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American Society for Biochemistry and Molecular Biology

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Journal of Biological Chemistry

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DOI: 10.1074/jbc.271.38.23121


Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity. We investigated the location of these thrombin binding sites by assessing the binding of thrombin to fibrin lacking or containing γ′ chains, which are fibrinogen γ chain variants that contain a highly anionic carboxyl-terminal sequence. We found the high affinity thrombin binding site to be located exclusively in D domains on γ′ chains (Ka, 4.9 × 106−1; n, 1.05 per γ′ chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 × 106−1; n, 1.69 per molecule). The amino-terminal β15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this sequence. The tyrosine-sulfated, thrombin exosite-binding hirudin peptide, S-Hir53-64 (hirugen), inhibited both low and high affinity thrombin binding to fibrin (IC50 1.4 and 3.0 μ, respectively). The presence of the high affinity γ′ chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin time measurements, and thrombin exosite binding to fibrin at either site did not inhibit its catalytic activity toward a small thrombin substrate, S-2238. We infer from these findings that there are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen. The high affinity thrombin binding site on γ′ chains is a constitutive feature of fibrin as well as fibrinogen.


Published version. Journal of Biological Chemistry, Vol. 271, No. 38 (September 1996): 23151-23125. DOI. © 1996 American Society for Biochemistry and Molecular Biology. Used with permission.

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