[Dental and oral surgical treatment of a B haemophilic patient with high inhibitor level. Case report]. / Magas inhibitor titeru B-haemophiliás beteg fogorvosi-szájsebészeti ellátása. Esetismertetés.

More than 1000 hemophilic male patients are registered in Hungary, from which only a trace number suffers from factor IX inhibitory hemophilia. For correct dental and oral surgical treatment of these patients mandatory cooperation is required among medical specialties, exerting multi-staged haemostatic principles. Authors represent in this case report the dental and oral surgical treatment of a B hemophilic patient with high inhibitor level and describe possible local haemostatic measures.

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide.

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells.

Most adenoviruses bind directly to the coxsackie and adenovirus receptor (CAR) on target cells in vitro, but recent research has shown that adenoviruses can also use soluble components in body fluids for indirect binding to target cells. These mechanisms have been identified upon addressing the questions of how to de- and retarget adenovirus-based vectors for human gene and cancer therapy, but the newly identified mechanisms also suggest that the role of body fluids and their components may also be of importance for natural, primary infections. Here we demonstrate that plasma, saliva, and tear fluid promote binding and infection of adenovirus type 5 (Ad5) in respiratory and ocular epithelial cells, which corresponds to the natural tropism of most adenoviruses, and that plasma promotes infection by Ad31. By using a set of binding and infection experiments, we also found that Ad5 and Ad31 require coagulation factors IX (FIX) or X (FX) or just FIX, respectively, for efficient binding and infection. The concentrations of these factors that were required for maximum binding were 1/100th of the physiological concentrations. Preincubation of virions with heparin or pretreatment of cells with heparinase I indicated that the role of cell surface heparan sulfate during FIX- and FX-mediated adenovirus binding and infection is mechanistically serotype specific. We conclude that the use of coagulation factors by adenoviruses may be of importance not only for the liver tropism seen when administering adenovirus vectors to the circulation but also during primary infections by wild-type viruses of their natural target cell types.

Variability in bleeding phenotype in Amish carriers of haemophilia B with the 31008 C-->T mutation.

The aim of this study was to characterize the variability of bleeding phenotype and its association with plasma factor IX coagulant activity (FIX:C) in haemophilia B carriers in a large Amish pedigree with a unifying genetic mutation, C-to-T transition at base 31008 of the factor IX gene (Xq27.1-27.2). A cross-sectional survey of haemophilia B carriers included a multiple choice questionnaire evaluating symptoms of mucocutaneous bleeding, joint bleeding and bleeding after haemostatic stress [menstruation, postpartum haemorrhage (PPH), dental extractions and invasive surgeries]. Severity of bleeding was graded as 0 to 4, 0 being no bleeding whereas 4 being severe bleeding. Association between total bleeding scores and the FIX:C was evaluated. Sixty-four haemophilia B carriers participated in this study. Median age: 18 years (range 1-70 years); median bleeding score: 1 (range 0-8). Besides PPH, isolated symptoms of bruising, epistaxis, menorrhagia and postsurgical bleeding including dental extraction were not associated with lower FIX:C. Bleeding score >/=3 was associated with involvement of at least two bleeding sites and a lower mean FIX:C of 42 +/- 10.3% (95% CI 36.4-47.7) while a score >3 had involvement of /=3. Phenotypic variability existed among the carriers of haemophilia B who belonged to a single pedigree carrying a single unifying mutation. The utility of bleeding scores to define bleeding phenotype precisely in haemophilia B carriers needs further evaluation.

Prophylactic administration of glycoPEGylated factor IX provides protection and joint outcome superior to recombinant factor IX after induced joint bleeding.

BACKGROUND: Following induced joint hemorrhage, hemophilia B results in the abnormal persistence of iron deposition, inflammation, and neovascularity of the synovial tissue, as well as deterioration of the bone articular surface and strength. Previously, we demonstrated that a factor IX (FIX) replacement protein with extended circulating FIX activity, glycoPEGylated FIX nonacog beta pegol (N9-GP), could improve synovial and osteochondral parameters in F9 knockout mice when administered after joint injury. OBJECTIVE: We explored the use of N9-GP prior to unilateral joint hemorrhage and compared to unmodified recombinant FIX (rFIX). METHODS: Pharmacodynamics, histology, and microcomputed tomography were used to assess the effects of prophylactic administration of glycoPEGylated FIX. RESULTS: In comparison to rFIX, N9-GP significantly improved soft tissue histological parameters, as well as bone outcome at 2 weeks post injury, while performing equally in reduction of blood present in the joint space assessed 1 day after injury. CONCLUSIONS: These results indicate that, in comparison to rFIX, the prophylactic use of extended half-life FIX provides superior protection from bleeding-induced joint damage, manifested by improved correction of histologic parameters.

Kinetics of Factor X activation by the membrane-bound complex of Factor IXa and Factor VIIIa.

Intrinsic tenase consists of activated Factors IX (IXa) and VIII (VIIIa) assembled on a negatively charged phospholipid surface. In vivo, this surface is mainly provided by activated platelets. In vitro, phosphatidylcholine/phosphatidylserine vesicles are often used to mimic natural pro-coagulant membranes. In the present study, we developed a quantitative mathematical model of Factor X activation by intrinsic tenase. We considered two situations, when complex assembly occurs on either the membrane of phospholipid vesicles or the surface of activated platelets. On the basis of existing experimental evidence, the following mechanism for the complex assembly on activated platelets was suggested: (i) Factors IXa, VIIIa and X bind to their specific platelet receptors; (ii) bound factors form complexes on the membrane: platelet-bound Factor VIIIa provides a high-affinity site for Factor X and platelet-bound Factor IXa provides a high-affinity site for Factor VIIIa; (iii) the enzyme-cofactor-substrate complex is assembled. This mechanism allowed the explanation of co-operative effects in the binding of Factors IXa, VIIIa and X to platelets. The model was reduced to obtain a single equation for the Factor X activation rate as a function of concentrations of Factors IXa, VIIIa, X and phospholipids (or platelets). The equation had a Michaelis-Menten form, where apparent V(max) and K(m) were functions of the factors' concentrations and the internal kinetic constants of the system. The equation obtained can be used in both experimental studies of intrinsic tenase and mathematical modelling of the coagulation cascade. The approach of the present study can be applied to research of other membrane-dependent enzymic reactions.

Gut epithelial cells as targets for gene therapy of hemophilia.

Gut epithelium is an attractive target for gene therapy of hemophilia due to the large number of rapidly dividing cells that should be readily accessible to a wide range of vectors by a noninvasive route of administration. We have performed in vitro tests to determine the suitability of gut epithelial cells for gene transfer, protein synthesis, and secretion of coagulation factors VIII and IX. The results with retroviral vectors indicate that transduced epithelial cells from human, rat, or porcine small or large intestine can synthesize significant amounts of factor VIII or factor IX and that two-thirds or more of the recombinant protein is secreted in a basolateral direction (i.e., away from the lumen and toward underlying capillaries and lymphatics). Furthermore, we have demonstrated that intestinal epithelial cells are susceptible to efficient gene transfer by lipofection and adenovirus vectors. In the case of factor IX, we have produced a high-titer adenovirus vector capable of transducing gut epithelial cells resulting in synthesis of factor IX. The results of our in vitro studies indicate that gene transfer targeting gut epithelium as a new approach to hemophilia gene therapy is rational and merits in vivo studies in hemophilia animal models.

Functional properties of factor VIIa/tissue factor formed with purified tissue factor and with tissue factor expressed on cultured endothelial cells.

Factor VIIa (F. VIIa)/tissue factor (TF) function was examined using purified human TF reconstituted into mixed phospholipid vesicles and TF expressed on cultured human umbilical vein endothelial cells (HUVEC) treated with thrombin. In reaction mixtures containing either type of TF, F. VIIa, 10 nM, either 3H-factor X or 3H-factor IX, 88 nM, and Ca2+, 5 mM, F. VIIa/TF activated factor X (F. X) several fold faster than it activated factor IX (F. IX). Adding heparin, 1 U/ml, increased rates of activation of both substrates and F. X remained the preferred substrate. Adding plasma at concentrations of 5% or above inhibited factor VIIa/TF catalytic activity. Inhibition was shown to require F. Xa as a cofactor, was prevented by antibodies to extrinsic pathway inhibitor (EPI), and was reversible by decalcification. Thus, with factor VIIa/TF formed with both types of TF, EPI appeared responsible for inhibition induced by plasma. Our data indicate that functional properties of factor VIIa/TF as delineated in reaction mixtures made with purified TF reconstituted into mixed phospholipid vesicles also hold for factor VIIa/TF activity on the surface of cultured HUVEC.

Biospecific interactions of vitamin K-dependent factors with phospholipid-like polystyrene derivatives. Part II: factor IX.

We previously demonstrated that phosphorylated polystyrene derivatives exhibit phospholipid-like behaviour and therefore are able to interact with factor II, one of the vitamin K-dependent coagulation factors. Under the same conditions as for factor II, we examined the interactions of factor IX with phosphorylated resins of various compositions in phosphate groups: these studies were carried out with or without albumin precoating of the polymers and either in the presence or absence of calcium ions. Adsorption experiments show that, in the absence of calcium ions, only one class of adsorption sites of factor IX can be evidenced with the interactions taking place through the formation of binary complexes, whereas in the presence of calcium ions, the affinity of factor IX for phosphorylated resins becomes very high and two types of adsorption sites have been evidenced with biospecific ternary complexes being formed. The domains of predominance of these complexes were determined. Moreover, the only functional groups borne by the phosphorylated polystyrene resins involved in factor IX-polymer interactions are phosphodiester groups. Comparison between factor II and factor IX adsorption onto the same polymers leads to the conclusion that the observed differences probably reflect the differences in the Gla domains of the vitamin K-dependent factors. Finally, this study demonstrates that phosphorylated polystyrene derivatives can be used as stationary phases for purification of factor IX by highly specific liquid biochromatography.

Anticoagulant mechanism of sulfonated polyisoprenes.

The influence of sulfonated polyisoprene (SPIP) on coagulation factors and human blood cells was investigated to elucidate and compare its anticoagulant mechanism with that of heparin. While the number of red cells was unaffected, the number of platelets decreased dramatically in the presence of SPIP due to aggregation. Using a synthetic peptide substrate to assay thrombin activity in the presence of its natural inhibitor, antithrombin (AT), we observed no stimulation by SPIP of AT-mediated inhibition. Nevertheless, thrombin cleavage of its natural substrate fibrinogen to fibrin peptide A was slightly inhibited. SPIP altered the electrophoretic mobility of fibrinogen and completely inhibited fibrinogen from clotting. We detected no significant influence of SPIP on factors II, VII, IX, and X, while factor XI and factors V and VIII were only slightly affected. Therefore, the main mechanism of SPIP's anticoagulant activity appears to be a strong interaction with fibrinogen and fibrin monomer, first, to prevent proteolytic conversion of the former to the latter and second, to inhibit polymerization of the fibrin monomer, once formed.

Further evaluation of a heparin neutralizer and its effect on factor IX in normal and coumadin-plasma.

Heparsorb, a commercial anion exchange resin capable of removing large amounts of heparin from heparinized plasma, has recently been introduced into the clinical diagnostic laboratory as a useful reagent for the evaluation of blood coagulation in heparinized blood and the evaluation of an unexpected prolongation of global coagulation tests. In this paper data are presented which indicate that Heparsorb has little or no effect on the activities of blood coagulation factors in heparinized plasma, except for a modest (22%) reduction in factor IX-activity. Maximal loss of factor IX-activity was observed after incubation of plasma with Heparsorb for 15 min at room temperature. Prolonged storage of plasma before or after incubation with Heparsorb and changes in plasma pH had no appreciable effect on the extent of factor IX-activity loss. Evidence is presented to substantiate earlier findings that factor IX-activity is not removed by Heparsorb from plasma of patients on coumadin therapy, and to indicate that this lack of effect of Heparsorb on factor IX-activity in coumadin plasma is due to reduced affinity of hypocarboxy-IX for the heparin neutralizing resin.

Alprolix (recombinant Factor IX Fc fusion protein): extended half-life product for the prophylaxis and treatment of hemophilia B.

Hemophilia B is a genetic disease caused by mutation of the gene for coagulation protein Factor IX. When severe, the disease leads to spontaneous life-threatening bleeding episodes. Current therapy requires frequent intravenous infusions of therapeutic recombinant or plasma-derived protein concentrates containing Factor IX. Alprolix™ (recombinant Factor IX Fc fusion protein), is a therapeutic Factor IX preparation that has been engineered for a prolonged half-life in circulation, has completed pivotal clinical trials and has been approved recently in the USA, Canada, Australia and Japan for use in the clinic for patients with hemophilia B. This promising therapy should allow patients to use fewer infusions to maintain appropriate Factor IX activity levels in all clinical settings, and its use may be indicated in both on demand and prophylactic treatments.

Coating of adenovirus type 5 with polymers containing quaternary amines prevents binding to blood components.

Adenovirus type 5 (Ad5) gene therapy vectors require protection against antibodies, complement proteins and blood cells if they are to be delivered intravenously to treat metastatic disease. Such protection can be achieved by chemically modifying Ad5 with polymers based on hydrophilic HPMA. Here, such polymers were designed to include side chains bearing reactive carbonyl thiazolidine-2-thione groups (TTs) to covalently modify available amino groups of the lysine residues in the Ad5 capsid. Furthermore, the inclusion of side chains bearing positively charged quaternary ammonium groups (QAs) was designed to improve electrostatic interaction of the polymers with negatively charged Ad5 hexon protein. Finally, to enable triggered uncoating and reactivation of the Ad5, either the TTs or both the TTs and the QAs were linked to polymer backbone via reductively degradable disulfide bonds. SDS-PAGE demonstrated that these polymers covalently modified Ad5 capsid proteins in a reduction reversible manner. In infection studies, polymers containing QAs prevented binding of coagulation factor X to Ad5. Furthermore, the antibody and complement mediated binding of Ad5 to erythrocytes was reduced by such polymers (>95% without polymer, 25% following coating). These data indicate that coating Ad5 therapeutics with such polymers will improve blood circulation half-life and deposition at disease sites.

Sustained and therapeutic levels of human factor IX in hemophilia B mice implanted with microcapsules: key role of encapsulated cells.

BACKGROUND: A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein was explored in this study. In order to prevent immune rejection of the delivered cells, they were enclosed in non-antigenic biocompatible alginate microcapsules prior to being implanted intraperitoneally into mice. We have shown that encapsulated C2C12 myoblasts can temporarily deliver therapeutic levels of factor IX (FIX) in mice, but the C2C12 myoblasts elicited an immune response to FIX. In this study we report the use of mouse fetal G8 myoblasts secreting hFIX in hemophilia mice. METHODS: Mouse G8 myoblasts were transduced with MFG-FIX vector. A pool of recombinant G8 myoblasts secreting approximately 1500 ng hFIX/10(6) cells/24 h in vitro were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into immunocompetent C57BL/6 and hemophilic mice. RESULTS: Circulating levels of hFIX in treated mice reached approximately 400 ng/ml for at least 120 days (end of experiment). Interestingly, mice treated with encapsulated G8 myoblasts did not develop anti-hFIX antibodies. Activated partial thromboplastin time (APTT) of plasmas obtained from treated hemophilic mice was reduced from 107 to 82 sec on day 60 post-treatment, and whole blood clotting time (WBCT) was also corrected from 7-9 min before treatment to 3-5 min following microcapsule implantation. Further, mice were protected against bleeding following major trauma. Thus, the FIX delivery in vivo was biologically active. CONCLUSIONS: Our findings suggest that the type of cells encapsulated play a key role in the generation of immune responses against the transgene. Further, a judicious selection of encapsulated cells is critical for achieving sustained gene expression. Our findings support the feasibility of encapsulated G8 myoblasts as a gene therapy approach for hemophilia B.

Viral inactivation of vesicular stomatitis virus in normal human serum by cross-linked polyvinylpyrrolidone.

Methods for inactivating virus contaminants in serum, cryoprecipitate-poor plasma, and protein concentrates need to be identified. In this study, vesicular stomatitis virus (VSV)-spiked human serum and cryoprecipitate-poor plasma were treated with cross-linked povidone iodine (XLPVPI) at concentrations of 0, 4, 6, 8, and 10 mg/mL up to 120 min at 4 and 24 degrees C. The activities of virus and relevant proteins were examined. The results indicated that XLPVPI at concentrations that inactivate > 5 logs of VSV in serum decreased factor IX and protein C activities by < 10% in cryoprecipitate-poor plasma. At concentrations up to 10 mg of XLPVPI/mL, < 10% of protein C and factor IX activity was lost after incubation for 5 min at 24 degrees C. In addition, < 10% loss in protein C and factor IX activity was observed at 4 degrees C after treatment with < or = 6 mg of XLPVPI/mL for 20 min. Treatment of human serum with 6 mg of XLPVPI/mL at 4 degrees C and 8 mg of XLPVPI/mL at 24 degrees C for 5 min provided inactivation of > 5 logs of VSV.

Analysis of the generation and inhibition of factor Xa. Area under generation curves is independent of enzyme generation rate.

The activation of factor X in the presence of antithrombin has been studied in order to determine the parameters that control the area under the resulting factor Xa generation curve. Generation curves were analyzed using a model containing three parameters: the total generation of factor Xa, Emax; the rate of factor Xa generation, expressed as a first-order rate constant, kappa 1; and the rate of inhibition, expressed as another first-order rate constant, kappa 2. Using factor IXa-VIIIa to activate factor X, we found the area under the generation curve to be proportional to Emax, which was varied by varying the factor IXa concentration, and inversely proportional to kappa 2, which was varied by varying the antithrombin concentration. With this activator, however, kappa 1 varied in parallel with Emax, resulting in a correlation between integrated area and kappa 1. In order to determine whether Emax or kappa 1, or both, was a controlling parameter, similar activations were done with varying concentrations of the factor X-activating enzyme of Russell's viper venom. With this activator it was possible to vary Emax and kappa 1 independently, again at varying antithrombin concentrations. These results showed the integrated area to be proportional to Emax and inversely proportional to kappa 2, as before, but independent of the activation rate, kappa 1. In this system, therefore, the area under the factor Xa generation curve is controlled by the amount of factor Xa generated and its rate of inhibition but is independent of the rate of factor Xa generation.