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
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) (Sup(0), 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 Sup(0) 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 Sup(0). 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 Sup(0). 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.
Beumer
S
University Hospital Utrecht, Department of Hematology, Utrecht, Netherlands.
Heijnen-Snyder
GJ
IJsseldijk
MJ
de Groot PG
Sixma
JJ