STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF PROTEOLYTIC FRAGMENTS DERIVED FROM THE C-TERMINAL REGIONS OF BOVINE FIBRINOGEN

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been ch aracterized by sequence analysis, differential scanning calorimetry an d circular dichroism. Determination of the extremities of the polypept ide chains forming individual fragments allowed the scheme of proteoly sis and the borders between domains in the D region of fibrinogen to b e established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prep ared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectiv ely, strongly supporting a coiled-coil structure for this region of th e fibrinogen molecule. The D-X and D-Z fragments, prepared from a chym otryptic digest of the D-LA fragment, were found to be similar to the D-L and D-Y fragments, respectively, except for an internal cleavage a t K393-T394 in their beta chains. This cleavage leads to destabilizati on of all thermolabile domains, indicating interaction between them. T he D-L and D-Y fragments, containing only one polymerization site in t heir beta chains, were able to inhibit fibrin polymerization at high c oncentration. However, these same fragments failed to bind to fibrin-S epharose under conditions where their structural analogues, D-X and D- Z, were tightly bound, indicating that cleavage after K393 substantial ly increases the affinity of this site.