Modulation of Extravascular Binding of Recombinant Factor IX Impacts the Duration of Efficacy in Mouse Models.

BACKGROUND: There is an emerging concept that in addition to circulating coagulation factor IX (FIX), extravascular FIX contributes to hemostasis. OBJECTIVE: Our objective was to evaluate the efficacy of extravascular FIX using animal models of tail clip bleeding and ferric chloride-induced thrombosis. METHODS: Mutant rFIX proteins with described enhanced (rFIXK5R) or reduced (rFIXK5A) binding to extracellular matrix were generated and characterized using in vitro aPTT, one-stage clotting, and modified FX assays. Using hemophilia B mice, pharmacokinetic (PK) parameters and in vivo efficacy of these proteins were compared against rFIX wild-type protein (rFIXWT) in a tail clip bleeding and FeCl3-induced thrombosis model. Respective tissue disposition of FIX was evaluated using immunofluorescence. RESULTS: In vitro characterization demonstrated comparable clotting activity of rFIX proteins. The PK profile showed that rFIXK5A displayed the highest plasma exposure compared to rFIXWT and rFIXK5R. Immunofluorescence evaluation of liver tissue showed that rFIXK5R was detectable up to 24 hours, whereas rFIXWT and rFIXK5A were detectable only up to 15 minutes. In the tail clip bleeding model, rFIXK5R displayed significant hemostatic protection against bleeding incidence for up to 72 hours postintravenous administration of 50 IU/kg, whereas the efficacy of rFIXK5A was already reduced at 24 hours. Similarly, in the mesenteric artery thrombus model, rFIXK5R and rFIXWT demonstrated prolonged efficacy compared to rFIXK5A. CONCLUSION: Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding (rFIXK5R) to extravascular space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, whereas rFIXK5A rapidly lost its efficacy despite higher plasma exposure.

Targeting the Human ßc Receptor Inhibits Contact Dermatitis in a Transgenic Mouse Model.

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (ßc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit ßc, a property that has made ßc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of ßc has precluded testing of inhibitors of human ßc in mouse models. To overcome this problem, we developed a human ßc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human ßc instead of mouse ßc. Human ßc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human ßc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human ßc receptor transgenic mouse as a unique platform to test the inhibitors of ßc in vivo.

Kin discrimination in social yeast is mediated by cell surface receptors of the Flo11 adhesin family.

Microorganisms have evolved specific cell surface molecules that enable discrimination between cells from the same and from a different kind. Here, we investigate the role of Flo11-type cell surface adhesins from social yeasts in kin discrimination. We measure the adhesion forces mediated by Flo11A-type domains using single-cell force spectroscopy, quantify Flo11A-based cell aggregation in populations and determine the Flo11A-dependent segregation of competing yeast strains in biofilms. We find that Flo11A domains from diverse yeast species confer remarkably strong adhesion forces by establishing homotypic interactions between single cells, leading to efficient cell aggregation and biofilm formation in homogenous populations. Heterotypic interactions between Flo11A domains from different yeast species or Saccharomyces cerevisiae strains confer weak adhesive forces and lead to efficient strain segregation in heterogenous populations, indicating that in social yeasts Flo11A-mediated cell adhesion is a major mechanism for kin discrimination at species and sub-species levels. These findings, together with our structure and mutation analysis of selected Flo11A domains, provide a rationale of how cell surface receptors have evolved in microorganisms to mediate kin discrimination.

Interactions by the Fungal Flo11 Adhesin Depend on a Fibronectin Type III-like Adhesin Domain Girdled by Aromatic Bands.

Saccharomyces cerevisiae harbors a family of GPI-anchored cell wall proteins for interaction with its environment. The flocculin Flo11, a major representative of these fungal adhesins, confers formation of different types of multicellular structures such as biofilms, flors, or filaments. To understand these environment-dependent growth phenotypes on a molecular level, we solved the crystal structure of the N-terminal Flo11A domain at 0.89-Å resolution. Besides a hydrophobic apical region, the Flo11A domain consists of a ß sandwich of the fibronectin type III domain (FN3). We further show that homophilic Flo11-Flo11 interactions and heterophilic Flo11-plastic interactions solely depend on the Flo11A domain and are strongly pH dependent. These functions of Flo11A involve an apical region with its surface-exposed aromatic band, which is accompanied by acidic stretches. Together with electron microscopic reconstructions of yeast cell-cell contact sites, our data suggest that Flo11 acts as a spacer-like, pH-sensitive adhesin that resembles a membrane-tethered hydrophobin.

Uptake of xenosiderophores in Bacillus subtilis occurs with high affinity and enhances the folding stabilities of substrate binding proteins.

Siderophores play an essential role in a multitude of microbial iron acquisition pathways. Many bacteria use xenosiderophores as iron sources that are produced by different microbial species in their habitat. We investigated the capacity of xenosiderophore uptake in the soil bacterium Bacillus subtilis and found that it employs several substrate binding proteins with high specificities and affinities for different ferric siderophore species. Protein-ligand interaction studies revealed dissociation constants in the low nanomolar range, while the protein folding stabilities were remarkably increased by their high-affinity ligands. Complementary growth studies confirmed the specificity of xenosiderophore uptake in B. subtilis and showed that its fitness is strongly enhanced by the extensive utilization of non-endogenous siderophores.