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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:e16.)
© 2000 American Heart Association, Inc.

ATVB Electronic Pages

Fibronectin in an Extracellular Matrix of Cultured Endothelial Cells Supports Platelet Adhesion via Its Ninth Type III Repeat

A Comparison With Platelet Adhesion to Isolated Fibronectin

Sara Beumer; Glenda J. Heijnen-Snyder; Martin J. W. IJsseldijk; Philip G. de Groot; Jan J. Sixma

From University Hospital Utrecht, Department of Hematology, Utrecht, Netherlands.

Correspondence to Dr J.J. Sixma, University Hospital Utrecht, Department of Hematology (G03.647), Heidelberglaan 100/PO Box 85500, 3584 CX Utrecht/3508 GA Utrecht, Netherlands. E-mail jsixma{at}laboratory.azu.nl

Abstract

Abstract—We investigated the involvement of different domains of fibronectin in mediating platelet adhesion to fibronectin in the extracellular matrix (ECM) of cultured endothelial cells under flow conditions. Polyclonal anti-fibronectin antibodies were absorbed with Sepharose to which no protein, intact fibronectin, or different fibronectin fragments had been coupled to obtain supernatants (Sups) (Sup⁰, Sup^FN, and Sup^{name of the fragment}, respectively) from which a specific part of the antibodies had been removed. Treatment of the ECM before perfusion with Sup⁰ resulted in a 36% decrease in platelet coverage, whereas treatment with Sup^FN resulted in maximal adhesion. Treatment of the ECM with supernatants from which antibodies directed against the gelatin- or heparin-binding domain had been removed showed the same inhibition as treatment with Sup⁰. Removal of antibodies directed to the 120-kDa cell-binding domain resulted in a level of adhesion equal to the level found when the ECM was treated with Sup^FN. Further analysis of this central region showed that only treatment with supernatants from which antibodies directed to the ninth type III repeat (III-9) of fibronectin had been removed resulted in a significantly higher adhesion than treatment with Sup⁰. Studies of adhesion to the fragments themselves showed that only fragments containing III-10 were able to support adhesion. Mutation of the Arg-Gly-Asp (RGD) sequence into Arg-Gly-Glu (RGE) in one of those fragments resulted in a complete loss of adhesive capacity. These data suggest that platelet adhesion to fibronectin in the ECM depends on III-9, whereas III-10 does not seem to be required. For platelet adhesion to isolated fibronectin, an intact RGD sequence seems to be crucial.

Key Words: platelet adhesion • fibronectin • endothelial cell matrix