Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

Coagulation factors IX/X-binding protein is an intertwined dimer with a central loop projecting into the adjoining subunit. Excluding this loop, each subunit has a fold similar to rat mannose-binding protein.

The ␥-carboxyglutamic acid (Gla) domain of blood coagulation factors is responsible for Ca 2؉ -dependent phospholipid membrane binding. Factor X-binding protein (X-bp), an anticoagulant protein from snake venom, specifically binds to the Gla domain of factor X. The crystal structure of X-bp in complex with the Gla domain peptide of factor X at 2.3-Å resolution showed that the anticoagulation is based on the fact that two patches of the Gla domain essential for membrane binding are buried in the complex formation. The Gla domain thus is expected to be a new target of anticoagulant drugs, and X-bp provides a basis for designing them. This structure also provides a membrane-bound model of factor X.

Snake venom contains a number of the hemostatically active C-type lectin-like proteins, which affect the interaction between von Willebrand factor (vWF) and the platelet glycoprotein (GP) Ib or platelet receptor to inhibit/induce platelet activation. Flavocetin-A (FL-A) is a high-molecular mass C-type lectin-like protein (149 kDa) isolated from the habu snake venom. FL-A binds with high affinity to the platelet GP Ibalpha-subunit and functions as a strong inhibitor of vWF-dependent platelet aggregation. We have determined the X-ray crystal structure of FL-A and refined to 2.5 A resolution. This is a first elucidation of a three-dimensional structure of the platelet GP Ib-binding protein. The overall structure reveals that the molecule is a novel cyclic tetramer (alphabeta)(4) made up of four alphabeta-heterodimers related by a crystallographic 4-fold symmetry. The tetramerization is mediated by an interchain disulfide bridge between cysteine residues at the C-terminus of the alpha-subunit and at the N-terminus of the beta-subunit in the neighboring alphabeta-heterodimer. The high affinity of FL-A for the platelet GP Ib alpha-subunit could be explained by a cooperative-binding action through the multiple binding sites of the tetramer.

No abstract

The coagulation factor IX/factor X-binding protein (IX/X-bp) from the venom of Trimeresurus flavoviridis is a heterogeneous two-chain protein, and the structure of each chain is similar to that of the carbohydrate-recognition domain of C-type lectins, such as asialoglycoprotein receptors, pancreatic stone protein, and the Fc epsilon receptor for immunoglobulin E. Analysis of the binding properties of IX/X-bp revealed that it binds to the gamma-carboxyglutamic acid (Gla)-containing domains of factors IX and X [Atoda, H. et al. (1994) Eur. J. Biochem. 224, 703-708]. In the present study, we isolated another anticoagulant protein that binds to factor IX but is not to factor X. This protein, designated IX-bp, inhibited factor IXa-induced clotting but not factor Xa-induced clotting, whereas IX/X-bp inhibits both. The concentration of IX-bp for half-maximal binding to solid-phase bovine factor IX was 0.4 nM whereas IX-bp did not bind to factor X even at 40 nM. The binding of IX-bp to solid-phase factor IX was inhibited by the addition of Gla-domain peptide of factor IX, indicating that IX-bp binds to the Gla-domain region of factor IX. IX-bp had two Ca(2+)-binding sites with different affinities for Ca2+ ions. At pH 7.5, the apparent Kd values for these sites were 14 and 130 microM, respectively. IX-bp was a two-chain protein (27.5-kDa band before reduction and 16.8- and 15.7-kDa bands after reduction on SDS-PAGE) and it reacted with immunoglobulin G against IX/X-bp. The complete amino acid sequence of IX-bp was determined. The 16.8-kDa chain (A chain) of IX-bp consisted of 129 residues, of which 19 were different from those in the A chain of IX/X-bp (129 residues). The sequence of the 15.7-kDa chain (B chain) was identical to that of the B chain of IX/X-bp (123 residues). We conclude that IX-bp is a protein that is structurally similar to but functionally different from IX/X-bp. The difference of binding specificity between IX-bp and IX/X-bp presumably arises from the sequence differences in the A chains.

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.

The indispensable role of Ca2+ ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation factors has been definitively established but the participation of Mg2+ ions, another alkaline-earth metal that is present abundantly in blood plasma, in such a process is not yet understood. We show here that the Ca(2+)-stabilized conformation of coagulation factor IX undergoes a further conformational change upon binding of Mg2+ ions using three independent structural probes. The probes we used were (i) IX/X-bp, a snake venom anticoagulant that recognizes the Gla domains in coagulation factors IX and X, (ii) conformation-specific polyclonal antibodies against bovine factor IX, and (iii) monoclonal antibodies against the Gla domain of human factor IX. The binding of all these probes had an absolute requirement for Ca2+ ions, and Mg2+ ions alone were ineffective. However, when added together with Ca2+ ions, Mg2+ ions at physiological concentrations greatly augmented the binding of these probes to factor IX; the required concentration of Ca2+ ions was much reduced, and the affinity of each probe for factor IX was increased even in the presence of an excess of Ca2+ ions. These results suggest the presence of a Mg(2+)-specific binding site that does not interact with Ca2+ ions in factor IX. Furthermore, Mg2+ ions potentiated the susceptibility of factor IX to activation by factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca2+ concentration was reduced by Mg2+ ions, and the rate of conversion to factor IXa was increased by Mg2+ ions in the presence of an excess of Ca2+ ions. At a saturating concentration of Ca2+ ions (5 mM), addition of 1 mM Mg2+ reduced the apparent Km value for factor IX from 0.31 to 0.18 microM, and in the presence of a physiological concentration of Ca2+ ions (1 mM), the reduction in Km by Mg2+ ions was far more striking (from 0.91 to 0.24 microM). The apparent Vmax values were hardly affected by Mg2+ ions. Our present data reveal a hitherto novel physiological role of the Mg2+ ions in plasma. Not only Ca2+ ions but also Mg2+ ions are important regulators of the stabilization of the native conformation of factor IX as well as of its efficient activation.