| Abstract
| - Interaction of fibrin with endothelial cells stimulates capillary tube formation thus promotingangiogenesis. This interaction occurs via endothelial cell receptor VE-cadherin and fibrin β chain 15−42regions [Bach, T. L., et al. (1998) J. Biol. Chem. 272, 30719−30728]. To clarify the mechanism of thisinteraction, we expressed in Escherichia coli a number of recombinant fibrin(ogen) fragments containingthe β15−42 region or the VE-cad(1−2) and VE-cad(1−4) fragments encompassing two and fourextracellular NH2-terminal domains of VE-cadherin, respectively, and tested interaction between them bysurface plasmon resonance and ELISA. Neither the recombinant Bβ1−57 or Bβ1−64 fragments, norβ15−57 or β15−64 prepared from the latter fragments by thrombin treatment to remove fibrinopeptidesB, 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 arrangementof the β chains in fibrin, bound VE-cad(1−4) well, but not VE-cad(1−2); no binding was observed withthe 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 blottingand surface plasmon resonance. No binding was observed with the H16A and R17Q single mutants andthe H16P, P18V double mutant while the P18A and D20N single mutants bound VE-cad(1−4) with thesame affinity as the thrombin-treated wild-type dimer. These results indicate that the VE-cadherin bindingsite in fibrin includes NH2-terminal regions of both fibrin β-chains, that His16 and Arg17 are critical forthe binding, and that the third and/or fourth extracellular domains of VE-cadherin are required for thebinding to occur.
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