The interface between the EGF2 domain and the protease domain in blood coagulation factor IX contributes to factor VIII binding and factor X activation

The light chain of activated factor IX (FIXa) is involved in a number of functional properties, including FIXa enzymatic activity. This suggests the existence of a functional link between the FIXa light chain and the catalytic domain. The FIXa structure includes a few putative interactions between EGF2 and the protease domain. The role thereof has been addressed in this study. Recombinant FIX variants FIX-N92A, FIX-N92H, FIX-Y295A, and FIX-F299A were produced in 293 cells. After activation, the purified mutants were analyzed for a variety of functional parameters. None of these substitutions had a major effect on the interaction with antithrombin or the cleavage of the chromogenic substrate CH(3)SO(2)-d-CHG-Gly-Arg-p-nitroanilide. All FIXa mutants, however, exhibited a reduced level of factor X (FX) activation. Defective proteolytic activity occurred both in the absence and in the presence of activated factor VIII (FVIIIa). All mutants also exhibited a reduced level of FX activation in the absence of phospholipids. This suggests that putative interdomain contacts involving residues Asn(92), Tyr(295), and Phe(299) affect reactivity toward FX. Detailed kinetic studies in the presence of phospholipids and FVIIIa revealed substrate inhibition, particularly for mutants FIXa-N92A and FIXa-N92H. Surface plasmon resonance demonstrated that the same replacements weaken the association with the isolated factor VIII (FVIII) A2 domain and the FVIII light chain. This implies a defect in the formation of the FX-activating complex that is membrane-independent. We conclude that contacts between EGF2 and the protease domain of FIXa are crucial for FIXa enzymatic activity and for the assembly of the FX-activating complex.     

Backbone resonance assignments of the C2 domain of coagulation factor VIII

Factor VIII (FVIII, other clotting factors are named similarly) is a glycoprotein that circulates in the plasma bound to von Willebrand factor. During the blood coagulation cascade, activated FVIII (FVIIIa) binds to FIXa and activates FX in the presence of calcium ions and phospholipid membranes. The C1 and C2 domains mediate membrane binding that is essential for activation of the FVIIIa–FIXa complex. Here, ¹H, ¹³C, and ¹⁵N backbone chemical shift assignments are reported for the C2 domain of FVIII, including assignments for the residues in solvent-exposed loops. The NMR resonance assignments, along with further structural studies of membrane-bound FVIII, will advance understanding of blood-clotting protein interactions.     

An antibody specific for coagulation factor IX enhances the activity of the intrinsic factor X-activating complex

During hemostasis the zymogen factor X (FX) is converted into its enzymatically active form factor Xa by the intrinsic FX-activating complex. This complex consists of the protease factor IXa (FIXa) that assembles, together with its cofactor, factor VIIIa, on a phospholipid surface. We have studied the functional properties of a FIXa-specific monoclonal antibody, 224AE3, which has the potential to enhance intrinsic FX activation. Binding of the antibody to FIXa improved the catalytic properties of the intrinsic FX-activating complex in two ways: (i) factor VIIIa bound to the FIXa-antibody complex with a more than 18-fold higher affinity than to FIXa, and (ii) the turnover number (kcat) of the enzyme complex increased 2- to 3-fold whereas the Km for FX remained unaffected. The ability of 224AE3 to increase the FXa-generation potential (called the "booster effect") was confirmed in factor VIII (FVIII)-depleted plasma, which was supplemented with different amounts of recombinant FVIII. In the presence of antibody 224AE3 the coagulant activity was increased 2-fold at physiological FVIII concentration and up to 15-fold at low FVIII concentrations. The booster effect that we describe demonstrates the ability of antibodies to function as an additional cofactor in an enzymatic reaction and might open up a new principle for improving the treatment of hemophilia.     

Structural and functional characterization of platelet receptor-mediated factor VIII binding

Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.     

Role of the C2 domain of factor VIIIa in the assembly of factor-X activating complex on the platelet membrane

Optimal rates of factor X (FX) activation require binding of factor IXa (FIXa), factor VIII(a) [FVIII(a)], and FX to activated platelet receptors. To define the FVIIIa domains that mediate platelet interactions, albumin density gradient washed, gel-filtered platelets (3.5 x 10(8)/mL) activated by the thrombin receptor peptide, SFLLRN (25 microM), were incubated with 125I-labeled FVIII C2 domain, or 125I-FVIIIa, or 125I-FVIII((LC)), or peptides from the C2 domain region, with or without anti-C2 domain monoclonal antibodies (MoAb), ESH4 or ESH8. FVIIIa (Kd approximately 1.7 nM), FVIII((LC)) (Kd approximately 3 nM), and the C2 domain (Kd approximately 16 nM) all interacted with approximately 700-800 binding sites/platelet. Unlike FVIIIa, the C2 domain did not respond to the presence of excess EGR-FIXa (45 nM) and FX (1.5 microM) with enhanced binding stoichiometry and affinity. Both the MoAb ESH4 and a synthetic peptide corresponding to FVIII residues 2303-2332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2 domain peptide corresponding to residues 2248-2285 (epitope for the FVIII MoAb, ESH8) failed to inhibit FVIIIa binding. Thus, a major platelet-binding site resides within residues 2303-2332 in the C2 domain of FVIIIa, and an additional site within residues 2248-2285 increases the stoichiometry and affinity of FVIIIa binding to activated platelets only in the presence of FIXa and FX but does not directly mediate FVIIIa binding to the platelet surface.     

Calmodulin interacts with cyclic nucleotide phosphodiesterase and calcineurin by binding to a metal ion-independent hydrophobic region on these proteins

Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions.     

MEP chromatography of antibody and Fc-fusion protein using aqueous arginine solution

MEP HyperCel resin, one of the Protein-A mimetic columns, is designed to bind antibodies at physiological pH and elutes the bound antibodies at mildly acidic pH. We have tested aqueous arginine solution for washing and elution of the resin. To our surprise, bound antibody and Fc-fusion protein eluted at pH 7.0 using 1 M arginine solution. Various solvent additives were then examined at pH 7.0. Among the tested additives, urea and arginine were the only additives that were effective in elution. Thus, urea and arginine at low concentrations were effectively used for washing the resin. NaCl and MgCl₂ at 0.1–1 M and ethanol at 5–20% were not effective. Based on these observations, it appears that protein binds to MEP resin through both polar and hydrophobic interactions with some contribution of electrostatic interaction, which can be simultaneously reduced by arginine or urea. On the other hand, Mabsorbent, another Protein-A mimetic column, appears to be more non-specific and non-selective.     

Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.     

Characterization of virus adsorption by using DEAE-sepharose and octyl-sepharoser

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and phiX174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included phiX174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.     

Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns.

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.     

Characterization of Virus Adsorption by Using DEAE-Sepharose and Octyl-Sepharose†

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and X174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included X174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.     

Factor VIII A3 domain residues 1793–1795 represent a factor IXa-interactive site in the tenase complex

BackgroundFactor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811–1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790–1798 form a V-shaped loop, and juxtapose residues 1811–1818 on the extended surface of FVIIIa.AimTo examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790–1798.Methods and resultsSpecific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790–1798 and 1811–1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC₅₀; 19.2 and 42.9 μM, respectively), in keeping with a possible role for the 1790–1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5–2.2-fold greater K_D compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the K_m by 1.6–2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the K_m was increased by 3.4-fold and the V_max was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction.ConclusionThe 1790–1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site.     

Na+ site in blood coagulation factor IXa: effect on catalysis and factor VIIIa binding

During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a Na+ site. Comparative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (chymotrypsin numbering). Kinetic data obtained at several concentrations of Na+ and Ca2+ with increasing concentrations of a synthetic substrate (CH3-SO2-d-Leu-Gly-Arg-p-nitroanilide) were fit globally, assuming rapid equilibrium conditions. Occupancy by Na+ increased the affinity of FIXa for the synthetic substrate, whereas occupancy by Ca2+ decreased this affinity but increased k(cat) dramatically. Thus, Na+-FIXa-Ca2+ is catalytically more active than free FIXa. FIXa(Y225P), a Na+ site mutant, was severely impaired in Na+ potentiation of its catalytic activity and in binding to p-aminobenzamidine (S1 site probe) validating that substrate binding in FIXa is linked positively to Na+ binding. Moreover, the rate of carbamylation of NH2 of Val16, which forms a salt-bridge with Asp194 in serine proteases, was faster for FIXa(Y225P) and addition of Ca2+ overcame this impairment only partially. Further studies were aimed at delineating the role of the FIXa Na+ site in macromolecular catalysis. In the presence of Ca2+ and phospholipid, with or without saturating FVIIIa, FIXa(Y225P) activated FX with similar K(m) but threefold reduced k(cat). Further, interaction of FVIIIa:FIXa(Y225P) was impaired fourfold. Our previous data revealed that Ca2+ binding to the protease domain increases the affinity of FIXa for FVIIIa approximately 15-fold. The present data indicate that occupancy of the Na+ site further increases the affinity of FIXa for FVIIIa fourfold and k(cat) threefold. Thus, in the presence of Ca2+, phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic activity by approximately 12-fold, implicating its role in physiologic coagulation.     

Domain organization of membrane‐bound factor VIII

Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane‐bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane‐bound state is critical for the biological efficiency of FVIII. Here, we present our cryo‐electron microscopy (EM) and structure analysis studies of human FVIII‐LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII‐LC membrane‐bound structure supports aspects of our previously proposed FVIII structure from membrane‐bound two‐dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane‐bound helical and 2D crystal structures based on EM data, and the existing X‐ray structures. This flexibility of the FVIII‐LC domain organization in different states is discussed in the light of the FVIIIa–FIXa complex assembly and function. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 448–459, 2013.     

Residues 88-109 of factor IXa are important for assembly of the factor X activating complex.

Activated platelets and phospholipid vesicles promote assembly of the intrinsic factor X (FX) activating complex by presenting high-affinity binding sites for blood coagulation FIXa, FVIIIa, and FX. Previous reports suggest that the second epidermal growth factor (EGF)-like domain of FIXa mediates assembly of the FX activating complex (Ahmad, S. S., Rawala, R., Cheung, W. F., Stafford, D. W., and Walsh, P. N. (1995) Biochem. J. 310, 427-431; Wong, M. Y., Gurr, J. A., and Walsh, P. N. (1999) Biochemistry 38, 8948-8960). To identify important residues, we prepared several chimeric FIXa proteins using homologous sequences from FVII: FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127), and point mutants (FIXaR94D and FIXa(loop1)G94R). In the presence and absence of FVIIIa, a 2- to 10-fold reduced V(max) of FX activation (nm FXa min(-1)) was observed for FIXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1)G94R, whereas FIXa(loop3) and FIXaR94D were normal. For all of the FIXa proteins, K(m)((app)) values were normal as were EC(50) values for interactions with FVIIIa. However, K(d)((app)) (in nm) for the FX activating complex assembled on phospholipid vesicles was increased for FIXa(FVIIEGF2) (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +/- 2.90) relative to FIXa(N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(loop3) (4.5 +/- 0.20), and FIXaR94D (2.2 +/- 0.09) suggesting that reduced V(max) is a result of impaired complex assembly. These data indicate that residues 88-109 (but not Arg(94)) are important for normal assembly of the FX activating complex on phospholipid vesicles.     

Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: A computational protein–protein docking and molecular dynamics refinement study

Coagulation factor X (FX) zymogen activation by factor IXa (FIXa) enzyme plays a critical role in the middle-phase of coagulation cascade. The activation process is catalytically inert and requires FIXa binding and complex formation with co-factor VIIIa (FVIIIa). In order to understand the structural details of the FVIIIa:FIXa complex, we employed knowledge-driven protein–protein docking and aqueous-phase MD refinement methods to develop a stable structural complex between FVIIIa and FIXa. The model shows that all four domains of FIXa wrap across FVIIIa that spans the co-factor binding surface of A2, A3 and C1 domains. The region surrounding the 558-helix of the A2-domain of FVIIIa is predicted to be the key interaction site with the helical segments of Lys293–Lys301 and Asp332–Arg338 residues of the serine-protease domain of FIXa. The hydrophobic helical stack between the GLA and EGF1 domains of FIXa is predicted to be primary interacting region with the A3–C2 domain interface of FVIIIa.     

A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

A recent chemical footprinting study in our laboratory suggested that region 1803–1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803–1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803–1810 and 1811–1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811–1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811–1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803–1810 and FVIII/FV 1811–1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811–1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811–1818 contributes to FIXa binding. Both regions 1803–1810 and 1811–1818 contribute to FVIIIa stability.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters.

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

The N-terminal epidermal growth factor-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor.

Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal gamma-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIa*TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa*TF complex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIX(WT)), FIX(Q50P), FIX(PCEGF1) (EGF1 domain replaced with that of protein C), FIX(DeltaEGF1) (EGF1 domain deleted), FX(WT), and FX(PCEGF1). Complexes of FVIIa with TF as well as with soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa*TF or FVIIa*sTF activated each mutant significantly more slowly than the FIX(WT) or FX(WT). Importantly, in ligand blot assays, FIX(WT) and FX(WT) bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments revealed that the affinity of the mutants for sTF was reduced 3-10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF with K(i) of approximately 60 microm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEGF1) normally, which is interpreted to mean that the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.