Interaction of the Factor XIII Activation Peptide with α-Thrombin

Abstract

The serine protease thrombin proteolytically activates blood coagulation factor XIII by cleavage at residue Arg 37 ; factor XIII in turn cross-links fibrin molecules and gives mechanical stability to the blood clot. The 2.0-Å resolution x-ray crystal structure of human ␣-thrombin bound to the factor XIII-(28 -37) decapeptide has been determined. This structure reveals the detailed atomic level interactions between the factor XIII activation peptide and thrombin and provides the first high resolution view of this functionally important part of the factor XIII molecule. A comparison of this structure with the crystal structure of fibrinopeptide A complexed with thrombin highlights several important determinants of thrombin substrate interaction. First, the P1 and P2 residues must be compatible with the geometry and chemistry of the S1 and S2 specificity sites in thrombin. Second, a glycine in the P5 position is necessary for the conserved substrate conformation seen in both factor XIII-(28 -37) and fibrinopeptide A. Finally, the hydrophobic residues, which occupy the aryl binding site of thrombin determine the substrate conformation further away from the catalytic residues. In the case of factor XIII-(28 -37), the aryl binding site is shared by hydrophobic residues P4 (Val 34 ) and P9 (Val 29 ). A bulkier residue in either of these sites may alter the substrate peptide conformation.The serine protease thrombin plays a central role in the blood coagulation process (1, 2). It proteolytically activates blood coagulation and plasma factors such as factor V, factor VIII, factor XIII, and protein C (3). Its proteolytic activity is also responsible for catalyzing the conversion of fibrinogen to fibrin (4) by the cleavage of fibrinopeptides A and B from the N termini of the fibrinogen ␣ and ␤ chains, respectively. In addition, thrombin is the most potent stimulator of platelet aggregation (5). Thrombin achieves this diverse yet specific recognition of substrates with a deep active site cleft and by exploiting an apolar binding site near the catalytic residues as well as an anion binding exosite distant from the active site cleft (6).Crystal structures of ␣-thrombin complexed to a number of natural and synthetic inhibitors have been studied in detail and characterized to near atomic resolution (for example, see . These structures have provided a wealth of information about the interactions between inhibitors and enzyme. However, more limited knowledge about the atomic level interactions between substrate and thrombin comes mainly from the crystal structures of thrombin bound to fibrinopeptide A. Structures of human and bovine ␣-thrombin bound to the Nterminal peptide of the fibrinogen ␣-chain (15,16) show that the C-terminal region of the peptide runs anti-parallel to the Ser 214 -Glu 217 segment of thrombin and that the arginine at the cleavage site occupies the S1 specificity pocket. The N-terminal region of the peptide adopts a compact ␣-helical conformation, folding back toward the active site cleft to bring the hydrop...

Crystal Structure of Thrombin Bound to the Uncleaved Extracellular Fragment of PAR1

“…3). These interactions are analogous to those observed for Pro residues at the P2 positions of PAR4 (15), factor XIII (20), and H-D-Phe-Pro-Arg-CH 2 Cl (3, 17). The P1 residue of PAR1, Arg 41 , is involved in extensive H-bonding interactions that are observed in practically all trypsin-like proteases bound to substrate (43)(44)(45).…”

“…Indeed, the catalytic activity of thrombin depends little on the nature of the P3 residue (48). Nonetheless, the H-D-Phe-Pro-Arg-CH 2 Cl-inhibited structure reveals interactions that are relevant to recognition of natural substrates like fibrinogen (18), PAR4 (15,19), and factor XIII (20,21). The structure of thrombin in complex with the potent natural inhibitor hirudin has revealed how thrombin recognizes ligands that bridge the active site and exosite I (41).…”

“…These findings, along with the current structure of the thrombin-PAR1 complex, suggest that the tethered ligand may be "under tension" in the uncleaved form of PAR1, ready to snap back onto the receptor surface and away from the enzyme as soon as cleavage at Arg 41 engages the backbone N of Leu 38 . These intramolecular interactions result in a single 3 10 -helix turn reminiscent of the architecture seen in fragments of fibrinogen and factor XIII bound to thrombin (18,20). The C-terminal portion of the cleavage site presents the tethered ligand within 4 Å from the thrombin surface.…”

“…Structural and site-directed mutagenesis data document the important role of exosite I in the interaction of thrombin with fibrinogen (6 -9) and the protease-activated receptors (PARs) 2 PAR1 (7, 10 -13) and PAR3 (7,14,15). Likewise, abundant structural and functional data document how thrombin recognizes substrates at the active site (3,16,17), including fibrinogen (18), PAR4 (15,19), and factor XIII (20,21). On the other hand, structural elucidation of how substrates bridge the active site and exosite I in their binding to thrombin has been challenging.…”

Engineering Thrombin for Selective Specificity toward Protein C and PAR1

“…All constructs could be expressed to homogeneity and characterized in terms of their interaction with key physiological substrates. Trp 215 is highly conserved in trypsin-like proteases (3) and is located within a hydrophobic patch ( in recognition of the procoagulant substrates factor XIII (34,35) and PAR4 (17,36). Trp 215 also plays a key role in the conformational transition of thrombin from the inactive E* to the active E form (7,18) by relocating more than 10 Å within the active site.…”