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PubMed1990

Direct sequencing of the activation peptide and the catalytic domain of the factor IX gene in six species.

By means of RNA amplification with transcript sequencing (RAWTS) under low stringency conditions, sequence was obtained directly without cloning for the activation peptide and the catalytic domain of factor IX from six species--sheep, pig, rabbit, guinea pig, rat, and mouse. The data presented demonstrate that, by the appropriate design of oligonucleotides and by performance of a nested PCR under appropriate conditions, it is possible to obtain sequence on a battery of species with a minimum of oligonucleotide primers. A total of 5.2 kb of cross-species sequence was generated with RAWTS. The results indicate that (1) 69% of the amino acids in the catalytic domain, but only 23% of the amino acids in the activation peptide, are identical in humans and the six species; (2) the catalytic domain evolves at a slower rate, but the extent and pattern of conservation of amino acids in the activation peptide suggest that the peptide functions as more than a cleavage spacer that separates the heavy and light chains in the catalytically inactive zymogen; (3) 37% of the amino acids in the activation peptide and 34% of the amino acids in the catalytic domain are factor IX-specific; i.e., they are either identical or changed in a highly conservative fashion in factor IX, but not in other related coagulation proteases; (4) these conserved factor IX-specific amino acids fall into three clusters, which are candidates for involvement in the protein interactions specific to factor IX; (5) there is a human-specific deletion after lysine 142 and a rodent-specific insertion after alanine 161; (6) in guinea pig, the insertion is associated with a seven-amino-acid repeat that corresponds to a perfect repeat of a 21-bp sequence; (7) humans have lost a potential N-glycosylation site that is conserved in the other species; (8) in each species, a few nonconservative changes occur in amino acids that are otherwise completely conserved, suggesting that compensatory mutations may have occurred; and (9) when compared to that of mouse, the amino acid identity with guinea pig factor IX is no greater than that found for the non-rodent species, a result compatible with the postulated increased rate of evolution in rodents.

Amino Acid Sequence↗
PubMed2005

An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice.

The factors responsible for the removal of injected factor IX (fIX) from the blood of individuals with haemophilia B are only partly understood, and may include binding to endothelial or subendothelial sites, passive extravasation related to size or charge, or interactions requiring fIX activation. To investigate these issues, we have produced and characterised recombinant fIX proteins with amino acid changes: delta155-177, an internal deletion which removes most of the activation peptide while retaining the activation cleavage sites; S365A, which inactivates the serine protease activity of fIXa; and K5A, previously shown to eliminate fIX binding of endothelial/subendothelial collagen IV. All proteins were expressed in stably transfected HEK 293 cells, purified by immunoaffinity chromatography, and compared to the wild type HEK 293-derived protein (fIX (WT)). Mutant fIX proteins K5A and delta155-177 exhibited 72 and 202% of the specific activity of fIX (WT), respectively; S365A was without activity. Following intravenous injection in haemophilia B (fIX knockout) mice, recoveries did not differ for fIX (WT) and delta155-177, but were higher for K5A and S365A. The terminal catabolic half-life of delta155-177, alone among the mutants, was increased, by 45% versus fIX (WT). Nine hours post-injection, the observed areas under the clearance curve (AUCs) of delta155-177 and K5, but not S365A, were elevated 2-fold. delta155-177 was equally effective as fIX (WT) in reducing blood loss following tail vein transection in haemophilia B mice. Our results suggest that deletion of the multiple sites of fIX post-translational modification found within the activation peptide eliminated important fIX clearance motifs.

Amino Acid Motifs↗
PubMed1993

Factor VIII, factor IX and fibrinogen content in cryoprecipitate, fresh plasma and cryoprecipitate-removed plasma.

In order to provide accurate information for physicians, factor VIII, factor IX and fibrinogen content were determined in 40 bags of cryoprecipitate, fresh plasma and cryoprecipitate-removed plasma. A cryoprecipitate bag with a volume of 21.8 +/- 5.3 ml contained 139.5 +/- 42.9 units of factor VIII and 200.0 +/- 80.0 mg of fibrinogen. Fresh plasma with a volume of 208.0 +/- 22.5 ml contained 180.9 +/- 45.3 of factor IX, significantly higher than in cryoprecipitate-removed plasma. It was also found in this study that group O blood showed a significantly lower level of factor VIII.

ABO Blood-Group System↗
PubMed1987

Studies on the effect of heat treatment on the thrombogenicity of factor IX concentrates in dogs.

This study examines the effects of heat treatment for 72 h at 80 degrees C on the potential thrombogenicity of lyophilized human coagulation factor IX concentrates. Since heating generated minor amounts of thrombin, concentrate was prepared with antithrombin III addition prior to heat treatment. Changes in coagulation parameters were followed prior to and after infusion of 100 iu/kg of heated and unheated concentrates to dogs. All batches produced a transient fall in platelet count during infusion and a delayed rise in plasma fibrinopeptide A, accompanied by a minor prolongation of the activated partial thromboplastin time. Such changes were less marked for heated batches. Control infusion of a 'failed' factor IX concentrate showed an additional fall in fibrinogen, rise in fibrin degradation products and a more rapid rise in fibrinopeptide A, while thrombin infusion caused an even more dramatic intravascular coagulation. These studies indicated no increase in the potential thrombogenicity of freeze dried factor IX concentrates as a result of heat treatment.

Albumins↗
PubMed1992

An A to T transversion at position -5 of the factor IX promoter results in hemophilia B.

The molecular pathology that results in hemophilia B has been determined by many studies to be highly variable. Several point mutations have been identified in a 40-bp region within the Factor IX promoter. These include mutations at nucleotides -20, -6, +8, and +13. Here we report an A to T transversion at position -5 of the Factor IX gene. There is strong circumstantial evidence to support an important role for this region of the Factor IX promoter in the developmental regulation of the gene through the binding of specific transcription factors.

DNA Mutational Analysis↗
PubMed2002

MonoFIX-VF, a new mono-component factor IX concentrate: a single-centre continuous-infusion study.

MonoFIX-VF, a monocomponent factor IX concentrate, has replaced the use of Prothrombinex-HT as the treatment of choice for patients with factor IX deficiency in Australia. The haemostatic effect of MonoFIX-VF, administered by continuous infusion, was assessed in four subjects being treated for 10 bleeding episodes including five surgical procedures. MonoFIX-VF was found to be a safe and effective treatment for patients with haemophilia B.

Drug Evaluation↗
PubMed1999

Haemophilia B carrier detection by factor IX:C analysis; no impact of the type of mutation or severity of disorder.

Haemophilia B, an X-linked recessive bleeding disorder characterized by lack or deficiency of factor IX, has been shown to be caused by any of a variety of DNA abnormalities (partial or total deletions, nonsense or missense mutations). Since in most countries carrier detection is based on factor IX coagulant activity (FIX:C) assay, this study was designed to determine whether carriers' FIX:C values are dependent on the severity of haemophilia (mild, moderate or severe) or on the genetic anomaly in the family. FIX:C concentrations were studied in 28 obligate carriers, 39 women known to carry the mutation and 33 verified noncarriers subgrouped by severity of disorder or genetic anomaly. No significant subgroup differences in FIX:C values were found, thus suggesting the level of FIX:C concentrations in carriers to be unaffected by the severity of haemophilia, or by its expression (i.e. deficient or dysfunctional factor IX). The specificity and sensitivity of FIX:C analysis for the purpose of carrier diagnosis was judged by receiver operating characteristic curve analysis, where an FIX:C cut-off level of 75 IU dL-1 was found to be optimal (sensitivity 93% and specificity 88%).

Adolescent↗
PubMed1976

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.

Adolescent↗
PubMed2004

Myocardial infarction during factor IX infusion in hemophilia B: case report and review of the literature.

There are very few reports in the literature of acute myocardial infarction (MI) occurring during infusion of factor concentrates, particularly cryosupernatant in patients with hemophilia B. We describe a case of a 61-year-old man with hemophilia B who suffered an acute MI while receiving cryosupernatant infusion as factor replacement therapy. Cryosupernatant is rich in coagulation factor IX and contains low levels of fibrinogen and von Willebrand's factor. Factor IX and other factors present in cryosupernatant can possibly become activated during the manufacturing process causing thrombus formation in patients who are prone to it.

Aged↗
PubMed1994

Ca(2+)-dependent interactions between Gla and EGF domains in human coagulation factor IX.

The Ca(2+)-induced interaction between the Gla and EGF domains of human factor IX was investigated by means of three fragments: 6-kDa Gla, 19-kDa (EGF)2, and 25-kDa Gla-(EGF)2. Size-exclusion chromatography and spectroscopic measurements revealed that the Gla-EGF interaction is rather strong; it can be reconstituted by mixing the 6-kDa and 19-kDa fragments which form a stable 1:1 heterocomplex in the presence of Ca2+. By itself, the 6-kDa Gla self-associates in these conditions. The Gla-EGF interaction can be disrupted in 5 M urea where the compact structure of both domains is preserved. Binding of Ca2+ to 19-kDa (EGF)2 occurred with a Kd of 71 microM in the absence and 108 microM in the presence of 5 M urea and stabilized the first EGF domain, increasing its Tm by 12 degrees C. Addition of Ca2+ to the 6-kDa and 25-kDa fragments produced biphasic changes in their fluorescence; the intensity increased slightly at low Ca2+ concentration and then decreased in a monotonic manner. In 5 M urea, only the decrease occurred, with apparent Kds of 0.33 and 0.30 mM for 6-kDa Gla and 25-kDa Gla-(EGF)2, respectively. Thus, in 5 M urea in the presence of Ca2+, the isolated Gla domain has a compact structure and Ca2+ binding properties similar to those in the 25-kDa fragment. In the absence of urea, the Gla domain interacts either with itself, when isolated, or with the first EGF domain when present, as in the 6-kDa/19-kDa heterocomplex, in the 25-kDa fragment and presumably intact factor IX.

Calcium↗
PubMed1985

A factor IX gene probe: its use in carrier detection, antenatal diagnosis and characterisation of the molecular basis for hemophilia B.

Of hemophilia B carriers, 67% were shown to have informative restriction fragment length polymorphisms (Taq I or Xmn I) associated with the factor IX gene. Analysis of DNA for these polymorphisms can enable the detection of the hemophilia B carrier state in females and of hemophilia B in the male fetus in the first trimester. Extensive mapping of the factor IX gene in one hemophiliac who developed antibodies to factor IX failed to detect structural abnormalities in his gene. A non-deletional basis for hemophilia B is proposed in this instance.

Chromosome Mapping↗
PubMed1996

Recombinant factor IX secreted by transduced human keratinocytes is biologically active.

We have been investigating the use of human keratinocytes as potential target cells for gene therapy for haemophilia B, with the aim of curing haemophilia by means of a factor IX secreting skin graft. Previous studies indicated that keratinocytes might be suitable cells, although a potential problem was that the recombinant factor IX secreted by transduced keratinocytes was found to be only 40% biologically active. We now report, using an alternative assay to test for activity, that the secreted factor IX appears to be almost fully active.

Factor IX↗
PubMed1981

Activated factor IX complex in treatment of surgical cases of hemophilia A with inhibitors.

Three patients with severe hemophilia A with inhibitors to factor VIII were treated with activated factor IX complex. Bleeding was controlled adequately during surgical procedures involving each of the three. Partial thromboplastin times showed a variable shortening and prothrombin times were significantly shortened to values less than normal. Hemostasis was substantiated by the use of epsilon aminocaproic acid. Neither anamnestic responses nor thrombotic complications were observed. A transient hypertension developed in two patients shortly after infusion with the activated factor IX complex.

Adult↗
PubMed1982

The role of factor IX in tissue thromboplastin induced coagulation.

Tissue thromboplastin apoprotein was partially purified from human brain. The apoprotein was recombined with mixed phospholipids to yield active thromboplastin. The recombined thromboplastin induced proteolytic activation of isolated human factor IX in the presence of factor VII and Ca2+. The clotting times of various deficient plasmas were determined as a function of apoprotein concentration, keeping the phospholipid concentration constant. The clotting times of a factor XII-deficient plasma were the same as those of a factor XII/factor IX-deficient plasma, except at very low apoprotein concentrations. However, under those conditions the difference in clotting times was independent of the presence of anti-factor VII serum. Similar observations were made for factor XI-deficient plasma in comparison with factor XI/factor IX-deficient plasma. These results indicate that activation of factor IX by factor VII/tissue thromboplastin does not significantly contribute to plasma coagulation.

Apoproteins↗
PubMed2001

Deletion of the factor IX gene as a result of translocation t(X;1) in a girl affected by haemophilia B.

A balanced de novo translocation t(X;1) is described in a girl with severe haemophilia B. The translocated X was shown cytologically to be preferentially active, and methylation analysis of the DXS255 locus confirmed the skewed X-inactivation with the paternal allele being the active one. Cytogenetic and molecular analysis showed that this chromosomal rearrangement led to the deletion of at least part of the factor IX gene. Therefore, the girl was heterozygous for factor IX deficiency and expression of her clinical phenotype was the result of the inactivation of the normal maternal X chromosome. The localization of one of the X chromosome translocation breakpoints in YAC clone 957F9, that was demonstrated to map distally to the factor IX gene, revealed the complexity of this chromosomal rearrangement.

Blotting, Southern↗