Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin

Citation
Ts. Panetti et al., Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin, J BIOL CHEM, 274(1), 1999, pp. 430-437
Citations number
59
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
274
Issue
1
Year of publication
1999
Pages
430 - 437
Database
ISI
SICI code
0021-9258(19990101)274:1<430:IORPAP>2.0.ZU;2-I
Abstract
Many properties have been assigned to the procollagen and properdin (Type I ) modules of thrombospondin-1 (TSP1) based on activities of large proteolyt ic fragments of TSP1 or peptides containing TSP1-derived sequences. To exam ine the activities of the modules more exactly, we expressed the first prop erdin module (P1); the third properdin module (P3); the first and second pr operdin modules (P12); the first, second, and third properdin modules (P123 ); and the procollagen module with the first, second, and third properdin m odules (CP123) in the GELEX expression vector (GE1) using the baculovirus s ystem. 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 e xpressed by insect cells, secreted into the culture medium, and purified by gelatin-agarose affinity chromatography, P123 shared with TSP1 a resistanc e 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 s odium chloride. In cross-linking experiments employing activated recombinan t factor XIII and the transglutaminase cross-linking site in the fibronecti n-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 th e complex indicated that cross-linking was to the portion of the fibrin alp ha-chain remaining in the D-dimer of plasmin digests. P123 also cross-linke d to the Aa-chain of unclotted fibrinogen. P123 competed for I-125-TSP1 inc orporation into the fibrin clot. P123 did not crosslink to plasminogen, his tidine-rich glycoprotein, fibronectin, or plasma globulins other than fibri nogen/ fibrin, These results indicate that the properdin modules of TSP1 sp ecifically interact with fibrinogen/ fibrin but not with heparin under phys iologic conditions.