ENDOTHELIAL-CELL VE-CADHERIN FUNCTIONS AS A RECEPTOR FOR THE BETA-15-42 SEQUENCE OF FIBRIN

The content of fibrin with the apical surface of human umbilical vein endothelial cells (HUVEC) can induce capillary tube formation via the interaction of fibrin beta 15-42 with a putative cell receptor (Chalup owicz, D. G., Chowdhury, Z. A., Bach, T. L., Barsigian, C., and Martin ez, J. (1945) J. Cell Biol. 130, 207-215). To characterize this intera ction, we studied the binding of the thrombin-cleaved N-terminal disul fide knot of fibrin (NDSK II), a dimeric fragment with exposed beta 15 -42, to HUVEC in three separate assay systems. Time-course binding of I-125-NDSK II to HUVEC monolayers or suspensions revealed that binding was specific at 50-60%, as determined by the addition of unlabeled ND SK II. Specific binding of I-125-NDSK II to HUVEC was 70% reversible b y dilution or by competition, and was found to be divalent cation-inde pendent. Binding plateaued after 10 min at a saturation of 15-20 nM. S catchard analysis using the LIGAND computer program defined a single p opulation of receptors with a K-D of 7.7 +/- 1.6 nM and approximately 21,000 +/- 7000 binding sites/cell. N-terminal disulfide knot derivati ves in which beta 15-42 was absent (NDSK 325) or unexposed (NDSK, NDSK I) did not show specific binding. Specific binding of I-125-NDSK II c ould not be inhibited by RGDS or by antibodies to the alpha(v)beta(3) or beta(1) integrins, PECAM-1, ICAM-1, or N-cadherin, In contrast, a s ynthetic beta 15-42/ovalbumin conjugate inhibited total I-125-NDSK II binding by 47 +/- 19% (corresponding to 95% of specific I-125-NDSK II bound) and a monoclonal antibody to vascular endothelial cadherin (VE- cadherin) inhibited binding by 35 +/- 8% (corresponding to 70% of spec ific 125I-NDSK II bound). Another assay was based on the capture of ca dherins from HUVEC lysates by a polyclonal pan-cadherin antibody immob ilized on plastic dishes. Binding of NDSK II to the captured cadherins was 89 +/- 5% specific, while specific binding of NDSK 325 and NDSK w as negligible. An immortalized line of human adipose-derived microvasc ular endothelial cells, which express N-cadherin but not VE-cadherin, demonstrated no specific binding of NDSK II by the capture assay. Thes e data define a novel interaction of fibrin with VE-cadherin, which is mediated by the fibrin N-terminal beta 15-42 sequence, and may contri bute to the mechanism through which fibrin induces angiogenesis.