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A patient manifesting acquired factor IX deficiency in association with the nephrotic syndrome received prothrombin complex concentrate and demonstrated an accelerated plasma disappearance rate of factors II, IX and X. Amelioration of proteinuria and the plasma coagulation defect followed therapy with corticosteroids and azathioprine.
This report describes three patients with a prolonged bleeding time and reduced platelet retention on glass beads, two of the three established criteria for conventional von Willebrand's disease (vWd). However, the third standard, the diminution of factor VIII, was replaced by a decrease of factor IX or XI in the first two instances, respectively, and combined factor VII and IX deficiency was present in the last patient. These associations suggest that the vW (bleeding time/platelet retention) factor is not necessarily homogeneous, may not be dependent on a single genetic determinant, and may be present in a molecular complex composed not only of factor VIII but of other blood clotting proteins as well. Although simulating classic vW, these homologues may more appropriately be called von Willebrand syndromes. Finally, unusual as they may be, their clinical recognition is important to insure that such patients receive not only factor VIII concentrates in treatment since these materials do not contain factors IX and XI and may not even be a suitable source of platelet retention factor under these modified circumstances.
We gave danazol (600 mg per day orally for 14 days), an attenuated androgen, to four adults with classic hemophilia and one adult with Christmas disease. The levels of factor VIII in the patients with classic hemophilia ranged from 1 to 3 per cent before treatment and rose to 3 to 8 per cent during the treatment period. The level of factor IX in the patient with Christmas disease rose from 5 to 14 per cent. The rise in clotting-factor activity was usually observed within five to six days after the initiation of therapy and peaked between 7 and 13 days. The drug had no untoward effects. During the 70 patient-days of therapy, only two patients required plasma products, each on one occasion. These data suggest that danazol therapy may decrease the hemorrhagic tendency and reduce the need for transfusions of plasma products in classic hemophilia and Christmas disease. Controlled clinical trials will be required to establish its value in these applications.
In utero somatic gene therapy in the later stages of pregnancy may allow targeting of organ systems which are difficult to reach later in life and to prevent the development of tissue damage otherwise caused by the early onset of inherited diseases. We report here on the percutaneous delivery of two adenoviral vectors, containing the beta-galactosidase reporter gene and the human Factor IX gene respectively, to the fetal liver and circulation by ultrasound-guided umbilical vein puncture similar to procedures used in human pregnancy. Vector spread, as detected by PCR analysis for the beta-galactosidase encoding vector, was found in almost all fetal and neonatal organs and in the maternal liver. Expression of the beta-galactosidase transgene was detected in many fetal tissues by RT-PCR. High beta-galactosidase production was shown by immuno-histochemistry predominantly in the liver, where about 30percent of the hepatocytes stained positive, and in the adrenal cortex. Production of factor IX was determined by ELISA in the plasma of treated fetuses and newborn lambs and reached at birth up to 80percent of the normal human plasma concentration. This demonstrates a very hopeful proof of principle for the development of prenatal treatment of many genetic diseases but also requires more detailed investigations with respect to the observed systemic spread of the vector.
Combined severe deficiencies of blood clotting factors IX and X were observed in 2 patients who suffered from systemic amyloidosis. This unique deficiency state was marked by refractoriness to Vitamin K as well as to transfusion therapy. Increased antithrombin activity was present in both individuals and corresponded in time to the emergence of a monoclonal IgG kappa light chain paraprotein in 1. Both patients demonstrated profound bleeding disorders. It is hypothesized that the Vitamin K dependent factors have special affinity for amyloid deposits due to an unusual amino acid (gamma-carboxyglutamic acid) present in these factors.
As an extension of our earlier work, procoagulant activity of erythrocyte [red blood cell (RBC)] membrane was examined using biochemical and rheological techniques. Western blot analysis of coagulation factors incubated with erythrocytes (RBCs) showed that only factor IX (FIX) was activated by RBC membranes in the presence of calcium ions. A fluorogenic assay suggested that activated FIX is capable of activating factor X. A preliminary crude extraction of the substance from RBC membranes suggested that a FIX-activating enzyme may be located on the RBC membrane. The initiation of FIX activation by RBCs was enhanced by an elevation in hematocrit. Moreover, the rate of FIX activation by RBCs from normal pregnant women and diabetic patients was much faster than that from normal subjects. In addition, glucose treatment of normal RBCs resulted in the increase in procoagulant activity. It is suggested that FIX activation by RBC membranes may serve as a triggering mechanism for blood coagulation, although further study will be required to clarify the putative FIX activating enzyme on the RBC membrane and to permit more extensive physiological experiments to be performed.
The transcription start sites of the human gene coding for the coagulation factor IX have been identified. Three major transcription initiation sites within a small area of approximately 30 nucleotides were found by S1 nuclease analysis and primer extension studies.
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To improve production of functional fully gamma-carboxylated recombinant human clotting factor IX (r-hFIX), cell lines stably overexpressing r-hFIX have been engineered to also overexpress proteins of the gamma-carboxylation system. Here we demonstrate that siRNA silencing of calumenin, an inhibitor of the gamma-carboxylation system, enhances production of functional r-hFIX produced by engineered BHK21 cells. The production yield of functional r-hFIX was 80% in engineered cells where calumenin had been silenced 78%. We propose that this high-yield expression system can easily be adapted to overproduce functional forms of all members of the vitamin K-dependent protein family.
The risk of an immune response to the coagulation factor IX (F.IX) transgene product is a concern in gene therapy for the X-linked bleeding disorder hemophilia B. In order to investigate the mechanism of F.IX-specific lymphocyte activation in the context of adeno-associated viral (AAV) gene transfer to skeletal muscle, we injected AAV-2 vector expressing human F.IX (hF.IX) into outbred immune-competent mice. Systemic hF.IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4+ T and B cells. ELISPOT assays documented robust responses to hF.IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF.IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in approximately 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX.
Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000). Thus, it should be possible to maintain transgene expression within the therapeutic range. Therefore, we inserted elements of the tetracycline (Tet)-dependent Tet-On gene regulatory system into replication deficient adenovectors. The new system consists of two adenovectors: a response vector expressing hFIX (Ad5.TRE.hFIX), and a regulator vector expressing a second generation reverse tetracycline transactivator controlled by a CMV- (Ad5.CMV.rtTA) or human alpha1-antitrypsin-promoter (Ad5.hAAT.rtTA). Expression studies in four human cell lines showed high expression of hFIX from Ad5.TRE.hFIX in all cell lines in combination with Ad5.CMV.rtTA regulator vector, but only high specific expression in HepG2-cells in combination with Ad5.hAAT.rtTA regulator vector. Additionally, up- and down-regulation of hFIX expression could be demonstrated in vitro with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination and modulating doxycycline concentrations. When SCID-mice were infected with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination, up- and down-regulation of hFIX expression was achieved by oral doses of doxycycline for a period of at least two months. Replacement of the Ad5.CMV.rtTA vector by the Ad5.hAAT.rtTA vector showed minimal expression of hFIX in vivo. Although hFIX expression showed a slow and gradual decrease over time in vivo with the Ad5.CMV.rtTA vector, it remained within the therapeutic range. To date, regulation of hFIX has not been described in this way.
A series of in vitro studies designed to ascertain the potential in vivo thrombogenicity of human factor IX-containing concentrates is described. Using concentrates obtained from several different Centres the fibrinogen clotting time with some preparations was less than 6 h and/or the recalcification time of normal plasma was shortened. In some preparations, however, the plasma recalcification time was lengthened. Further studies revealed that all diluted factor-IX concentrates generated thrombin after recalcification, and that the rate of thrombin generation appeared to be characteristic of a particular preparation. This characteristic has been designated the TGt50, which is the incubation period in minutes, after recalcification, required to obtain a 50 s clotting time of a fibrinogen substrate. The TGt50 was found to correlate most strongly with recalcification time of celite exhausted plasma (P less than 0.001), but no correlation was observed between it and the immunological antithrombin III or factor-VIII antigen levels. Evidence is presented which suggests that the thrombin generation test and recalcification time of celite exhausted plasma may represent suitable in vitro quality control assays for factor-IX concentrates.
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Transgenic mutation-detection systems have been pioneered in mice, but the approach is applicable to any species in which transgenic animals can be generated. The observed mutations seen in mutation-detection systems are influenced by the underlying pattern of mutation, i.e., the mutational pattern that occurs in wild-type organisms in endogenous segments of DNA that are not under selective pressure. Unfortunately, the biology of most genes and assays markedly skew the underlying pattern of mutation. Herein, we raise multiple issues that must be addressed in order to estimate the underlying pattern of spontaneous mutation from transgenic mouse mutation-detection systems. If these issues can be addressed, the underlying pattern of spontaneous mutation can then be deduced for multiple cell types and for transgenes integrated into different parts of the genome. Even though transgenic methodology cannot be applied directly to humans, it is likely that comparable data on the underlying pattern of spontaneous mutation will be available in humans. Such data are currently available for germline mutations in the factor IX gene. These data are reviewed because of their relevance to two of the multiple issues that must be addressed in transgenic mouse mutation-detection systems: (1) How can the underlying pattern of mutation be deduced from the observed pattern? and (2) How similar are the underlying patterns of mutation in humans and in mice? The analysis of recent germ-line mutation in the factor IX gene yield estimates of the mutation rates per base pair per generation. In brief, the mutation rates vary from 0.037 x 10(-10) for deletions (> 20 bp) to 360 x 10(-10) for transitions at the dinucleotide CpG. If these mutation rates are extrapolated to the entire genome, the aggregate mutation rate is estimated to be 36 x 10(-10). This implies that the diploid genome of each person contains about 21 de novo mutations. In the future, the underlying pattern of spontaneous mutation will be deduced for multiple human genes and these will serve as benchmarks to assess the similarity of the mutational process in humans and in mice.
A case of myocardial infarction (MI) in a hemophilia B patient with a factor IX (FIX) inhibitor (6 BU) is described. MI occurred after two infusions of FEIBA concentrate. Unexpectedly, these infusions resulted in a neutralization of the inhibitor and a consistent plasma FIX activity which may have increased the thrombotic risks. Four days later, a psoas hematoma was suspected. At that time the inhibitor remained undetectable, allowing a therapy with purified FIX concentrates. No recurrence of thrombotic complication was observed. This is an additional illustration of the thrombotic risks associated with the use of activated prothrombin complex concentrates, especially in patients having pre-existing risk factors for thrombosis. The management of bleeding episodes in hemophilia B patients with inhibitor represents an especially difficult challenge.
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