Endothelial Cell VE-cadherin Functions as a Receptor for the β15–42 Sequence of Fibrin

doi:10.1074/jbc.273.46.30719

Endothelial Cell VE-cadherin Functions as a Receptor for the β15–42 Sequence of Fibrin*

From the Cardeza Foundation for Hematologic Research and Division of Hematology, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Abstract

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 β15–42 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 β15–42, to HUVEC in three separate assay systems. Time-course binding of¹²⁵I-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 aK _D of 7.7 ± 1.6 nm and approximately 21,000 ± 7000 binding sites/cell. N-terminal disulfide knot derivatives in which β15–42 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 α_vβ₃ or β₁ integrins, PECAM-1, ICAM-1, or N-cadherin. In contrast, a synthetic β15–42/ovalbumin conjugate inhibited total¹²⁵I-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 specific¹²⁵I-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 β15–42 sequence, and may contribute to the mechanism through which fibrin induces angiogenesis.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant HL-20092 (to J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Dunglison M.D./Ph.D. candidate; recipient of a predoctoral fellowship from the American Heart Association, Southeastern Pennsylvania Affiliate.
↵§ To whom correspondence should be addressed: Cardeza Foundation for Hematologic Research, Thomas Jefferson University, 1015 Walnut St., Philadelphia, PA 19107. Tel.: 215-955-8458; Fax: 215-923-3836; E-mail:jos.martinez{at}mail.tju.edu.
↵2 T. L. Bach, C. Barsigian, C. H. Yaen, and J. Martinez, unpublished observations.
Abbreviations:

HUVEC

human umbilical vein endothelial cells

VE-cadherin

vascular endothelial cadherin

PECAM-1

platelet endothelial cell adhesion molecule-1

Ig

immunoglobulin family of cell adhesion molecules

ICAM-1

intercellular cell adhesion molecule-1

HADMEC

human adipose-derived microvascular endothelial cells

NDSK

N-terminal disulfide knot of fibrinogen

TBS

Tris-buffered saline.
- Received March 11, 1998.
- Revision received July 28, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.