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.

Thrombospondin is a member of a super class of proteins referred to as adhesive glycoproteins; these adhesive proteins are important mediators of cell-to-cell as well as cell-to-matrix interactions. The interaction of platelets with one another and with selected products of the vessel wall are mediated in part by fibrinogen (FG), fibronectin (FN), von Willebrand factor (vWF), and thrombospondin (TSP). 1 Each of these glycoproteins is only minimally interactive with resting platelets, but specific binding sites are expressed on the activated platelet surface. 2 ' 5 The structure of each of these glycoproteins shares a common tripeptide sequence, ArgGly-Asp-(RGD), which is thought to mediate the interaction of FG, FN, and vWF with the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) complex. 6 " 8 ' 47 Current information indicates that TSP, although it shares the RGD sequence, 9 does not adhere to the activated platelet via the GPIIb/IIIa complex. 8 ' 10 TSP is a high molecular weight glycoprotein; the estimated molecular mass is dependent on the method of analysis and ranges from 145,000 d 11 to 185,000 d 12 for each of the apparently identical chains of the triple-chained protein. 9,13 TSP has been shown to interact with platelets, 14-16 heparin, 17,18 type V collagen, 19 glycolipids, 20,21 as well as such soluble proteins as FN, 22 plasminogen, 23 histidine-rich glycoprotein (HRGP), 24 and FG. [25][26][27][28][29] The functional regions of the TSP molecule have been examined in a preliminary manner through a combination of limited proteolysis and through the use of monoclonal antibodies. 9,30,31 The aminoterminal 23 kd region of the TSP molecule (the single-chain molecular mass of 180 kd is used for TSP), which can be generated by thrombin, plasmin, thermolysin, and chymotrypsin proteolysis of TSP, 32-35 (see also Lawler and Hynes, Frazier et al, and Aiken et al in this issue of Seminars), is the heparin-binding domain of TSP. Chymotryptic digestion of TSP in the absence of calcium yields a 70 kd core structure, which contains the disulfide cross-linking sites 36,37 (see Lawler and Hynes and Frazier et al in this issue of Seminars); this region contains the peptide segment or segments that bind type V collagen, FN, plasminogen, FG, and laminin in solid-phase binding assays. 36,37 Utilizing a series of monoclonal antibodies (Mabs) that recognize calcium-dependent structures of human TSP, Dixit et al 38 have developed at least four functionally significant Mabs. The first of these, designated A2.5, 38 reacts with the heparin-binding, amino-terminal 25 kd domain. The second, A6.1, recognizes the 70 kd chymotryptic resistant core fragment. The third, D4.6, recognizes a 40 kd fragment that is present in the COOH-terminal half of the TSP molecule. The fourth, C6.7, recognizes the COOH-terminal 18 kd peptide of TSP, which has been shown to contain the platelet and red blood cell interactive domain. 38,39 We have begun an examination of the binding characteristics of TSP using a blot-binding assay. 29 This work is based on ...

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.

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.