Study of a proposed international standard for blood coagulation factor IX

An International Collaborative Study was organized to establish a standard for factor IX. Two freeze-dried concentrate preparations, C1 and C2, and one freeze-dried plasma P were compared with each other, with fresh normal plasmas and with local standards in 13 laboratories. One of the concentrate preparations (C1) contained heparin and this gave rise to non-parallel assays in laboratories testing concentrate C1 in dilutions containing more than 0.05 i.u. of heparin per ml. Assays of factor IX showed good precision for both plasma and concentrate in all laboratories; no systematic effect of method, operator or day of assay was detected. The plasma preparation P and the concentrate preparation C2 were compared with 59 individual fresh normal plasma samples, and a mean potency ratio of 0.78 (95% confidence limits 0.73-0.84) for plasma and 5.62(95% confidence limits 5.13-6.16) for the concentrate C2 obtained. Only 21 estimates of concentrate C1 in terms of fresh plasma were obtained giving a mean potency ratio of 3.85 (95% confidence limits 1.87-7.92). The estimated loss of potency for freeze-dried plasma stored at -20 degrees C is approximately 0.4% per year. The concentrate C2 is apparently more stable and only very small losses occurred even at higher storage temperatures. All participants agreed that the preparation C2 would be suitable to serve as an International Standard for factor IX; they also agreed that the figure assigned for the unitage should be based on the number of ml of 'average fresh normal plasma' estimated to contain the factor IX activity of one ampoule of the preparation. It is proposed to recommend to the World Health Organization that the preparation of factor IX concentrate C2, in ampoules coded 72/32, be considered for establishment as the International Standard for factor IX, and that the international unit for factor IX be assigned on the basis of 5.62 units per ampoule of this preparation.     

Coagulation factor X-binding protein from Deinagkistrodon acutus venom is a Gla domain-binding protein

Factor IX/factor X-binding protein (IX/X-bp) is an anticoagulant isolated from the venom of Trimeresurus flavoviridis (habu snake) and binds predominantly to factor IX. In this study, we isolated IX/X-bp-like proteins from the venom of Deinagkistrodon acutus (hundred pace snake) with binding characteristics different from those of IX/X-bp. The complete amino acid sequence and binding characteristics of the main anticoagulant protein, named X-bp, were investigated. The concentrations of X-bp at half-maximal binding to solid-phase factors X and IX were 0.4 and 3 nM, respectively. The binding of X-bp to solid-phase factor X was inhibited by 50% by 6- and 9-fold excess concentrations of factor X and Gla domain (GD) peptide 1-44, respectively, but was not influenced by GD peptide 1-41 and Gla domainless factor X. X-bp bound two Ca2+ ions per molecule with Kd values of 16 +/- 0.7 (mean +/- SE, n = 6) and 103 +/- 10 microM. X-bp was a heterodimer of C-type lectin-like subunits. The 16 kDa chain (A chain) consisted of 129 amino acid residues and was 68% identical to the sequence of the A chain of IX/X-bp. The 15 kDa chain (B chain) consisted of 123 amino acid residues and was 87% identical to IX/X-bp. Three-dimensional model construction from the known fold of IX/X-bp showed that amino acid residues different from those of IX/X-bp are mostly on the molecular surface. Some of these are concentrated on a part of the concave surface which is considered to be the coagulation factor-binding site, presumably acting as a discriminator for ligand binding. These results indicated that X-bp isolated from D. acutus venom was a GD-binding protein, and the C-terminal region of GD peptide was critical for folding of the peptide.     

In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells

Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.     

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.     

Factor IX of the blood coagulation system: a review

Factor IX is a factor of the blood coagulation system. Its activation occurs on the surface of phospholipid membranes. It can be activated by the factor VIIa-TF (tissue factor)-Ca2+ complex via an extrinsic pathway and by factor XIa in the presence of Ca2+ via the intrinsic pathway of blood coagulation system activation. The activated factor IXa is a serine proteinase. The main function of the activated factor IXa in complex with factor VIIIa and phospholipids in presence of Ca2+ consists of the activation of factor X. Factor IX is synthesized in the liver and is subject to a number of posttranslational modifications including gamma-carboxylation, beta-hydroxylation, and glycosylation. It forms a subgroup of vitamin K-dependent plasma proteins including factors VII and X and protein C characterized by identical domain structures having high levels of homology. Factor IX consists of an NH2-terminal Gla domain, two epidermal growth factor (EGF)-like domains, and a C-terminal domain containing Ser in its active site. Factor IX deficiency in human plasma results in the disease known as hemophilia B.     

Role of the propeptide and gamma-glutamic acid domain of factor IX for in vitro carboxylation by the vitamin K-dependent carboxylase

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the processive carboxylation of specific glutamates in a number of proteins related to blood coagulation and bone. To address the independent importance of the propeptide, gamma-carboxyglutamic acid (Gla) domain and elements beyond the Gla domain of factor IX in vitamin K-dependent carboxylation, we have examined the kinetics of carboxylation of peptides containing (1) propeptide and Gla domain, (2) the Gla domain alone, (3) uncarboxylated bone Gla protein, (4) propeptide followed by the entire uncarboxylated factor IX molecule, and (5) the factor IX propeptide followed by a non-Gla domain sequence. Our studies indicate that peptides with a covalently linked propeptide have Km values similar to the physiological substrate of the carboxylase. In contrast, the Gla domain of factor IX has a >/=230-fold higher Km for the carboxylase than the corresponding peptide with a covalently linked propeptide. This contrasts with bone Gla protein, another vitamin K-dependent protein, which appears not to require a covalently linked propeptide for high-affinity binding to the carboxylase. Analysis of the carboxylation products of a propeptide/non-Gla domain substrate indicate that it is carboxylated multiple times in a processive manner. These studies show that the perceived binding affinity of the carboxylase substrate and processivity is conferred by the propeptide without requiring the conserved Gla domain sequences and that factor IX and bone Gla protein may have distinct mechanisms of interacting with the carboxylase.     

Purification and characterization of human coagulation factor V

We have purified human coagulation Factor V 6,000-fold to homogeneity from citrated plasma using polyethylene glycol 6000 precipitation, adsorption of Factor V to barium citrate, DEAE-Sepharose chromatography, and gel filtration on Ultrogel AcA 34 (yield 21%). Human Factor V is a single polypeptide chain before and after disulfide bond reduction with an apparent Mr = 335,000 as determined by electrophoresis on 5% acrylamide sodium dodecyl sulfate gels. Human Factor V is a glycoprotein containing 13% of weight carbohydrate and there is a high content of sialic acid (86 residues/mol) compared to the other sugars. When human Factor V is treated with thrombin, coagulation activity increases 25- to 30-fold to a specific activity of 1.7 to 2.0 units/microgram. Thrombin activation is accompanied by the cleavage of three bonds in the Factor V molecule. We have detected activation intermediates with apparent Mr = 295,000 and 248,000 and final products with apparent Mr = 150,000, 121,000, and a doublet at 95,000-91,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final products of thrombin activation of human Factor V and bovine Factor V are similar, yet the intermediates observed are different. This suggests that cleavages are made at similar locations in bovine and human Factor V, but that they occur in a different sequence. When human Factor V is treated with the Factor V activator from Russell's viper venom, it is split into two components with apparent Mr = 303,000 and 95,000-91,000 and is fully activated. The increase in coagulation activity observed upon treatment of human Factor V with thrombin or the Factor V activator from Russell's viper venom seems to correlate with the generation of the doublet Mr = 95,0090-91,000 component.     

Identification of a factor IX binding site on the third apple domain of activated factor XI

Activated factor XI (factor XIa) participates in blood coagulation by activating factor IX. Previous work has demonstrated that a binding site for factor IX is present on the noncatalytic heavy chain of factor XIa (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). Recombinant factor XI proteins were expressed in which each of the four apple domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding domain from the homologous but functionally distinct protease prekallikrein (PK). To identify the site of factor IX binding, the chimeric proteins were activated with factor XIIa and tested for their capacity to activate factor IX in plasma coagulation and purified protein assays. The chimera with the substitution in the third apple domain (factor XI/PKA3) had <1% of the coagulant activity of wild type factor XIa in a plasma coagulation assay, whereas the chimeras with substitutions in A1, A2, and A4 demonstrated significant activity (68-140% of wild type activity). The Km for activation of factor IX by factor XIa/PKA3 (12. 7 microM) is more than 30-fold higher than the Km for activation by wild type factor XIa or the other factor XI/PK chimeras (0.11-0.37 microM). Two monoclonal antibodies (2A12 and 11AE) that recognize epitopes on the factor XI A3 domain were potent inhibitors of factor IX activation by factor XIa, whereas antibodies against the A2 (1A6) and A4 (3G4) domains were poor inhibitors. The data indicate that a binding site for factor IX is present on the third apple domain of factor XIa.     

Retroviral vector-modified bone marrow stromal cells secrete biologically active factor IX in vitro and transiently deliver therapeutic levels of human factor IX to the plasma of dogs after reinfusion

Canine bone marrow stromal cells (BMSCs), transduced ex vivo with retroviral vectors, expressed and secreted biologically active human and canine coagulation factor IX (hFIX and cFIX) in vitro, and on autologous reinfusion expressed hFIX into the circulation of normal (nonhemophiliac) dogs. Human FIX, when expressed in vitro by BMSCs of two dogs at 1.22 and 1.39 microg/10(6) cells/24 hr in medium supplemented with vitamin K, respectively, exhibited 28.1 and 27.3% normal biological activity as determined on the basis of a one-stage clotting assay. BMSCs of two additional dogs expressed 1.54 and 4.81 microg of cFIX/10(6) cells/24 hr in vitamin K-supplemented medium and the expressed cFIX possessed 58.4 and 32.9% normal activity, respectively. Between 2.33 and 3.35 x 10(8) transduced BMSCs, expressing 1.22 and 2.61 microg of hFIX/10(6) cells/24 hr or 3.24 and 7.82 microg of cFIX/10(6) cells/24 hr were reintroduced into the four donor dogs by intravenous infusion. Human FIX was detected in plasma for 7 or 12 days after BMSC reinfusion, with peak levels of 85.8 and 233.0 ng/ml observed at 2 days. Canine anti-hFIX antibodies, which were detected as early as 2-4 days after reinfusion of BMSCs expressing hFIX, may have masked potentially longer duration expression in vivo. Peak plasma levels of hFIX represented 2.1 and 5.8% normal human hFIX levels. When adjusted for percent normal one-stage clotting activity determined in vitro, these levels represented 0.6 and 1.6% normal human hFIX activity levels. Thus, we have demonstrated that retroviral vector-modified BMSCs can deliver human therapeutic levels of hFIX to the circulation of dogs.     

Extravascular administration of factor IX: potential for replacement therapy of canine and human hemophilia B

Current therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availability of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (s.c.), intramuscular (i.m.), intraperitoneal (i.p.) or intravenous (i.v.) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical i.v. dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the i.v. route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the i.m. injection in the canine resulted in a bioavailability of 82.8%, while the s.c. injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with i.v. administration. These data show that significant levels of F.IX may be obtained via s.c. injection in canine and human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.     

Mutational analysis of antistasin, an inhibitor of blood coagulation factor Xa derived from the Mexican leech Haementeria officinalis

Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.     

Activation of factor VII during alimentary lipemia occurs in healthy adults and patients with congenital factor XII or factor XI deficiency, but not in patients with factor IX deficiency

Factor VII activity (FVIIc), a risk marker for coronary heart disease, is increased during postprandial lipemia. Factor VII activation accompanies lipolysis of triglyceride-rich lipoproteins, but the nature of this association and whether it is causal remain uncertain. To explore this issue, four patients with homozygous factor XII deficiency, four with complete factor XI deficiency, six with factor IX deficiency, and their respective age- and sex-matched controls were given two isocaloric dietary regimens, one providing on average 136 g fat and the other 19 g fat. Blood was taken before breakfast, immediately before lunch at 195 minutes, and at completion of the study at 390 minutes. All samples for each subject and matched control were assayed as one batch for FVIIc, activated factor VII, and factor VII antigen (FVIIag). Activation of factor VII was observed with the high-fat regimen but not with the low-fat regimen in all controls, factor XII-deficient patients, and factor XI-deficient patients. No factor VII activation was observed during either regimen in factor IX-deficient patients, but a normal postprandial responsiveness of factor VII to dietary fat was restored in one patient who replicated the study after factor IX therapy. Plasma FVIIag was not altered postprandially in either regimen in any group of patients or controls. Factor IX apparently plays an obligatory role in the postprandial activation of factor VII, although the mechanism remains to be determined.     

Mass-spectrometric identification and sequence location of the ten residues of the new amino acid (gamma-Carboxyglutamic acid) in the N-terminal region of prothrombin

The detailed mass-spectrometric evidence for our original findings [Magnusson et al. (1974) FEBS Lett. 44, 189-193] of ten gamma-carboxyglutamic acid residues in the N-terminal calcium-binding polypeptide of prothrombin is presented. The identification and sequence location of gamma-carboxyglutamic acid was made by electron-impact and field-desorption studies on acetyl permethyl peptide derivatives, and on the free amino acid. Details of the derivatives formed, and how this new amino acid may be easily recognized and sequenced from the mass spectrum, are given as a basis for future work.     

Neutrophil elastase cleavage of human factor IX generates an activated factor IX-like product devoid of coagulant function

In preliminary studies, the generation of thrombin in vivo was found to induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F.IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost 69:1127, 1993 [abstr]). These changes were associated with evidence of increased elastase availability. To study the possibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F.IX by human neutrophil elastase (HNE) was performed in vitro. An activated partial thromboplastin time (aPTT) clotting assay demonstrated that, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that could not be distinguished from the respective heavy and light chain peptides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators. In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the active site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3) activate human factor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal sequencing indicated that HNE cleaved human F.IX at Thr140, Thr144, Ile164, Thr172, and Val181. The cleavages at Thr140/Thr144 and at Thr172/Val181 are both very close to the normal F.XIa alpha-(Arg145) and beta-(Arg180) cleavage sites, respectively. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of improper active site formation. These in vitro observations support the possibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as disseminated intravascular coagulation (DIC).     

The second exon-encoded factor XII region is involved in the interaction of factor XII with factor XI and does not contribute to the binding site for negatively charged surfaces

Contact system activation, in vitro, is triggered by activation of factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located within the amino acid residues 1-28 by identifying the epitope recognized by a monoclonal antibody (MoAb), B7C9, which inhibits kaolin-induced clotting activity. To further elucidate the role of the amino terminal binding site in the regulation of FXII activation, we have characterized a FXII recombinant protein (rFXII-triangle up19) deleted of the amino acid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-triangle up19 was constructed and expressed in HepG2 cells by using vaccinia virus. Purified rFXII-triangle up19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharose, thus confirming that it lacked the epitope for this MoAb, and had no amidolytic activity towards the chromogenic substrate S-2302 in the absence of activator. rFXII-triangle up19 specific clotting activity was lower (44%) than that of native FXII. The activation rate of rFXII-triangle up19 by kallikrein in the absence of dextran sulfate was about four times higher than that of full-length FXII and was increased in the presence of dextran sulfate. However, rFXII-triangle up19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-triangle up19 bound to kaolin, which binding was equally well inhibited by either, rFXII-triangle up19 or full-length FXII (IC50 = 7.2 +/- 2.2 nmol/L for both proteins). Accordingly, a synthetic peptide corresponding to FXII amino acid residues 3-19 did not inhibit the binding of labeled full-length FXII to kaolin. rFXII-triangle up19 generated a similar amount of FXIIa- and kallikrein-C1-inhibitor complexes in FXII-deficient plasma in the presence of kaolin, as did full-length FXII; but generated less factor XIa-C1-inhibitor complexes (50%) than full-length FXII. This impaired factor XI activation by rFXII-triangle up19a was also observed in a purified system and was independent of the presence of high molecular weight kininogen. Furthermore, the synthetic peptide 3-19, preincubated with factor XI, inhibited up to 30% activation of factor XI both in the purified system as well as in plasma. These results together indicate that amino acid residues 3-19 of FXII are involved in the activation of factor XI and do not contribute to the binding of FXII to negatively charged surfaces.     

Purification of human factor IX by chromatography of a coagulation factor concentrate

A purification procedure for, and some properties of, coagulation factor IX are described. The coagulation factor concentrate used for the treatment of hemophilia B patients was employed as the starting material. The isolation procedure consists of chromatography in DEAE-cellulose, two chromatographies in hydroxyapatite gel and two gel filtrations in Sephadex G-200. Only trace amounts of factors II, VII and X were present in the final preparation and the specific activity of factor IX was 159 corresponding 10,300 times purification from plasma. The molecular weight was estimated to be 76,000 in gel filtration and 86,000 in sodium dodecyl sulfate disc gel electrophoresis. Three activity peaks with pIs 4.15, 4.25 and 4.40 were obtained by isoelectric focusing.     

Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (10(12) to 10(13) particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1alpha promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 10(10) particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1alpha-F. IX of 2.7 x 10(11) particles resulted in plasma levels of 700 to 3, 200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1alpha-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.     

Role of gamma-carboxyglutamic acid. Cation specificity of prothrombin and factor X-phospholipid binding

Divalent cations are required for two roles in prothrombin-phospholipid interaction. The first role, catalysis of a prothrombin protein transition has a reaction half-life of 100 min at 0 degrees and is a prerequisite to phospholipid binding. The binding sites required for the transition have a very low cation specificity. All di- and trivalent cations tested were effective in this role with the exception of beryllium. Barium catalyzed the transition but only at high concentrations (6.6 mM was required for half-reaction). Blood-clotting Factor X, another gamma-carboxyglutamic acid-containing protein, also undergoes a cation-catalyzed protein transition which is a prerequisite to Factor X-phospholipid binding. In both proteins, the transition can be monitored by a decrease in the protein's intrinsic fluorescence. Compared to prothrombin, the Factor X transition occurs much more rapidly, has a somewhat greater specificity for cations, and requires higher concentrations of cations. This indicates that the cation binding sites provided by gamma-carboxyglutamic acid are not completely uniform in all proteins. The second role of divalent cations in prothrombin-phospholipid interaction is in the actual protein-phospholipid binding. This interaction was studied by protein fluorescence quenching resulting from excitation energy transfer to a chromophore attached to the phospholipid membrane. Only strontium and barium satisfactorily replaced calcium in this role. A number of other cations form protein-phospholipid complexes but of the wrong structure. These cations inhibit the prothrombinase complex (Factor Xa, calcium, phospholipid, Factor V). The cation specificity for Factor X-phospholipid binding is the same as for prothrombin except that higher concentrations of cations are required. Factor Xa (generated by action of Russell's viper venom on Factor X) displayed the same calcium requirements for the protein transition and phospholipid interaction as Factor X. The cation requirements of the prothrombinase complex correlate with the cation requirements of prothrombin and Factor X-phospholipid binding. Strontium is the only cation that will singly replace calcium. Barium is ineffective alone because the concentrations required to catalyze the protein transitions cause precipitation of the phospholipid. Combination of certain other cations with barium will, however, substitute for calcium. The other cations (specifically magnesium or manganous ion) catalyze the protein transitions and barium forms the correct protein-phospholipid complexes.     

Ex vivo fibroblast transduction in rabbits results in long-term (>600 days) factor IX expression in a small percentage of animals

Delivery of human factor IX to the circulation was analyzed in rabbits by ex vivo fibroblast transduction followed by subcutaneous implantation. Kinetic studies of human factor IX in rabbits demonstrated a half-life of approximately 16 hr and a volume distribution of 22%, where intraperitoneal and subcutaneous bioavailability was three- to sevenfold lower than by intravenous administration. Ex vivo retroviral transduction of autologous fibroblasts was performed on 15 animals. After subcutaneous injection of fibroblast-collagen mixtures, the expression of human factor IX in rabbit plasma was followed by ELISA. Of 15 rabbits injected, expression of human factor IX was detected in 2 animals, and expression was long term (>600 days). One animal had stable levels of human factor IX, at 20 ng/ml, while the second animal had lower and gradually decreasing levels of human factor IX. There were no gross differences in pathology at the injection sites, when comparing animals with human factor IX in plasma and those without. Immunological studies demonstrated antibody formation in response to injection mixture components (including human factor IX), but again there was no correlation with immune response and long-term factor IX production in animals. Tissues at the implantation sites were positive for factor IX DNA by PCR analysis, regardless of whether there was detectable plasma factor IX or not. Small numbers of PCR-positive cells were detected in the internal organs of the long term-expressing rabbits while similar tissues were negative in nonexpressing animals.     

Immunosuppressive treatment in haemophiliacs with inhibitors to factor VIII and factor IX

9 patients with severe haemophilia A and inhibitors (inhibitor levels between 0.1 to 5.8 U/ml) and 3 patients with severe haemophilia B and inhibitors (inhibitor levels between 0.1 to 11 U/ml) were treated on a total of 16 and 13 occasions, respectively, with a large dose of antigen (factor VIII or factor IX) and cyclophosphamide (10-15 mg/kg b.w. i.v. initially and then 2-3 mg/kg b.w. orally for 7-10 days) in connection with severe bleeding and surgery. All the patients had proved not to respond to treatment with factor VIII or factor IX concentrate alone, and all except one had shown strong secondary antibody increases. In 6 of the patients with haemophilia A the treatment (11 occasions) had a satisfactory haemostatic effect and even permitted neurosurgery without bleeding complications. The inhibitor level remained at zero for 5-10 days, after which it gradually began to return towards its original level. In these cases it was possible to give factor VIII in amounts which neutralised the inhibitor and afterwards raised the factor VIII initially to at least 50%. In the 3 patients with haemophilia B treatment (13 occasions) was successful except on one occasion, and surgery was performed without abnormal bleeding. The factor IX level was initially raised to at least 50% except in the one failure. The inhibitor level remained at zero for 12 days to 3 months, after which it gradually rose towards its original level. One patient was treated on 8 occasions.