RECOMBINANT POLYPEPTIDES DERIVED FROM THE FIBRIN BINDING DOMAIN OF FIBRONECTIN ARE POTENTIAL AGENTS FOR THE IMAGING OF BLOOD-CLOTS

Thrombus formation in the circulation is accompanied by covalent linka ge of fibronectin (FN) through transglutamination of glutamine no. 3 i n the fibrin binding amino terminal domain (FED) of FN. We have exploi ted this phenomenon for thrombus detection by the employment of radioa ctively-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 respe ct to their ability to attach to fibrin clots in vitro and in vivo. Th e accessibility of Gin-3 in these molecules was demonstrated by the in corporation of stoichiometric amounts of C-14- putrescine in the prese nce of plasma transglutaminase. Competitive binding experiments to fib rin have indicated that, although the binding affinities of the FBD mo lecules are lower than that of FN, substantial covalent linkage was ob tained in the presence of transglutaminase, and even in the presence o f excess FN or heparin. The biological clearance rates of radioactivel y labelled FBD molecules in rats and rabbits were much higher than tho se 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 k Da 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 mo del in rabbits, using either [I-125] or [In-111]-labelled materials. A t 24 h. clot-to-blood ratios ranged between 10 and 22 for [I-125]-12 k Da FBD and 40 and 60 for [In-111]-12 kDa FBD. Ln the rat model, hepari n did not inhibit the uptake of FBD. Taken together, the results indic ate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.