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
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.