Functional changes in the conformation of thrombospondin-1 during complexation with fibronectin or heparin

Thrombospondin-l (TSP-1) interacts specifically with heparin and fibronecti n in vitro and colocalizes with fibronectin and heparan sulfate in the extr acellular matrix (ECM). Its conformation is strongly dependent on Ca2+ conc entration. We have previously shown that both heparin and fibronectin have two binding sites on the TSP-1 subunit which may require conformational cha nge for their occupancy (R. Dardik and J. Lahav, 1987, fur. J. Biochem. 168 , 347; ibid 1989, 185, 581). To investigate the effect of TSP-I binding to fibronectin and heparin on its functional conformation, TSP-1 was subjected to proteolysis in the presence and absence of ligands and of Ca2+. We foun d that while trypsin cleavage of free TSP-1 resulted in the inactivation of ligand binding, TSP-1 bound to either fibronectin or heparin remained stab ly associated with these ligands. Cleavage by thrombin or tissue plasminoge n activator (tPA) showed that Ca2+-depleted TSP-1, when bound to fibronecti n or to heparin, yielded proteolytic cleavage patterns typical of the Ca2+- containing form. Cleavage by chymotrypsin was not affected by binding to fi bronectin or heparin; hence loss of proteolytic susceptibility was not due to steric hindrance by the ligands. Taken together, these results indicate that: (A) binding of TSP-I to fibronectin or heparin is a two-step mechanis m where binding to one site leads to conformational changes that enable bin ding to the second site; (B) TSP-1 in complex with fibronectin or heparin a dopts the Ca2+-containing conformation in the absence of Ca2+; and (C) such complexes are highly resistant to cleavage by tPA and, if cleaved by other enzymes, the TSP-1 fragments remain bound to other ECM components. These c haracteristics have profound significance for platelet adhesion and cell mi gration into wounds where Ca2+ concentrations are reduced. (C) 1999 Academi c Press.