Interaction of Fibrin(ogen) with the Endothelial Cell Receptor VE-Cadherin: Mapping of the Receptor-Binding Site in the NH2-Terminal Portions of the Fibrin β Chains

Abstract

Interaction of fibrin with endothelial cells stimulates capillary tube formation thus promoting angiogenesis. This interaction occurs via endothelial cell receptor VE-cadherin and fibrin β chain 15−42 regions [Bach, T. L., et al. (1998) J. Biol. Chem. 272, 30719−30728]. To clarify the mechanism of this interaction, we expressed in Escherichia coli a number of recombinant fibrin(ogen) fragments containing the β15−42 region or the VE-cad(1−2) and VE-cad(1−4) fragments encompassing two and four extracellular NH₂-terminal domains of VE-cadherin, respectively, and tested interaction between them by surface plasmon resonance and ELISA. Neither the recombinant Bβ1−57 or Bβ1−64 fragments, nor β15−57 or β15−64 prepared from the latter fragments by thrombin treatment to remove fibrinopeptides B, bound the recombinant VE-cadherin fragments. At the same time, a dimeric recombinant thrombin-treated (β15−66)₂ fragment, which had been disulfide-linked via Cys65 to mimic the dimeric arrangement of the β chains in fibrin, bound VE-cad(1−4) well, but not VE-cad(1−2); no binding was observed with the untreated (Bβ1−66)₂ dimer. We next mutated several residues in the dimer, His16, Arg17, Pro18, and Asp20, and tested the interaction of the thrombin-treated mutants with VE-cad(1−4) by ligand blotting and surface plasmon resonance. No binding was observed with the H16A and R17Q single mutants and the H16P, P18V double mutant while the P18A and D20N single mutants bound VE-cad(1−4) with the same affinity as the thrombin-treated wild-type dimer. These results indicate that the VE-cadherin binding site in fibrin includes NH₂-terminal regions of both fibrin β-chains, that His16 and Arg17 are critical for the binding, and that the third and/or fourth extracellular domains of VE-cadherin are required for the binding to occur.