactions.
(A & B) Steady state binding analysis curves of Req versus concentration of rhVim or rhRod to either P-sel/Fc or PSGL-1/Fc. rhVim and rhRod both have strong binding to P-sel/Fc regardless of whether the immobilized compound is (A) rhVim or rhRod (on AR2G sensors) or (B) P-sel/Fc or PSGL-1/Fc (on AHC sensors). Neither rhVim nor rhRod binds to PSGL-1/Fc. (C) Increasing concentrations of rhRod blocked PSGL-1/Fc binding to immobilized P-sel/Fc.
Table 1.
KD determination by biolayer interferometry.
Fig 5.
rhRod blocks leukocyte adhesion to platelets, inflamed endothelium, and P-selectin-coated channels.
(A) Mepacrine-labeled whole blood perfusing over fibrin(ogen)-coated channels at 5 dyn/cm2. rhRod did not affect platelet adhesion to fibrin(ogen)-coated channels, based on mean fluorescence intensity. (B) rhRod decreased leukocyte adhesion to fibrin(ogen)-captured platelets similar to rhVim at equal molar (1.3 μM) and mass (40 μg/mL) concentrations. (C) rhRod decreased leukocyte adhesion to IL1β/IL4-co-stimulated HUVEC. (D) rhRod also decreased isolated PMN adhesion to IL1β/IL4-co-stimulated HUVEC. (E) Finally, rhRod decreased isolated PMN adhesion to P-selectin coated channels in a dose dependent fashion. Leukoocyte adhesion was measured at shear stress of 2 dyn/cm2. (F) Representative images of mepacrine-labeled whole blood leukocytes perfused over IL1β/IL4 co-stimulated HUVEC in the presence of control buffer (top) and rhRod 40 μg/mL (bottom). Each subject is represented by a unique symbol. Scale bar = 50 μm. *p<0.05, **p<0.01, ***p<0.005, & ****p<0.001.
Fig 6.
rhRod co-localizes with P-selectin and attenuates neutrophil accumulation in the liver sinusoids in endotoxemic mice.
(A) Montage image from intravital microscopy of mouse liver sinusoids after intraperitoneal Atto 550-labeled rhRod (top) or control (bottom) followed by endotoxin injection. rhRod (green) and P-selectin (red) colocalize (yellow) along the sinusoid wall. Neutrophils were labeled with anti-Ly-6G antibody (cyan) and the sinusoids were identified using 150,000 Da-Dextran (gray). (B) rhRod administration decreased the number of firmly adherent (<1 cell body movement over 1 minute) neutrophils per vessel area in liver sinusoids. (C) Representative images of vimentin expression (brown; black arrows) with hematoxylin counterstain in the liver of mice 4 hours after saline or endotoxin intraperitoneal injection. (D) There was no difference in the percent area of vimentin staining between the two groups. Scale bar = 50 μm. *p<0.05.