INTERACTION OF PLATELET-DERIVED GROWTH-FACTOR WITH THROMBOSPONDIN-1

Key factors that mediate vascular smooth muscle cell proliferation and migration are platelet-derived growth factor (PDGF) and thrombospondi n 1 (TSP1). We now report that PDGF(BB) bound tightly and specifically to TSP1, that this interaction was markedly dependent on the disulphi de bond arrangement in TSP1, and that binding of PDGF(BB) to TSP1 did not preclude PDGF(BB) from binding to its receptor on rat aortic vascu lar smooth-muscle cells. At physiological ionic strength and pH, PDGF( BB) bound to Ca2+-depleted TSP1 with a dissociation constant of 11 +/- 2 nM and to Ca2+-replete TSP1 with a dissociation constant of 32 +/- 5 nM. Binding was specific, as both soluble TSP1 and unlabelled PDGF(B B) competed for binding of iodinated PDGF(BB) to immobilized TSP1, whe reas other platelet alpha-granule proteins did not compete. The tertia ry structure of TSP1 is regulated by intramolecular disulphide interch ange; we found that catalysis of disulphide interchange in TSP 1 by pr otein disulphide isomerase ablated the binding of PDGF(BB). The intera ction of PDGF(BB) with TSP1 was weakened by increasing salt concentrat ion and essentially ablated at 0.65 ionic strength; it was inhibited b y heparin with a half-maximal effect at 20 i.u./ml, implying that the binding was mediated largely by ionic interactions. An anti TSP1 monoc lonal antibody decreased the binding of iodinated PDGF(BB) to PDGF rec eptor on rat aortic Vascular smooth-muscle cells by 37 +/- 2.0;, where as platelet TSP1 non-competitively inhibited binding of iodinated PDGF (BB). Uncomplexed PDGF(BB) bound to PDGF receptor with an affinity 5 /- 2 times that of PDGF(BB)-TSP1 complexes. These results suggest that TSP1 might assist in the targeting of PDGF to its receptor on vascula r smooth-muscle cells.