Identification and Characterization of the Thrombin Binding Sites on Fibrin

Abstract

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 gamma' chains, which are fibrinogen gamma 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 gamma' chains (Ka, 4.9 x 10(6) M-1; n, 1.05 per gamma' chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 x 10(6) M-1; n, 1.69 per molecule). The amino-terminal beta15-42 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 microM respectively). The presence of the high affinity gamma' 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 gamma' chains is a constitutive feature of fibrin as well as fibrinogen.

Identification and characterization of novel mutations implicated in congenital fibrinogen disorders

“…Interaction of thrombin‐cleaved EA regions with the D regions, as well as interactions between D regions (D:D) allow for end‐to‐middle and end‐to‐end assembly of the fibrin molecule, respectively 1, 2. Mature fibrin supports platelet aggregation, provides structural stability to the clot, promotes interactions with platelets and endothelial cells, and serves as a binding site for circulating plasma factors such as factor XIII, thrombin, and fibrinolytic proteins (eg, tissue‐type plasminogen activator [tPA] and plasminogen) 1, 3, 4…”

Genetic analyses of novel compound heterozygous hypodysfibrinogenemia, Tsukuba I: FGG c.1129+62_65 del AATA and FGG c.1299+4 del A

“…The major A-chain (98-99% of the A-chain) contains 610 residues (translated from the first five exons of FGA), while the alternative A-chain (1-2% of the A-chain) contains 846 residues and is translated from all six exons of FGA [4]. The predominant form of the -chain, A (85-93% of the -chain) contains 411 residues, and the variant form of the -chain, ' (7-15% of the -chain) consists of 427 residues [5,6]. The A-chain is produced by the translation of all 10 FGG exons, while the '-chain is produced by alternative splicing and polyadenylation of the -chain mRNA transcript, resulting in the translation of exons 1-9 of FGG [7,8].…”

“…The A-chain is produced by the translation of all 10 FGG exons, while the '-chain is produced by alternative splicing and polyadenylation of the -chain mRNA transcript, resulting in the translation of exons 1-9 of FGG [7,8]. A major fraction (84%) of plasma fibrinogen contains a homodimer referred to as (A-B-A)2orA/A,and the minor fraction contains a heterodimer (15% of plasma fibrinogen) referred to as (A-B-A(A-B-') orA/' and a homodimer (1% of plasma fibrinogen) referred to as (A-B-')2 or '/' [5,6]. 5 More than 300 different mutations in the fibrinogen genes, FGA, FGB, and FGG, have been associated with the phenotype of afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, renal amyloidosis, or fibrinogen storage disease, as listed in the fibrinogen variant data base [9].…”

“…A major fraction (84%) of plasma fibrinogen contains a homodimer referred to as (A-B-A)2orA/A,and the minor fraction contains a heterodimer (15% of plasma fibrinogen) referred to as (A-B-A(A-B-') orA/' and a homodimer (1% of plasma fibrinogen) referred to as (A-B-')2 or '/' [5,6]. 5 More than 300 different mutations in the fibrinogen genes, FGA, FGB, and FGG, have been associated with the phenotype of afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, renal amyloidosis, or fibrinogen storage disease, as listed in the fibrinogen variant data base [9]. Hypofibrinogenemia or afibrinogenemia has been defined as reduced or negligible levels of immunologically determined fibrinogen in plasma, and dysfibrinogenemia has been defined as reduced levels of functionally determined fibrinogen with normal levels of immunologically determined fibrinogen in plasma.…”

“…As a consequence, we detected a high-molecular-weight aberrant '-chain in the Western blot analysis of fibrinogen from the plasma of our patient. Although the mechanism underlying alternative splicing for '-chain mRNA production [7,8] and the regulation of plasma '-chain levels have not yet been elucidated in detail, plasma fibrinogen contains approximately 7 to 15% '-chains and exists as three types of molecules: AA (84% of total plasma fibrinogen), A' (15%), and '' (1%) [5,6]. The normal '-chain is synthesized at markedly smaller amounts than the normal A-chain.…”