Immunological Studies of Factor VIII (Antihaemophiliac Globulin) in Haemophilia A

PATIENTS with haemophilia A have recently been divided into two groups; one characterized by a functionally defective factor VIII (AHG) molecule in the plasma, which is immunologically similar to normal factor VIII but lacks procoagulant activity^1–3, while the other seems to represent a true deficiency of factor VIII, for both procoagulant and immunological properties of factor VIII are absent. These conclusions are based on the ability of normal and some haemophiliac plasma to neutralize human antibodies from patients with spontaneously occurring inhibitors against factor VIII or from multi-transfuscd haemophiliacs who have developed circulating inhibitors directed against factor VIII. These studies have divided haemophilia A into those with cross reactive material (CRM +) and those without (CRM ?). Other methods of characterizing this genetic polymorphism in haemophilia have not been reported. We have used immunoelectrophoresis and antibody neutralization with a heterologous antibody to show a similar division of haemophilia A into a small group of CRM+ (15%) and a larger group of CRM? (85%). Immunoelectrophoresis and antibody neutralization studies have also been described in factor X polymorphism⁴.     

Factor VIII Activity in Haemophilic Plasma unmasked by Synthetic Organic Anions

HAEMOPHILIA A is generally regarded as a hereditary deficiency of antihaemophilic globulin (factor VIII). Some investigators, however, believe that antihaemophilic globulin may be inhibited rather than deficient. Most recent studies with monospecific antisera against antihaemophilic globulin have shown that patients with haemophilia A possess normal amounts of factor VIII, but which is biologically inactive^2,3. Furthermore, treating plasma from patients with haemophilia A and von Willebrand's disease with succinic acid produced protein fractions with antihaemophilic activity separable by chromatography⁴. These observations indicate that haemophilia A might well be caused by a defective or inhibited factor VIII molecule.     

Peptide map analysis of normal plasma and platelet factor VIII antigen

Factor VIII antigen from platelet intracellular granules was immunoprecipitated using a monospecific rabbit antibody to normal plasma factor VIII antigen. The factor VIII antigen in the immunoprecipitate was isolated on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with ¹²⁵I, trypsinized, and subjected to peptide mapping using two dimensional high voltage electrophoresis and thin layer chromatography. The platelet protein was compared to purified plasma factor VIII antigen. The two dimensional tryptic ¹²⁵I peptide map of platelet granule factor VIII antigen was similar but not identical to the plasma factor VIII antigen peptide map. The platelet and plasma proteins shared approximately 34 radioactive peptide spots. Seven plasma factor VIII antigen peptides were not detected in platelet factor VIII antigen. The reason for the structural differences of plasma and platelet granule factor VIII antigen are unknown. The possibility is raised that proteolysis has altered the platelet protein in vitro. It is also possible that factor VIII antigen synthesized by megakaryocytes differs from the plasma protein.     

Isolation of Catenated Forms of R Factor DNA from Minicells

STUDIES^1–6 of the molecular nature of antibiotic resistance (R) factors in Escherichia coli have shown that several of them consist of covalently closed molecules of circular DNA. Use was made of this property in the separation of the R factor from chromosomal DNA of E. coli, which has a similar nucleotide-base composition. In selecting for covalently circular R factor DNA molecules, however, the procedures used in these earlier experiments-caesium chloride-ethidium bromide centrifuga-tion⁷ and bulk nitrocellulose adsorption²—necessarily selected against isolation of other (non-circular) forms of R factor DNA that might have been present.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)⁶]- and [D-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH₂)⁶-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Summary Neb-TMOF, the trypsin modulating oostatic factor of gray fleshflyNeobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 byd-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X=NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) andd-Asn (XII). The influence of these peptides on trypsin biosynthesis inN. bullata was determinedin vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [d-His)⁶]- and [d-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performedin vitro on heart ofTenebrio molitor were found that-TMOF and [Phe(p-NH₂)⁶]-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Eliminatory Action of Glycine on Drug Resistance of Escherichia coli K12 Harboring an R Factor

Glycine, known to inhibit the synthesis of a peptidoglycan component of the bacterial cell wall, was effective in eliminating drug resistance of Escherichia coli K12 JE2100 strain harboring the R100–1 factor, although in lower frequencies than that of sodium dodecyl sulfate (SDS). The action of glycine was found to be less effective on the same R factor in JE177 strain, and not effective on the F factor in W6. Infection of R factors from R⁺ cells to R^– cells was found to take place in the glycine broth as efficiently as in broth without glycine. This might result in lowering the apparent efficiency of the action of glycine on those plasmids. The segregation patterns of drug-susceptible clones obtained by the glycine treatment were different from those obtained after the SDS treatment. These results coupled with other evidences suggest that the mode of action of glycine on R⁺ cells may be different from those of other curing agents and may involve mechanisms other than selection of R^– or drug-susceptible segregants that are present in R⁺ culture.     

Protein <Emphasis Type="Italic">trans</Emphasis>-splicing based dual-vector delivery of the coagulation factor VIII gene

A dual-vector system was explored for the delivery of the coagulation factor VIII gene, using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally. A pair of eukaryotic expression vectors, expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII), was constructed. With transient co-transfection of the two vectors into 293 and COS-7 cells, the culture supernatants contained (137±23) and (109±22) ng mL⁻¹ spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL⁻¹ for 293 and COS-7 cells, respectively. The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes. The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting. The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.     

Concanavalin A as an Inducer of Human Lymphocyte Mitogenic Factor

IT is likely that pharmacological products of antigen : lymphocyte interaction (“lymphokines”) act as mediators and regulators of a variety of cellular immune responses^1,2. This view is strengthened by demonstrations that phytomitogen lectins induce lymphocytes to generate products with similar biological activities^3,4 and physicochemical characteristics⁵, to the lymphokines. Increasing evidence suggests that mitogenic lymphokines may mediate lymphocyte transformation responses in vitro and facilitate lymphoid cell cooperation in vivo (refs. 1,2,6–9). The study of mitogenic factor production by phytomitogens which may predominantly activate thymus-dependent lymphocytes (Concanavalin A (ConA))^8,9 provides a model approach to the investigation of lymphokine function in man. Powles et al.⁴ have described a ConA-induced mitogenic factor which stimulated autologous human lymphocytes only, whereas antigen-induced lymphocyte factors generally stimulate both allogeneic and syngeneic lymphocytes^11–13. Interest in ConA as an inducer of human lymphocyte mitogenic factors would be widened if conditions were found in which ConA stimulated human lymphocytes to generate products which were mitogenic for both allogeneic and autologous lymphocytes. As a lymphokine stimulant, ConA has the advantage that it is largely removed from culture fluids by absorption to cross-linked dextrans (‘Sephadex G-50’) or serum glycoproteins¹⁴. Here we demonstrate that a ‘Sephadex’-binding fraction of ConA (ConA- V) induces human lymphocytes to generate a mitogenic factor which activates both allogeneic and autologous lymphocytes.     

Factor X activator from Vipera lebetina snake venom,molecular characterization and substrate specificity

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6–9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly – factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly – factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that VLFXA cleaves the Arg⁵²-Ile⁵³ bond in the heavy chain of human factor X and the Arg²²⁶-Val²²⁷ bond in human factor IX precursor. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.     

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)¹ is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment².Coagulation Factor VIII (FVIII)³ is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting⁴. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete⁵. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa^6,7.In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.     

Functional and Structural Characterization of Factor Xa Dimer in Solution

Previous studies showed that binding of water-soluble phosphatidylserine (C6PS) to bovine factor Xa (FXa) leads to Ca²⁺-dependent dimerization in solution. We report the effects of Ca²⁺, C6PS, and dimerization on the activity and structure of human and bovine FXa. Both human and bovine dimers are 10⁶- to 10⁷-fold less active toward prothrombin than the monomer, with the decrease being attributed mainly to a substantial decrease in k_cat. Dimerization appears not to block the active site, since amidolytic activity toward a synthetic substrate is largely unaffected. Circular dichroism reveals a substantial change in tertiary or quaternary structure with a concomitant decrease in α-helix upon dimerization. Mass spectrometry identifies a lysine (K²⁷⁰) in the catalytic domain that appears to be buried at the dimer interface and is part of a synthetic peptide sequence reported to interfere with factor Va (FVa) binding. C6PS binding exposes K³⁵¹ (part of a reported FVa binding region), K²⁴² (adjacent to the catalytic triad), and K⁴²⁰ (part of a substrate exosite). We interpret our results to mean that C6PS-induced dimerization produces substantial conformational changes or domain rearrangements such that structural data on PS-activated FXa is required to understand the structure of the FXa dimer or the FXa-FVa complex.     

A novel approach in potential anticoagulants from peptides epitope 558–565 of A2 subunit of factor VIII

Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser⁵⁵⁸–Gln⁵⁶⁵ region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa–FIXa interaction. Based on this, a series of linear peptides, analogs of the 558–565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.     

The detection of mitotic and meiotic aneuploidy in yeast using a gene dosage selection system

Summary A system is described in which spontaneous and chemically-induced mitotic and meiotic hyperploidy can be assayed in the same diploid culture of Saccharomyces cerevisiae. Monitoring gene dosage changes at two loci on chromosome VIII, the test utilizes a leaky temperature-sensitive allele arg4-8 and low level copper resistance conferred by the single copy allele cup1 ^s. An extra chromosome VIII provides simultaneous increased dosage for both genes, resulting in colonies that are both prototrophic for arginine at 30° C and copper resistant. During mitotic cell divisions in diploids, spontaneous chromosome VIII hyperploids (trisomes and tetrasomes) occur at a frequency of 6.4×10^-6 per viable cell. Among ascospores, the spontaneous chromosome VIII disome frequency is 5.5×10^-6 per viable spore. The tubulin-binding reagent methyl benzimidazol-2-yl carbamate (MBC) elicits enhanced levels of mitotic and meiotic aneuploidy relative to control levels. The system represents a novel model for examining chromosome behavior during mitosis and meiosis and provides a sensitive and quantifiable procedure for examining chemically induced aneuploidy.     

Thymus and Bursa Dependence of Lymphocyte Mitogenic Factor in the Chicken

AMONG the soluble products generated during activation of rodent or human lymphocytes¹, lymphocyte-stimulating (‘mitogenic’) factors are detected by their ability to accelerate DNA metabolism of freshly cultured allogeneic or syngeneic lymphocytes^2–8. If such lymphocyte activation products function as mediators of cellular immunity^1,9–11, their activities will be generated independently of the antibody-forming system. In the chicken, antigen-stimulation of sensitized lymphocytes in vitro involves both thymus (T-) and bursa-(B-)-dependent cells^12–14. If antigen-induced lymphocyte transformations are generally mediated by lymphocyte mitogenic factors, both T-cells and B-cells should produce lymphocyte mitogenic factors when stimulated by specific antigen. We have therefore determined whether antigen-stimulated chicken lymphocytes generate a lymphocyte mitogenic factor and whether this response is affected by neonatal thymectomy or bursectomy.     

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.     

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555–571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cell-expressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K_d for A2. The second assay determined the K_d for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K_d and k_cat values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K_d values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K_d for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k_cat for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.     

Species-Specific Aggregation Factor In Sponges

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA -initiation phase the cyclic AMP - and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10⁶ cells to 0.3 pmol/10⁶ cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10⁶ cells. the activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. the soluble as well as the particulate enzyme activities were checked in vitro. the cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

The 1.7 ? X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.