Thrombin cleavage of fibrinogen at the N-termini of the A and B chains leads to the formation of a fibrin monomer capable of linear and lateral self-association, resulting in a fibrin clot . Fibrinogen is a substrate whose recognition by thrombin is directly mediated by active site and exosite I interactions to form a Michaelis complex defined by a Km in the micromolar range . Two mutagenesis studies [13,14] identified 15 thrombin residues which potentially mediate direct contact with fibrinogen: K36, R67, H71, R73, Y76, R77a, K81, and K109/110 in exosite I; D186a/K186d near the Na+-binding region; and, K60f and R173/R175/D178 which border the active site cleft. Several structural studies have cocrystallized thrombin with fibrinopeptide A, but with mixed results [15–19]. The most complete peptide structure is of bovine thrombin with an uncleavable analog of residues 7–19 of human fibrinopeptide A . The three monomers in the asymmetric unit revealed conserved contacts from P10 to P2′, with P3′ in two different orientations. The P10–P1 interactions were consistent with those seen in other structures [15,17–19], and involve the burying the hydrophobic face of a one-turn helix into the aryl-binding pocket of thrombin (of primary importance is P9 Phe), and normal interactions of the P1 Arg in the acidic S1 pocket. In 2004, the structure of human thrombin bound to the central E domain of human fibrin was solved, revealing the predicted exosite I interaction interface . Thrombin residues seen in direct contact with fibrin were F34, S36a, L65, Y76, R77a, I82, and K110. Although the interface interposes the basic exosite I of thrombin with an acidic surface of fibrin, the majority of the contacts, and presumably the major energetic contribution, derive from the apposition of hydrophobic surfaces on the two molecules (F34, L65, Y76 and I82 on thrombin and F35 and A68 on the Aα and Bβ chains of fibrin, respectively). Because the structural data agree with the mutagenesis data, a figure of the active site and exosite interactions between thrombin and fibrinogen was constructed using the structural data alone (Fig. 3C, active site contacts from 1UCY in white, and fragments of Aα, Bβ and γ chains colored magenta, cyan and yellow, respectively). It is possible that other contacts exist in the 17 residue stretch which links the C-terminus of the substrate peptide to the Aα chain of the E domain, but these are not expected to be important. The exosite interactions are predicted to be preserved in thrombin cleavage of the Bβ chain of fibrinogen , but no structural information is available on the active site interactions involved.
Figure 3. Crystallographic and mutagenic identification of thrombin exosite and active site interactions. Thrombin is shown in three orientations: in the standard orientation with the active site facing, center; rotated −90° with exosite I facing, left; and, rotated +90° with exosite II facing, right. The first two panels are colored according to electrostatic and hydrophobic potential, as before, to illustrate the nature of the active site and two exosites. All other panels are colored to indicate interaction surfaces on thrombin: blue indicates residues whose mutations specifically affect the rate of substrate cleavage, presumably through direct interaction; and, red indicates thrombin surfaces less than 4 Å distant from substrate or cofactor residues which have been observed crystallographically. This figure emphasizes thrombin's exosite interactions, and although it is understood that the active site is necessarily involved in all substrate recognition events, only active site interactions which have been crystallographically defined are shown. Details of individual panels are given in the appropriate section in the text.