Summary
Thrombus formation in the circulation is accompanied by covalent linkage of fibronectin
(FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal
domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the
employment of radioactively-labelled recombinant polypeptide molecules derived from
the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa (“2 fingers”),
18.5 kDa (“3 fingers”) and 31 kDa FBD (“5 fingers”), were prepared and compared to
native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots
in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated
by the incorporation of stoichiometric amounts of 14C- putrescine in the presence of plasma transglutaminase. Competitive binding experiments
to fibrin have indicated that, although the binding affinities of the FBD molecules
are lower than that of FN, substantial covalent linkage was obtained in the presence
of transglutaminase, and even in the presence of excess FN or heparin. The biological
clearance rates of radioactively labelled FBD molecules in rats and rabbits were much
higher than those of FN and fibrinogen, thus indicating their potential advantage
for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited
the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging
agents was examined in a stainless steel coil-induced thrombus model in rats and in
a jugular vein thrombus model in rabbits, using either [125I] or [111ln]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for
[125I]-12 kDa FBD and 40 and 60 for [luIn]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken
together, the results indicate that recombinant 12 kDa FBD is a good candidate for
the diagnosis of venous thrombosis.
