In blood coagulation, units of the protein fibrinogen pack together to form a fibrin clot, but a crystal structure for fibrinogen is needed to understand how this is achieved. The structure of a core fragment (fragment D) from human fibrinogen has now been determined to 2.9 A resolution. The 86K three-chained structure consists of a coiled-coil region and two homologous globular entitles oriented at approximately 130 degrees to each other. Additionally, the covalently bound dimer of fragment D, known as 'double-D', was isolated from human fibrin, crystallized in the presence of a Gly-Pro-Arg-Pro-amide peptide ligand, which simulates the donor polymerization site, and its structure solved by molecular replacement with the model of fragment D.

We have developed a model of the extrinsic blood coagulation system that includes the stoichiometric anticoagulants. The model accounts for the formation, expression, and propagation of the vitamin K-dependent procoagulant complexes and extends our previous model by including: (a) the tissue factor pathway inhibitor (TFPI)-mediated inactivation of tissue factor (TF)⅐VIIa and its product complexes; (b) the antithrombin-III (AT-III)-mediated inactivation of IIa, mIIa, factor VIIa, factor IXa, and factor Xa; (c) the initial activation of factor V and factor VIII by thrombin generated by factor Xa-membrane; (d) factor VIIIa dissociation/activity loss; (e) the binding competition and kinetic activation steps that exist between TF and factors VII and VIIa; and (f) the activation of factor VII by IIa, factor Xa, and factor IXa. These additions to our earlier model generate a model consisting of 34 differential equations with 42 rate constants that together describe the 27 independent equilibrium expressions, which describe the fates of 34 species. Simulations are initiated by "exposing" picomolar concentrations of TF to an electronic milieu consisting of factors II, IX, X, VII, VIIa, V, and VIIII, and the anticoagulants TFPI and AT-III at concentrations found in normal plasma or associated with coagulation pathology. The reaction followed in terms of thrombin generation, proceeds through phases that can be operationally defined as initiation, propagation, and termination. The generation of thrombin displays a nonlinear dependence upon TF, AT-III, and TFPI and the combination of these latter inhibitors displays kinetic thresholds. At subthreshold TF, thrombin production/ expression is suppressed by the combination of TFPI and AT-III; for concentrations above the TF threshold, the bolus of thrombin produced is quantitatively equivalent. A comparison of the model with empirical laboratory data illustrates that most experimentally observable parameters are captured, and the pathology that results in enhanced or deficient thrombin generation is accurately described.

Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-Å resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4°and 49.5°rotations are required to open the N-and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N-and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.The transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal in the body (1, 2). Human serum transferrin (hTF) 4 is synthesized in the liver and secreted into the plasma; it acquires Fe(III) from the gut and delivers it to iron requiring cells by binding to specific transferrin receptors (TFR) on their surface. The entire hTF⅐TFR complex is taken up by receptor-mediated endocytosis culminating in iron release within the endosome (3). Essential to the re-utilization of hTF, iron-free hTF (apo-hTF) remains bound to the TFR at low pH. When the apo-hTF⅐TFR complex is returned to the cell surface, apo-hTF is released to acquire more iron.Strong homologies exist, both between TF family members, and between the two lobes of any given TF (4, 5). Each N-and C-lobe is divided into two subdomains (designated N1 and N2, and C1 and C2) connected by a hinge that gives rise to a deep cleft containing the iron-binding ligands. Iron is coordinated by four highly conserved amino acid residues: an aspartic acid (the sole ligand from the N1-or C1-subdomain), a tyrosine in the hinge at the edge of the N2-or C2-subdomain, a second tyrosine within the N2-or C2-subdomain, and a histidine at the hinge bordering the N1-or C1-subdomain. In addition, the iron atom is bound by two oxygen atoms from the synergistic anion (carbonate), w...

Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe N hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.

In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fold increase in the rate of thrombin generation compared with factor Xa alone. Structurally, little is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a crystal structure of protein C inactivated factor Va (A1⅐A3-C1-C2) that depicts a previously uncharacterized domain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copperbinding site have both been identified. Surprisingly, neither shows a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffold for the future generation of models of coagulation cofactors.

Factor XIII-cross-linked fragment D (double-D) from human fibrin was crystallized in the presence of two different peptide ligands and the X-ray structure determined at 2.3 A. The peptide Gly-Pro-Arg-Pro-amide, which is an analogue of the knob exposed by the thrombin-catalyzed removal of fibrinopeptide A, was found to reside in the gamma-chain holes, and the peptide Gly-His-Arg-Pro-amide, which corresponds to the beta-chain knob, was found in the homologous beta-chain holes. The structure shows for the first time that the beta-chain knob does indeed bind to a homologous hole on the beta-chain. The gamma- and beta-chain holes are structurally very similar, and it is remarkable they are able to distinguish between these two peptides that differ by a single amino acid. Additionally, we have found that the beta-chain domain, like its gamma-chain counterpart, binds calcium.

The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.

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