IDENTIFICATION AND CHARACTERIZATION OF THE THROMBIN BINDING-SITES ON FIBRIN

Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity, We investigated the loca tion of these thrombin binding sites by assessing the binding of throm bin to fibrin lacking or containing gamma' chains, which are fibrinoge n gamma chain variants that contain a highly anionic carboxyl-terminal sequence, We found the high affinity thrombin binding site to be loca ted exclusively in D domains on gamma' chains (K-alpha, 4.9 x 10(6) M( -1); n, 1.05 per gamma' chain), whereas the low affinity thrombin bind ing site was in the fibrin E domain (K-alpha, 0.29 x 10(6) M(-1); n, 1 .69 per molecule), The amino terminal beta 15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this s equence. The tyrosine-sulfated, thrombin exosite-binding hirudin pepti de, S-Hir(53-64) (hirugen), inhibited both low and high affinity throm bin binding to fibrin (IC50 1.4 and 3.0 mu M, respectively), The prese nce of the high affinity gamma' chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin t ime measurements, and thrombin exosite binding to fibrin at either sit e did not inhibit its catalytic activity toward a small thrombin subst rate, S-2238. We infer from these findings that there are two low affi nity non substrate thrombin binding sites, one in each half of the dim eric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen, The high a ffinity thrombin binding site on gamma' chains is a constitutive featu re of fibrin as well as fibrinogen.