EGF domain of coagulation factor IX is conducive to exposure of phosphatidylserine.

Lipid rafts are an initiation site for many different signals. Recently, we reported that an EGF domain in activated coagulation factor IX (EGF-F9) increases lipid raft formation and accelerates cell migration. However, the detailed mechanism is not well understood. This study aimed to evaluate the effects of EGF-F9 on the cell membrane. A431 cells (derived from human squamous cell carcinoma) were treated with recombinant EGF-F9. Cells were immunocytochemically stained with probes for lipid rafts or phosphatidylserine (PS). After 3 min of treatment with EGF-F9, cholera toxin subunit B (CTxB) binding domains emerged at the adhesive tips of filopodia. Subsequently, CTxB staining was observed on the filopodial shaft. Finally, large clusters of CTxB domains were observed at the edge of cell bodies. Markers for lipid rafts, such as caveolin-1 and a GPI anchored protein, co-localized with CTxB. Staining with annexin V and XII revealed that PS was exposed at the tips of filopodia, translocated on filopodial shafts, and co-localized with CTxB at the rafts. Immunocytochemistry showed that scramblase-1 protein was present at the filopodial tips. Our data indicates that EGF-F9 accelerates PS exposure around the filopodial adhesion complex and induces clustering of lipid rafts in the cell body. PS exposure is thought to occur on cells undergoing apoptosis. Further study of the function of the EGF-F9 motif in mediating signal transduction is necessary because it is shared by a number of proteins.

Coagulation factor IX regulates cell migration and adhesion in vitro.

Coagulation factor IX is thought to circulate in the blood as an inactive zymogen before being activated in the coagulation process. The effect of coagulation factor IX on cells is poorly understood. This study aimed to evaluate the effects of intact coagulation factor IX and its cleavage fragments on cell behavior. A431 cells (derived from human squamous cell carcinoma), Pro5 cells (derived from mouse embryonic endothelial cells), Cos7 cells, and human umbilical vein endothelial cells were utilized in this study. The effects of coagulation factor IX and its cleavage fragments on cell behavior were investigated in several types of experiments, including wound-healing assays and modified Boyden chamber assays. The effect of coagulation factor IX depended on its processing; full-length coagulation factor IX suppressed cell migration, increased adhesion to matrix, and enhanced intercellular adhesion. In contrast, activated coagulation factor IX enhanced cell migration, suppressed adhesion to matrix, and inhibited intercellular adhesion. An activation peptide that is removed during the coagulation process was found to be responsible for the activity of full-length coagulation factor IX, and the activity of activated coagulation factor IX was localized to an EGF domain of the coagulation factor IX light chain. Full-length coagulation factor IX has a sedative effect on cells, which is counteracted by activated coagulation factor IX in vitro. Thus, coagulation factor IX may play roles before, during, and after the coagulation process.

Evidence for factor IX-independent roles for factor XIa in blood coagulation.

BACKGROUND: Factor XIa is traditionally assigned a role in FIX activation during coagulation. However, recent evidence suggests this protease may have additional plasma substrates. OBJECTIVE: To determine whether FXIa promotes thrombin generation and coagulation in plasma in the absence of FIX, and to determine whether FXI-deficiency produces an antithrombotic effect in mice independently of FIX. METHODS: FXIa, FXIa variants and anti-FXIa antibodies were tested for their effects on plasma coagulation and thrombin generation in the absence of FIX, and for their effects on the activation of purified coagulation factors. Mice with combined FIX and FXI deficiency were compared with mice lacking either FIX or FXI in an arterial thrombosis model. RESULTS: In FIX-deficient plasma, FXIa induced thrombin generation, and anti-FXIa antibodies prolonged clotting times. This process involved FXIa-mediated conversion of FX and FV to their active forms. Activation of FV by FXIa required the A3 domain on the FXIa heavy chain, whereas activation of FX did not. FX activation by FXIa, unlike FIX activation, was not a calcium-dependent process. Mice lacking both FIX and FXI were more resistant to ferric chloride-induced carotid artery occlusion than FXI-deficient or FIX-deficient mice. CONCLUSION: In addition to its predominant role as an activator of FIX, FXIa may contribute to coagulation by activating FX and FV. As the latter reactions do not require calcium, they may make important contributions to in vitro clotting triggered by contact activation. The reactions may be relevant to FXIa's roles in hemostasis and in promoting thrombosis.

Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice.

Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-targeted integration-competent and -defective lentiviral vectors. The hyperfunctional FIX transgenes increased FIX activity reconstituted in the plasma without detectable adverse effects, allowing correction of the disease phenotype at lower vector doses and resulting in improved hemostasis in vivo. The combined effect of codon optimization with the hyperactivating FIX-R338L mutation resulted in a robust 15-fold gain in potency and therefore provides a promising strategy to improve the efficacy, feasibility, and safety of hemophilia gene therapy.

Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application.

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

Prolonged activity of factor IX as a monomeric Fc fusion protein.

Treatment of hemophilia B requires frequent infusions of factor IX (FIX) to prophylax against bleeding episodes. Hemophilia B management would benefit from a FIX protein with an extended half-life. A recombinant fusion protein (rFIXFc) containing a single FIX molecule attached to the Fc region of immunoglobulin G was administered intravenously and found to have an extended half-life, compared with recombinant FIX (rFIX) in normal mice, rats, monkeys, and FIX-deficient mice and dogs. Recombinant FIXFc protein concentration was determined in all species, and rFIXFc activity was measured in FIX-deficient animals. The half-life of rFIXFc was approximately 3- to 4-fold longer than that of rFIX in all species. In contrast, in mice in which the neonatal Fc receptor (FcRn) was deleted, the half-life of rFIXFc was similar to rFIX, confirming the increased circulatory time was due to protection of the rFIXFc via the Fc/FcRn interaction. Whole blood clotting time in FIX-deficient mice was corrected through 144 hours for rFIXFc, compared with 72 hours for rFIX; similar results were observed in FIX-deficient dogs. Taken together, these studies show the enhanced pharmacodynamic and pharmacokinetic properties of the rFIXFc fusion protein and provide the basis for evaluating rFIXFc in patients with hemophilia B.

Expression of functional recombinant human factor IX in milk of mice.

Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk beta-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.

Structural analysis of factor IX protein variants to predict functional aberration causing haemophilia B.

Factor IX (FIX) is a component protein of blood coagulation pathway, which activates factor X through interaction with factor VIII and Ca(2+). Defective FIX protein resulting from mutation in the corresponding gene causes an X-linked bleeding disorder known as haemophilia B. The aim of the present study was to speculate the potential detrimental effects of the FIX mutations upon the functionality of the protein, which could contribute to the comprehension of the mechanism underlying haemophilia B. In this report, we examined the effect of point mutation on the crystal structure of the native factor IX by measuring the change in the hydrogen-bonding pattern and electrostatic potential and explored the possibility of any correlation of the clinical severity of haemophilia B with the structural perturbation, by plotting the mutations of varying phenotype (severe and mild) on the crystal structure of FIX. Out of a total of 16 severe mutations 14 (88%) showed changes of hydrogen-bonding pattern to variable extent. Among the nine mild haemophilia B mutations, six (i.e. 66.66%) showed no change in hydrogen-bonding pattern. Our data suggest that there is a statistically significant correlation between the two groups of mutations as measured by change in the hydrogen-bonding pattern. Our study truly represents an initiation of an effort that would provide a framework for first evaluation of suspected mutations by in silico approaches, which might be further validated by other experimental techniques.

Update on the physiology and pathology of factor IX activation by factor XIa.

Factor IX is a key component of the plasma system that forms a fibrin clot at a site of vascular injury. Activation of factor IX by factor XIa is required in certain situations to prevent bleeding from premature clot degradation. Factor XIa is a coagulation protease comprised of two identical subunits. The biochemical and physiologic implications of this unusual structural feature are being actively investigated. Congenital factor XI deficiency causes a mild-to-moderate bleeding disorder, with hemorrhage typically involving the oral/nasal cavities and the urinary tract. Current treatment recommendations take this tissue-specific bleeding pattern into account and target factor replacement to certain types of procedures and clinical situations. Results from animal models and human population studies indicate that factor XI contributes to thromboembolic disease. This protease may therefore be a legitimate therapeutic target.

Optimization of self-complementary AAV vectors for liver-directed expression results in sustained correction of hemophilia B at low vector dose.

Self-complementary adeno-associated virus (scAAV) vectors can significantly minimize the vector load required to achieve sustained transgene expression. In this study, transcriptional regulatory elements were systematically screened to produce constitutive and liver-specific scAAV factor IX (FIX) expression cassettes. In addition, optimization of GC content, cis- regulatory elements, and codon usage in the human FIX (hFIX) transgene increased expression 4-20-fold. A vector was developed that was capable of expressing high FIX levels in comparison with the single-stranded (ss) AAV vector used in a recent clinical trial. The ssAAV and scAAV vectors display different transgene expression and genome stability patterns in the liver, as determined by immunohistochemical staining, in situ messenger RNA (mRNA) hybridization and vector genome quantitation. The ssAAV2 vector promoted strong FIX expression in only a subset of hepatocytes. The scAAV2-hFIX vector showed widespread ( approximately 80% of hepatocytes), moderate FIX expression levels similar to normal livers with correction of coagulation function in FIX-deficient mice. The ability of low dose scAAV-FIX vectors to achieve near-physiological expression may circumvent inflammatory responses in the liver. In addition to providing an improved scAAV vector for potential application in future hemophilia B clinical trials and liver-directed gene delivery, these studies underscore the need for rigorous analysis and optimization of vector genome cassettes.

Influence of factor IX on overall plasma coagulability and fibrinolytic potential as measured by global assay: monitoring in haemophilia B.

We sought to determine the influence of factor IX (FIX) deficiency upon overall coagulative and fibrinolytic capacities in plasma using the clot formation and lysis (CloFAL) assay, and to investigate the role of this global assay as an adjunctive monitoring tool in haemophilia B. CloFAL assay parameters were measured in vitro in platelet-poor plasma in relation to FIX activity and antigen (FIX:Ag), and were determined ex vivo among FIX-deficient patients (n = 41) in comparison to healthy individuals (n = 48). Supplementation of FIX-deficient plasma with FIX in vitro demonstrated a non-linear concentration dependence of FIX upon overall plasma coagulability. Ex vivo, coagulability was significantly decreased in FIX-deficient vs. healthy subjects among adults [median coagulation index (CI): 4% vs. 104% respectively; P < 0.001] and children (median CI: 9% vs. 63%; P < 0.001). Fibrinolytic capacity was increased in adult FIX-deficient vs. healthy subjects (median fibrinolytic index: 216% vs. 125%, respectively, P < 0.001), and was supported by a trend in shortened euglobulin lysis time (ELT). Severe haemophilia B patients showed heterogeneity in aberrant CloFAL assay waveforms, influenced partly by FIX:Ag levels. Patients with relatively preserved FIX:Ag (i.e. dysfunctional FIX) exhibited a shorter time to maximal amplitude in clot formation than those with type I deficiency. During patient treatment monitoring, markedly hypocoagulable CloFAL assay waveforms normalized following 100% correction with infused FIX. The CloFAL global assay detects FIX deficiency, demonstrates differences in coagulability between dysfunctional FIX and type I deficiency, and appears useful as an adjunctive test to routine FIX measurement in monitoring haemophilia B treatment.

La nueva cascada de la coagulación y su posible influencia en el difícil equilibrio entre trombosis y hemorragia / The new coagulation cascade and its possible influence on the delicate balance between thrombosis and hemorrhage

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Factor IXa inhibitors as novel anticoagulants.

Currently available anticoagulants are limited by modest therapeutic benefits, narrow clinical applications, increased bleeding risk, and drug-induced thrombophilia. Because factor IX plays a pivotal role in tissue factor (TF)-mediated thrombin generation, it may represent a promising target for drug development. Several methods of attenuating factor IX activity, including monoclonal antibodies, synthetic active site-blocked competitive inhibitors, oral inhibitors, and RNA aptamers, have undergone investigation. This review summarizes present knowledge of factor IX inhibitors with emphasis on biology, pharmacology, preclinical data, and early-phase clinical experience in humans.

Hemofilia: de la fisiología a la fisiopatología. Inhibidores / Haemophilia: from the physiology to the physiopathology. Inhibitors

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[The current feature of the study on human coagulation factor IX mutant].

Hemophilia B is an X-linked bleeding disease, caused by the mutations of human coagulation factor IX (hFIX) gene located in chromosome X. It results in a dramatic decline of hFIX quantity or clotting activity in plasma, and the intrinsic clotting pathway is affected seriously. In this article, the structure and function of hFIX gene as well as the protein encoded by this gene were reviewed. Various types of hFIX mutants found in hemophilia B were also described, including the mutations caused by founder effects, mutations in regulatory region, coding region, splicing sites of introns and two other special mutations. Meanwhile, the biological effects of the mutations described above were discussed. Finally, a mutation type (Arg-->Ala at point 338) that can increase the clotting activity of hFIX as well as the potential application was briefly introduced.

Mechanisms of Hedgehog gradient formation and interpretation.

Morphogens are molecules that spread from localized sites of production, specifying distinct cell outcomes at different concentrations. Members of the Hedgehog (Hh) family of signaling molecules act as morphogens in different developmental systems. If we are to understand how Hh elicits multiple responses in a temporally and spatially specific manner, the molecular mechanism of Hh gradient formation needs to be established. Moreover, understanding the mechanisms of Hh signaling is a central issue in biology, not only because of the role of Hh in morphogenesis, but also because of its involvement in a wide range of human diseases. Here, we review the mechanisms affecting the dynamics of Hh gradient formation, mostly in the context of Drosophila wing development, although parallel findings in vertebrate systems are also discussed.

Rheological analyses of coagulation of blood from different individuals with special reference to procoagulant activity of erythrocytes.

In our previous papers, we reported that factor IX (FIX), when activated by erythrocyte membranes, causes coagulation. We have identified and characterized the FIX-activating enzyme located in normal human erythrocyte membranes. In this paper, to examine physiological and pathological significances of procoagulant activity of erythrocytes, coagulation of blood obtained from different individuals was analyzed by means of a rheological technique. In more than 65% of subjects including normals and patients, the initiation of coagulation seemed to be governed by erythrocytes. Coagulation of whole blood and platelet-free plasma supplemented with erythrocytes had a tendency to occur rapidly in the elderly. It was suggested that the concentration of FIX-activating enzyme on erythrocyte membranes for aged donors was somewhat higher than that for young ages. Propagation reactions on erythrocyte membranes (i.e. factor X activation leading to thrombin generation after FIX activation) was slower than that on platelet membranes. Moreover, the propagation reaction on erythrocyte membranes was greatly dependent on individuals, whereas that on platelet membranes was not so much. Our study demonstrates that the activation of FIX by erythrocytes and subsequent propagation reaction on platelet membranes may be important for initiating and controlling blood coagulation reactions.

Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice.

Factor XI (FXI) and factor IX (FIX) are zymogens of plasma serine proteases required for normal hemostasis. The purpose of this work was to evaluate FXI and FIX as potential therapeutic targets by means of a refined ferric chloride (FeCl(3))-induced arterial injury model in factor-deficient mice. Various concentrations of FeCl(3) were used to establish the arterial thrombosis model in C57BL/6 mice. Carotid artery blood flow was completely blocked within 10 min in C57BL/6 mice by application of 3.5% FeCl(3). In contrast, FXI- and FIX-deficient mice were fully protected from occlusion induced by 5% FeCl(3), and were partially protected against the effect of 7.5% FeCl(3). The protective effect was comparable to very high doses of heparin (1000 units kg(-1)) and substantially more effective than aspirin. While FXI and FIX deficiencies were indistinguishable in the carotid artery injury model, there was a marked difference in a tail-bleeding-time assay. FXI-deficient and wild-type mice have similar bleeding times, while FIX deficiency was associated with severely prolonged bleeding times (>5.8-fold increase, P < 0.01). Given the relatively mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable strategy for treating or preventing thrombus formation.

A simple technique to reduce epistaxis and nasopharyngeal trauma during nasotracheal intubation in a child with factor IX deficiency having dental restoration.

Epistaxis and airway trauma are often associated with nasotracheal intubation. We describe a patient with Factor IX deficiency who required nasotracheal intubation. An inexpensive, nonproprietary, rapid technique was used to reduce the trauma of intubation.