CHARACTERIZATION OF THE ANTIPLASMIN ACTIVITY OF HUMAN THROMBOSPONDIN-1 IN SOLUTION

These studies demonstrate relatively rapid association of plasmin with thrombospondin and the effects of this interaction on plasmin activity towards D-Val-L-LeU-L-Lys p-nitroanilide hydrochloride (S-2251) and the proteinase inhibitors alpha2-antiplasmin (alpha2AP) and alpha2-macroglobulin (alpha2M). Binding of plasmin to thrombospondin reached an apparent reversible equilibrium within 3 min at 22-degrees-C. The amidase activity of bound plasmin was inhibited. An analysis of S-2251 hydrolysis indicated that thrombospondin is a linear mixed-type plasmin inhibitor. The dissociation constant (K(D)) for the binding of plasmin to thrombospondin was 0.5 muM, assuming one plasmin binding site per thrombospondin homotrimer. Plasmin and miniplasmin slowly cleaved thrombospondin, yielding products which were comparable with those generated by other proteinases. Tranexamic acid inhibited the digestion of thrombospondin by plasmin and miniplasmin, suggesting an important role for the kringle-5 domain in this process. When plasmin was incubated first with thrombospondin and then with alpha2AP, plasmin that was apparently bound to thrombospondin reacted with alpha2AP at a decreased rate; however, within 20 min, all of the plasmin was recovered in complex with alpha2AP. Similar results were obtained with alpha2M. Transfer of plasmin from thrombospondin to alpha2AP or alpha2M probably required plasmin-thrombospondin-complex dissociation. A low level of reaction of alpha2AP with thrombospondin-associated plasmin could not be ruled out. These results demonstrate that the activity of plasmin, when bound to thrombospondin, is greatly diminished or eliminated. The plasmin-thrombospondin complex, which is formed within 3 min, is fully reversible and the associated plasmin is in a latent form protected from proteinase inhibitors. Therefore, thrombospondin may regulate plasmin activity in a manner which is distinct from conventional proteinase inhibitors and other extracellular-matrix proteins.