Interaction of Recombinant Procollagen and Properdin Modules of Thrombospondin-1 with Heparin and Fibrinogen/Fibrin

Abstract

Many properties have been assigned to the procollagen and properdin (Type I) modules of thrombospondin-1 (TSP1) based on activities of large proteolytic fragments of TSP1 or peptides containing TSP1-derived sequences. To examine the activities of the modules more exactly, we expressed the first properdin module (P1); the third properdin module (P3); the first and second properdin modules (P12); the first, second, and third properdin modules (P123); and the procollagen module with the first, second, and third properdin modules (CP123) in the GELEX expression vector (GE1) using the baculovirus system. GE1 encodes the pre-pro sequence, the transglutaminase cross-linking site(s), the protease-sensitive site, and the gelatin binding domain from the amino terminus of rat fibronectin. All five recombinant proteins were expressed by insect cells, secreted into the culture medium, and purified by gelatinagarose affinity chromatography. P123 shared with TSP1 a resistance to trypsin unless reduced and alkylated. P12/GE1, P123/GE1, and CP123/GE1 bound poorly to heparin-agarose except in the absence of sodium chloride, whereas peptides based on P2 are known to bind to heparin in up to 150 mM sodium chloride. In cross-linking experiments employing activated recombinant factor XIII and the transglutaminase cross-linking site in the fibronectin-derived sequence, P12/GE1, P123/GE1, CP123/GE1, and P3/GE1 but not P1/GE1 became incorporated into a fibrin clot more than GE1 alone. Analysis of the complex indicated that cross-linking was to the portion of the fibrin ␣-chain remaining in the D-dimer of plasmin digests. P123 also cross-linked to the A␣-chain of unclotted fibrinogen. P123 competed for 125 I-TSP1 incorporation into the fibrin clot. P123 did not crosslink to plasminogen, histidine-rich glycoprotein, fibronectin, or plasma globulins other than fibrinogen/ fibrin. These results indicate that the properdin modules of TSP1 specifically interact with fibrinogen/ fibrin but not with heparin under physiologic conditions.

Thrombospondin 1 as a scavenger for matrix-associated fibroblast growth factor 2

“…Three lines of evidence suggest that inhibition of FGF-2 binding to the matrix is not caused by a competition with TSP-1 for binding to HSPGs: (1) preincubation of the matrix with TSP-1 before the addition of FGF-2, to allow TSP-1 binding to exposed HSPG, did not reduce the amount of bound FGF-2, as would be expected if TSP-1 masked the HSPG binding sites for FGF-2; (2) the 25-kDa fragment of TSP-1 containing the main heparin-binding domain was inactive in preventing FGF-2 binding that was instead inhibited by the 140-kDa fragment. This finding suggests that the high affinity HSPG binding site of TSP-1 is not involved in FGF-2 mobilization, although we cannot completely rule out the involvement of sequences in the type I repeat of TSP-1 described to interact with heparin with low affinity, whose actual biologic relevance is still debated 25,42,43 ; and (3) TSP-1 binds to FGF-2 with high affinity, similar to the affinity of FGF-2 for heparin. Altogether, this evidence supports the hypothesis that TSP-1 binding to FGF-2 might sequester the growth factor, preventing its binding to the matrix.…”

Thrombospondin Type 1 Repeats Interact with Matrix Metalloproteinase 2

“…Thrombospondins 1 and 2 share a number of structural similarities and have been reported to bind to a number of cell surface membrane and matrix proteins (17,42). In addition, unlike TSPs 3, 4, and 5, TSPs 1 and 2 inhibit angiogenesis.…”

Structure of von Willebrand Factor-cleaving Protease (ADAMTS13), a Metalloprotease Involved in Thrombotic Thrombocytopenic Purpura

“…Recombinant ADAMTS12 also binds to extracellular matrix (44). In another context, the TSP1 repeats of platelet thrombospondin appear to bind several glycoproteins and proteoglycans on cell surfaces and in extracellular matrix (46), as well as fibrinogen or fibrin (47). TSP1 repeats were discovered recently to bear two unusual post-translational modifications, an ␣-mannosyl group on the C-2 position of Trp and the disaccharide Glc-Fuc-O-Ser/ Thr in the motif CSX(S/T)CG (32).…”