| Autor: |
Bach TL; Cardeza Foundation for Hematologic Research and Division of Hematology, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA., Barsigian C, Yaen CH, Martinez J |
| Abstrakt: |
The contact of fibrin with the apical surface of human umbilical vein endothelial cells (HUVEC) can induce capillary tube formation via the interaction of fibrin beta15-42 with a putative cell receptor (Chalupowicz, D. G., Chowdhury, Z. A., Bach, T. L., Barsigian, C., and Martinez, J. (1995) J. Cell Biol. 130, 207-215). To characterize this interaction, we studied the binding of the thrombin-cleaved N-terminal disulfide knot of fibrin (NDSK II), a dimeric fragment with exposed beta15-42, to HUVEC in three separate assay systems. Time-course binding of 125I-NDSK II to HUVEC monolayers or suspensions revealed that binding was specific at 50-60%, as determined by the addition of unlabeled NDSK II. Specific binding of 125I-NDSK II to HUVEC was 70% reversible by dilution or by competition, and was found to be divalent cation-independent. Binding plateaued after 10 min at a saturation of 15-20 nM. Scatchard analysis using the LIGAND computer program defined a single population of receptors with a KD of 7.7 +/- 1.6 nM and approximately 21,000 +/- 7000 binding sites/cell. N-terminal disulfide knot derivatives in which beta15-42 was absent (NDSK 325) or unexposed (NDSK, NDSK I) did not show specific binding. Specific binding of 125I-NDSK II could not be inhibited by RGDS or by antibodies to the alphavbeta3 or beta1 integrins, PECAM-1, ICAM-1, or N-cadherin. In contrast, a synthetic beta15-42/ovalbumin conjugate inhibited total 125I-NDSK II binding by 47 +/- 19% (corresponding to 95% of specific 125I-NDSK II bound) and a monoclonal antibody to vascular endothelial cadherin (VE-cadherin) inhibited binding by 35 +/- 8% (corresponding to 70% of specific 125I-NDSK II bound). Another assay was based on the capture of cadherins from HUVEC lysates by a polyclonal pan-cadherin antibody immobilized on plastic dishes. Binding of NDSK II to the captured cadherins was 89 +/- 5% specific, while specific binding of NDSK 325 and NDSK was negligible. An immortalized line of human adipose-derived microvascular endothelial cells, which express N-cadherin but not VE-cadherin, demonstrated no specific binding of NDSK II by the capture assay. These data define a novel interaction of fibrin with VE-cadherin, which is mediated by the fibrin N-terminal beta15-42 sequence, and may contribute to the mechanism through which fibrin induces angiogenesis. |
