The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal g chain fragment complexed with the peptide Gly-Pro-Arg-Pro (fibrinogenyblood coagulation)

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the a chain of one fibrin molecule and the C-terminal region of a g chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) g chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the a chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction. Upon vascular injury, a series of protease activations culminates in the generation of thrombin, which mediates conversion of fibrinogen to fibrin by minor proteolysis (1). The resulting fibrin monomers polymerize spontaneously and noncovalently, forming two-stranded protofibrils that aggregate and form branches, creating a fibrin gel network. The fibrin polymers are stabilized further by the action of factor XIIIa, a transglutaminase that introduces covalent crosslinks into the network (2). The fibrin gel, together with activated platelets and other components of the blood coagulation system, forms a clot that staunches blood loss while the slower processes of tissue repair proceed. Disruption of this delicately balanced system can lead to bleeding or thrombotic disorders. Fibrinogen is a 340-kDa multichain protein composed of two a, two b, and two g chains that are interconnected by 29 disulfide bonds. In electron micrographs, fibrinogen (and the fibrin molecules comprising the initial protofibrils) appears as a trinodular structure (3). The N-terminal regions of the six chains are joined by five disulfide bonds between the (abg) half-molecules. This region constitutes the central nodule of the molecule. The three chains of each (abg) half-molecule extend outward in a symmetric fashion, forming two coiledcoil structures that terminate in globular domains in the distal two nodules. These distal nodules contain the C-terminal regions of the b and g chains of the protein (4, 5). Fibrinogen is cleaved by thrombin, resulting in the release of short peptides from the N termini of the a and b chains. This creates two new N termini that are designated the ‘‘A’’ and ‘‘B’’ polymerization sites, respectively (6). The a chain is cleaved first, leading to a fibrin protofibril with a half-staggered overlap between molecules. As the fibrils grow, new additional A sites bind to complementary ‘‘a’’ binding pockets on the g chains in the distal nodules of adjacent molecules (Fig. 1). The ‘‘a’’ binding pocket has been localized to residues 337 to 379 of the g chain (7), and close to Y363 (8). The growth of two-stranded protofibrils then proceeds until they reach a length of about 600 nm, at which point the protofibrils aggregate to form thicker, branched fibers (9). The b chain is cleaved by thrombin primarily during the extension of the protofibrils, leading to the binding of its new N terminus to the as-yet-unidentified complementary ‘‘b’’ pocket. This enhances the rate and extent of lateral association of protofibrils (10). Cleavage of the a chain alone by proteases such as batroxobin, however, also leads to gel formation (11), indicating that the initial association of the complementary ‘‘a’’ and A sites is sufficient to promote clot formation. Short peptides mimicking the a-chain A sequence of GPRVV. . . will bind to the ‘‘a’’ polymerization pocket in the g chain (8, 12–14). The peptides GPRP and GPRPamide bind even more tightly to the fibrinogen ‘‘a’’ site than does a peptide corresponding to the a chain native sequence GPRV (15). The complex between GPRP and fibrinogen has an increased resistance to degradation by plasmin (16), and the polymerization reaction is depressed by the addition of excess peptide (11, 12). The binding of calcium also stabilizes fibrinogen against plasmin digestion (17). The polymerization of fibrin monomers has been characterized extensively in biophysical and biochemical experiments over the last five decades (18–24). The polymerization reaction is fully reversible before the covalent crosslinking of the fibrin chains by factor XIIIa (25, 26). Fibrin polymerization is exothermic, unlike many polymerization and proteinassociation reactions, which are primarily entropy driven (27). The association and aggregation of fibrils appears to involve the formation of new hydrogen bonds between adjacent fibrin molecules (22). Fibrin is also a polar molecule, and the alignment of molecules within fibrinogen crystals can be enhanced by placing the crystals in a magnetic field (28). The rate of the polymerization reaction in vitro is strongly dependent upon the salt concentration of the medium (9). It is therefore apparent that relatively nonspecific, long-range electrostatic interactions between polar domains of adjacent fibrin monomers are important in the initial alignment of the molecules. The C-terminal region of the g chain of fibrinogen also is involved in other reactions that are important for the structure and function of the molecule. It contains a single calciumbinding site (29–31) that influences fibrin polymerization as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y947176-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Data deposition: The atomic coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank (accession nos. 2FIB and 3FIB). ‡To whom reprint requests should be addressed at: Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195. e-mail: [email protected].