An Investigation of Factor XIII Binding to Recombinant γ′/γ′ and γ/γ′ Fibrinogen.

Abstract

Increased levels of the alternatively spliced γ′ chain of fibrinogen have been measured in patients suffering from coronary artery disease (Lovely et al. Thromb. Haemost. 2002; 88:26–31). Others have suggested that the γ′ chain serves as a binding site for factor XIII (Siebenlist et al. Biochemistry 1996; 35:10448–10453), thus increasing the amount of cross-linking and therefore the stability of the γ′-containing clots (Falls and Farrell JBC 1997; 272:14251–14256). We have expressed recombinant γ′/γ′ homodimer and γ/γ′ heterodimer fibrinogen variants in CHO cells and characterized their polymerization by turbidity. Compared to normal recombinant γ/γ fibrinogen, γ′/γ′ fibrinogen displayed a slower rate of polymerization and a lower final absorbance. γ/γ′ Heterodimer fibrinogen had both a polymerization rate and final absorbance that was intermediate between γ/γ and γ′/γ′. These observations suggest that the presence of the γ′ chain slows lateral aggregation and leads to the formation of thinner fibers. These variants were then used to investigate the binding of factor XIII to fibrinogen. We designed an ELISA assay to measure this binding by immobilizing fibrinogen on a 96-well plate, incubating with factor XIII, and measuring bound factor XIII with an anti-factor XIII A-subunit antibody. Normal recombinant γ/γ fibrinogen bound factor XIII zymogen (A2B2) with a KD of 4.1 x 10−8 M +/− 1.3 x 10−8 M, similar to previous data for plasma fibrinogen. Surprisingly, both γ′/γ′ and γ/γ′ fibrinogen variants bound factor XIII zymogen with the same KD as did γ/γ fibrinogen. As shown by ELISAs with antibodies specific for either γ or γ′, the C-termini of both the γ and the γ′ chains are accessible on this immobilized fibrinogen. These findings suggest that the C-terminus of the γ′ chain is not a critical element in the binding of factor XIII zymogen. Our findings differ from previously reported findings. Further studies are in progress to reconcile these differences.