**3.1 Importance of Fg peptide 15-42 in Fg-endothelial cell interactions**

Fibrin(ogen) 15-42 sequences support a diverse array of biological functions mediated by fibrin(ogen). Although the primary structure of fibrinopeptide B (FPB) is poorly conserved across species, the fibrin 15-42 domain is highly conserved, implying evolutionary conservation of function (Courtney et al., 1994). The 15-42 region constitutes a cryptic domain in soluble Fg that is exposed in fibrin after thrombin cleavage (Odrljin et al., 1996b). Both the HBD and overlapping binding site for VE-cadherin are localized to 15-42. VE-

The Role of Fibrin(ogen) in Transendothelial Cell Migration During Breast Cancer Metastasis 193

However, the neutralizing antibodies (T2G1 and BV9) did not completely inhibit Fg-induced permeability (**Fig. 7B**), suggesting that additional cell recognition domains on Fg participate

Fig. 6. Fg-induced EC permeability involves Fg 15-42 and VE-cadherin. Cells were grown to confluency on Millicell™ 24-well cell culture inserts. Panel 6A, HUVEC were left untreated (control) or treated for 15 min with increasing concentrations of Fg or VEGF as indicated. Panel 6B, HUVEC were treated with 30 nM of Fg plus 1 mg/ml FITC-Dextran for the times indicated. The FITC-Dextran flux to the bottom chamber was measured by fluorometry and the data presented as the mean relative FITC-Dextran Flux ± SEM. Data points were derived from 3 or more independent experiments with the total number of replicates per condition ranging from 6-13. (Reprinted from (Sahni et al., 2009) with permission). P-values can be

Fig. 7. Fg-induced EC permeability involves Fg 15-42 sequences and VE-cadherin. Panel 7A, schematics of the aminoterminus of the fibrin(ogen) B chain and the domain structure of VE-cadherin are depicted. The arrow denotes the thrombin cleavage site for release of FPB. The 18C6 epitope maps to FPB, the T2G1 epitope maps to 15-21 and the VE-cadherin binding site on fibrin maps to 15-42. The epitope of the VE-cadherin-specific monoclonal antibody BV9 maps to the third and fourth extracellular domains (EC3-EC4). The fibrin 15-42 binding site on VE cadherin maps to EC3 near the EC3-EC4 junction. TM, transmembrane domain.

Panel 7B, all monoclonal antibodies used are IgG1 isotype murine antibodies and

in fibrin(ogen)-induced vascular permeability.

found in ref (Sahni et al., 2009).

cadherin mediates homophilic cell-cell adhesion critical for the maintenance of barrier integrity of the endothelium. Disruption of VE-cadherin-mediated endothelial barrier function leads to altered vascular permeability found in a number of diseases including ischemia-reperfusion (IR) injury, inflammation, angiogenesis, and cancer growth and metastasis (discussed in (Sahni et al., 2009)). Exposure of 15-42 and binding by VE-cadherin is also required for endothelial capillary tube formation in fibrin gels (Bach et al., 1998a; Chalupowicz et al., 1995); portions of the third extracellular domain (EC3) of VE-cadherin constitute a fibrin 15-42 receptor (Bach et al., 1998b; Yakovlev & Medved, 2009). Newly exposed chain residues, 15-GHRP-18, play a critical role in fibrin monomer aggregation during polymerization and clot formation during secondary hemostasis (Mosesson, 2005). Furthermore, exposure of the 15-42 domain mediates heparin-dependent fibrin binding to endothelial cell surfaces (Odrljin et al., 1996a); promotes endothelial cell adhesion and spreading (Bunce et al., 1992); promotes the release of endothelial cell-specific markers of endothelial activation (Ribes et al., 1989); and stimulates proliferation of endothelial cells, fibroblasts and cancer cells (Rybarczyk et al., 2003; Sahni et al., 2008; Sporn et al., 1995).
