Thrombospondin interaction with plasminogen. Evidence for binding to a specific region of the kringle structure of plasminogen

Abstract

Platelet thrombospondin interacts with plasminogen in a specific and saturable manner. Thrombospondin was found to specifically bind to plasminogen and the nonenzyme chain of plasmin. Preincubation of 125I- labeled thrombospondin with 30 mmol/L lysine was without effect in the binding of thrombospondin to immobilized plasminogen; preincubation of 125I-labeled plasminogen with 30 mmol/L lysine, on the other hand, significantly reduced the binding of plasminogen to immobilized thrombospondin, suggesting that the interaction of thrombospondin with plasminogen is not the direct result of the lysine binding sites of plasminogen. Arginine and benzamidine, ligands known to specifically bind to the kringle 5 domain of plasminogen, blocked the binding of thrombospondin to plasminogen. Limited elastase proteolysis of plasminogen and plasmin resulted in the generation of two distinct thrombospondin binding domains, one of which was retained on lysine- agarose. The isolation and amino-terminal analysis of these domains following elastase proteolysis of plasminogen identified them, respectively, as a domain containing kringle structures 4 and 5 and plasmin and the other domain consisting of kringle 5-plasmin. A 16- residue synthetic peptide, which represents the amino acids linking kringle 4 to kringle 5 (residues 435–450 of native plasminogen), was without effect in either binding to thrombospondin or blocking the binding of thrombospondin to plasminogen. Plasminogen, therefore, possesses a single thrombospondin interactive site that is independent of, but influenced by, the lysine binding site containing kringle structures and most likely is located within the kringle 5 domain.

Characterization of the antiplasmin activity of human thrombospondin-1 in solution

“…From among the kringle domains, we propose that K5 plays an important role, since TEA inhibited thrombospondin digestion by plasmin and miniplasmin similarly. This conclusion supports the work of DePoli et al [17].…”

“…Based on studies with lysine analogues, an important role for the high-affinity lysine-binding site (located in the kringle-I or KI domain of plasminogen) in mediating the plasmin(ogen)thrombospondin interaction was suggested. DePoli et al [17] proposed that the plasminogen-thrombospondin interaction occurs via kringle 5 (KS) since they were able to demonstrate binding of 1251-thrombospondin to an elastase-digestion fragment of plasminogen (miniplasminogen) which contains only K5 and the catalytic domain. The studies presented by Depoli et al [17] were performed using plasminogen which was denatured and/or blotted on nitrocellulose, and in some experiments binding was not inhibited by lysine.…”

“…DePoli et al [17] proposed that the plasminogen-thrombospondin interaction occurs via kringle 5 (KS) since they were able to demonstrate binding of 1251-thrombospondin to an elastase-digestion fragment of plasminogen (miniplasminogen) which contains only K5 and the catalytic domain. The studies presented by Depoli et al [17] were performed using plasminogen which was denatured and/or blotted on nitrocellulose, and in some experiments binding was not inhibited by lysine. Therefore, the importance of the various plasmin(ogen) kringle domains in the plasmin(ogen)thrombospondin interaction is not clearly resolved.…”

Binding of human plasminogen to basement-membrane (type IV) collagen

“…Although the interaction of plasminogen with intact ECMs as well as several isolated matrix protein components has been previously described (Salonen et al, 1985;Knudsen et al, 1986;DePoli et al, 1989;Preissner, 1990), the binding of plasminogen to the major ECM protein, type IV collagen, has remained uncharacterized. We have previously demonstrated that protein components of the ECM are capable of regulating plasminogen activation catalysed by tissue-associated plasminogen activators (Stack et al, 1990).…”

“…This binding is facilitated by a series of five triple-loop disulphide-bonded bridge structures referred to as kringles (Sottrup-Jensen et al, 1978), which are involved in targeting of the zymogen to the surface of the fibrin clot. In addition to fibrin, specific binding of plasminogen to isolated ECM components such as thrombospondin (DePoli et al, 1989), fibronectin (Salonen et al, 1985) and vitronectin (Preissner, 1990) has also been reported. Intact ECMs synthesized and secreted by endothelial cells were shown to bind plasminogen as well as enhance the efficiency of plasminogen activation (Knudsen et al, 1986).…”