Platelet thrombospondin (TSP) interacts with plasminogen in a specific and saturable manner. TSP can form a trimolecular complex with histidine-rich glycoprotein and plasminogen and the plasminogen within such complexes can reportedly be activated by tissue plasminogen activator. We have studied the interaction of TSP with plasminogen using Western blotting of plasminogen, reduced plasmin and the elastase-generated fragments of plasminogen and their binding of iodinated TSP. TSP was found to specifically bind to plasminogen and the heavy (non-enzyme) chain of plasmin in a calcium-independent manner. Binding could be blocked by preincubation of the immobilized plasminogen or plasmin with an excess of unlabeled TSP. Plasminogen domains (kringles) were generated by limited eTastase proteolysis. TSP bound specifically to a single 51 kDa plasminogen fragment. The elastase-generated fragments were separated by lysine-Sepharose chromatography and their identities established by amino acid composition and amino-terminal sequence analysis. The 51 kDa plasminogen fragment bound to lysine-Sepharose and had an amino-terminal sequence corresponding to kringle 4 (K4) and a composition consistent with that of K4-K5-plasmin. TSP binding to this fragment was not blocked by the presence of an excess of the fragment K1-K2-K3, K4, nor miniplasminogen (K5-plasmin). Binding does not appear to be directly dependent upon the specific high-affinity lysine binding site of the 51 kDa fragment. Our data suggests that thrombospondin interacts with plasminogen at a single distinct site, and that this recognition site is at or near the K4-K5 contiguous region of plasminogen.
