Cross-reactivity to porcine factor VIII of factor VIII inhibitors in patients with haemophilia in Australia and New Zealand

BACKGROUND: Inhibitory antibodies which neutralise factor VIII develop in 10-20% of individuals with inherited haemophilia A and rarely as autoantibodies in normal individuals to cause acquired haemophilia. The antibodies are directed against human factor VIII but cross-react to varying degrees with porcine factor VIII. Porcine factor VIII can be used for treatment in individuals with low cross-reactivity. AIMS: To determine the cross-reactivity of factor VIII inhibitors between human factor VIII and porcine factor VIII, in a population of patients with inherited and acquired haemophilia A. Also, to determine whether patients with inherited haemophilia and inhibitors have a higher incidence of factor VIII gene inversion in intron 22. METHODS: Samples and data sheets from 43 patients with inherited and ten with acquired haemophilia were submitted from hospitals in Australia and New Zealand. Inhibitor levels to human and porcine factor VIII were measured by the Bethesda method in 39 with inherited and nine with acquired haemophilia A. RESULTS: Of 39 patients with inherited haemophilia A, cross-reactivity was 0% in 17 patients, 1-19% in six, 20-39% in 11 and 40-80% in five. In six of nine patients with acquired haemophilia cross-reactivity was < or = 7%. In inherited severe haemophilia A, the frequency of the intron 22 inversion was not greater in 37 study patients than in 28 patients without an inhibitor. CONCLUSIONS: Many patients in Australia and New Zealand with inhibitors to human factor VIII presently show a low or absent level of cross-reactivity to porcine factor VIII. These may respond to treatment with this concentrate at least in the short term. There remains a group of patients with high cross-reactivity who will respond only to recombinant factor VIIa or prothrombin complex concentrates.     

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.     

Subunit structure and function of porcine factor Xa-activated factor VIII

Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 heterotrimer. We now report a stable porcine fVIIIaXa preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified subunits of fVIIIaXa revealed that factor Xa cleaves fVIII at Arg219 within the A1 domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVIIIaXa preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIaIIa [Lollar, P., & Parker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIaXa preparation contains five subunits, including fragments of the A1 and A2 domains that remain noncovalently bound. Fluorescence anisotropy measurements indicated there was no difference in the affinity of fVIIIaXa and fVIIIaIIa for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, the fVIIIaXa preparation bound dye-labeled factor IXa with 1:1 stoichiometry, indicating that all fVIIIaXa molecules in the preparation can bind factor IXa. However, fVIIIaXa had 4-fold less procoagulant activity than fVIIIaIIa. Kinetic analysis of fVIIIa cofactor activity using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIaIIa relative to fVIIIaXa within the intrinsic fXase complex, rather than a difference in their stabilities.     

Factor VIII inhibitors in patients with hemophilia A: epidemiology of inhibitor development and induction of immune tolerance for factor VIII

Factor (F) VIII inhibitor development remains one of the most serious complications in the treatment of hemophilia A. Former and recent studies on inhibitor development revealed that patients with severe hemophilia A and positive inhibitor family history are at highest risk of developing an inhibitor. Comparison of recent inhibitor incidence studies on previously untreated patients indicate that the risk of inhibitor development under treatment with recombinant FVIII concentrates is comparable to the inhibitor incidence under FVIII substitution by plasma-derived concentrates. However, longer observation periods are necessary to draw final conclusions. Since inhibitor development may result in inefficacy of FVIII concentrates in the treatment of severe bleedings, the induction of immune tolerance (IT) is still of main concern. Various regimens to induce IT by application of FVIII concentrates have been conducted up to now. Success rate appears to be influenced by low to high responder status, number of exposure days before onset of treatment, and dosage of therapeutic regimen. Especially, discontinuation of IT therapy seems to be associated with failure of therapy. Taking into account available data on IT therapy, we recommend early onset of a high dosage regimen in high responder patients as soon as possible after inhibitor detection, as this is associated with higher success rate and shorter elimination time.     

Acquired hemophilia secondary to factor VIII inhibitors after pregnancy

According to current concepts, the excitatory amino acid glutamate is involved in the pathogenesis of Parkinson's disease (PD). Overactivity of glutamatergic projection neurons and beneficial effect of antiglutamatergic substances in animal experiments suggest that excess supply of glutamate might contribute to the pathophysiology of PD. Reduced activity of the glutamate metabolizing enzyme glutamine synthetase (GS) leads to decreased uptake of glutamate and thus abundant glutamate. Here we report that PD patients and age-matched controls are comparable with respect to GS activity in peripheral blood mononuclear cells (PBMC). These results imply no systemic dysregulation of the enzyme GS in patients with PD.     

Prostate carcinoma associated spontaneous factor VIII:C inhibitor hemophilia. Successful therapy of severe hemorrhagic complication with porcine factor VIII in a 75-year-old patient

We have employed fluorescence in situ hybridization (FISH) in combination with standard morphology (MGG/FISH) to identify the clonal involvement of different bone marrow cell lineages in 20 AML patients (14 MDS-AML, 6 de novo AML). Even though the number of cells belonging to the abnormal clone varied between individual cases, the percentage of clonal blasts was similar in MDS-AML and de novo AML patients. The erythropoietic cells appeared to be part of the abnormal clone in 13 of 14 patients with MDS-AML, but only in 1 of 6 with de novo AML. Similarly, clonal granulocytes were detected in 13 of 14 patients with MDS-AML, compared to 2 of 6 with de novo AML. Lymphocytes consistently displayed normal, diploid karyotype. The results suggest that it is possible to distinguish between MDS-AML and de novo AML by the use of MGG/FISH; in de novo AML the abnormal chromosomal clone is generally confined to the immature myeloid cells, while in MDS-AML mature granulocytes and erythroid cells are of clonal origin. It is, however, not possible to conclude that MDS-AML is a "multipotent" type of leukaemia, since it cannot be ruled out that the chromosomally aberrant erythroid cells and granulocytes represent surviving cells from the original MDS clone.     

Effects of factor VIII concentrates on the immune system of patients with hemophilia

The introduction of high-purity factor VIII (FVIII) concentrates in the treatment of patients with hemophilia A has raised the issue that the use of these products may change on the immune system of the recipients. There is now clear evidence that high-purity concentrates, particularly those produced by immune-affinity chromatography or recombinant DNA technology, slow the fall in CD4 cells that occurs in HIV seropositive patients. It remains to be demonstrated that this biological effect results in clinical benefits and that the occurrence of AIDS is slowed or delayed by the use of high-purity concentrates. On the other hand, concern has been expressed about the possibility that high-purity products might render patients with hemophilia less immunotolerant, facilitating the onset of FVIII antibodies. Follow-up studies of previously untreated hemophiliacs infused for the first time with recombinant FVIII products have ignited this concern, because approximately one fourth of severe hemophiliacs developed inhibitors. However, most of the inhibitors were transient, so that ultimately they had little influence on the efficacy of replacement therapy. It was subsequently realized that inhibitors develop with high frequency even in hemophiliacs treated with less pure, plasma-derived products, provided testing is prospective and as frequent as for studies of recombinant FVIII. On the whole, these data have provided new insights on the natural history of inhibitor development in previously untreated hemophiliacs, showing that low-titer, short-lasting inhibitors develop more frequently than previously recognized.     

Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer's disease

Nerve growth factor (NGF) is important for the survival and maintenance of central cholinergic neurons, a signalling system impaired in Alzheimer's disease. We have treated 3 patients with Alzheimer's disease with a total of 6.6 mg NGF administered continuously into the lateral cerebral ventricle for 3 months in the first 2 patients and a total of 0.55 mg for 3 shorter periods in the third patient. The patients were extensively evaluated with clinical, neuropsychological, neurophysiological and neuroradiological techniques. Three months after the NGF treatment ended, a significant increase in nicotine binding was found in several brain areas in the first 2 patients and in the hippocampus in the third patient as studied by positron emission tomography. A clear cognitive amelioration could not be demonstrated, although a few neuropsychology tests showed slight improvements. The amount of slow-wave cortical activity as studied by electroencephalography was reduced in the first 2 patients. Two negative side effects occurred with NGF treatment: first, a dull, constant back pain was observed in all 3 patients, which in 1 patient was aggravated by axial loading resulting in sharp, shooting pain of short duration. When stopping the NGF infusion, the pain disappeared within a couple of days. Reducing the dose of NGF lessened the pain. Secondly, a marked weight reduction during the infusion with a clear weight gain after ending the infusion was seen in the first 2 patients. We conclude from this limited trial that, while long-term intracerebroventricular NGF administration may cause certain potentially beneficial effects, the intraventricular route of administration is also associated with negative side effects that appear to outweigh the positive effects of the present protocol. Alternative routes of administration, and/or lower doses of NGF, perhaps combined with low doses of other neurotrophic factors, may shift this balance in favor of positive effects.     

Surgery in hemophilia A patients with factor VIII inhibitor: 10-year experience

Patients with hemophilia A and circulating anticoagulant (factor VIII inhibitor) present a difficult, even unsolvable problem, particularly if they require surgical treatment and the inhibitor titer is high. During the 1986-1995 period 29 surgical procedures on inhibitor hemophilia A patients were performed in our center. Each of the cases had an individual character, and all demanded special clinical treatment. Based on this experience we present the possibilities of hemostasis maintenance during the perioperative period with high doses of human or porcine factor VIII, aPCC, plasmapheresis, and extracorporeal antibody adsorption to protein A-Sepharose. In some patients hemostasis maintenance requires combined treatment. To induce immunotolerance in patients with inhibitor is the gold standard treatment because it is then possible to achieve proper hemostasis after factor VIII infusion. Various methods of immunotolerance induction have been discussed and compared with our experience with immunotolerance induction in 11 patients with small factor VIII doses (25 IU/kg twice a week) and the modified Malm? protocol in 15 patients.     

Calcium inhibitors in the management of hypertensive patients on cyclosporine

An enzyme-linked immunosorbent assay (ELISA) for the detection of antigen secreted by viable Taenia solium metacestodes (Ag-ELISA) was applied to 43 pre-treatment and 47 follow-up cerebrospinal fluid (CSF) samples from Peruvian patients with neurocysticercosis demonstrated by computed tomography and enzyme-linked immunoelectrotransfer blot assay. The sensitivity of the assay was 86%. Negative pre-treatment results in the Ag-ELISA test were restricted to patients with only a single live cyst or only enhancing lesions. Patients with hydrocephalus had higher levels of circulating antigen. There was no difference between antigen levels in CSF taken before and immediately after treatment (day 14). Levels of parasite antigen were significantly positively correlated with the number of live cysts detected by tomography and were also proportional to the number and intensity of antibody reactions recognized by the immunoblot diagnostic test. In contrast, there was a negative correlation with the number of enhancing lesions revealed by tomography, supporting the hypothesis that enhancing lesions correspond to a terminal, moribund stage of the parasite. The use of antigen-detection tests specific for viable metacestodes has immediate utility in the clinical context, not only providing important information on the viability of the parasites but also leading to an improved understanding of the pathogenesis of neurocysticercosis before and after drug treatment.     

Treatment of haemophilia with factor VIII inhibitor by giving isoagglutinin-free factor VIII concentrate (author's transl)

Two nine-year-old boys with haemophilia A and circulating inhibitor against factor VIII were given large doses of commercial AHG concentrates to control bleeding. But the isoagglutinins in the AHG concentrates caused haemolysis in both patients. When AHF (Immuno) from AB donors - a cryoprecipate without isoagglutinins - was used no further inhibitor activity was detectable, previously observed anaphylactoid reactions no longer occurred and usual doses of AHG were now sufficient to control bleeding.     

Continuous intra-hepatic-arterial infusion of low dose 5-fluorouracil for colorectal cancer patients with unresectable liver metastases

Five cases of colorectal cancer with unresectable liver metastases treated from April 1992 to April 1993 in Osaka National Hospital were summarized in this paper. A silicone catheter was placed in the hepatic artery through the gastroduodenal artery by operative procedure and connected to a subcutaneously implanted reservoir. 5-FU was administered ambulatorily using Baxter Infusor (multi day type) according to a regimen of 5-day continuous infusion and subsequent 2-day rest. The patients were 4 men and 1 woman, and from 51 to 65 years old (average: 62.4 y.o.). According to criteria for antitumor effectiveness by CT scan, one patient was judged CR, two were PR, and one was PD. One case could not be estimated because of catheter obstruction. The total efficacy rate was 75%. The serum CEA level was reduced in 3 patients. As for complication, obstruction of catheter, damage to reservoir and segmental necrosis of liver were observed in 3 patients. In conclusion, our ambulatory therapy for colorectal cancer patients with liver metastases was considered to have a high potential of not only effectiveness for cancer lesion but also the improvement of patients' quality of life.     

A human alloantibody interferes with binding of factor IXa to the factor VIII light chain

Inhibitory antibodies directed against factor VIII develop in a substantial number of patients with hemophilia A as a consequence of factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of factor IXa to immobilized factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit factor VIII activity by interfering with factor IXa-factor VIIIa complex assembly.     

Raised factor VIII is associated with coronary thrombotic events

A 22-year-old woman had a normal full-term delivery 6 years after a successful allogeneic bone marrow transplantation (BMT) for acute myeloid leukemia (AML). Conditioning therapy consisted of cyclophosphamide (120 mg/kg) and total body irradiation (TBI) to a total of 1575 cGy in seven fractions (225 cGy x 7, at a dose rate of 3.5 cGy/min). Graft-versus-host disease prophylaxis was with methotrexate and cyclosporin A. Grade I acute GVHD developed after BMT but there was no chronic GVHD. She became amenorrhoeic after BMT and serial gonadal testing indicated hypergonadotrophic hypogonadism. She became pregnant and delivered a full-term, healthy baby 6 years after BMT. Successful pregnancy after TBI of more than 1200 cGy is extremely rare. This case, to the best of our knowledge, is the second patient who received a higher dose of TBI (1575 cGy) to have a successful pregnancy. This and previous reports indicate that normal pregnancy is possible after BMT with TBI in excess of 1200 cGy.     

Analysis of inversion in intron 22 of the factor F VIII:C gene in patients with hemophilia A in the Slovak population

Hemophilia A is the result of Factor F VIIIC (F8C) gene mutations. Predominating mutation is inversion, occurring in about 50% of patients with severe form of the disease. Inversion is the result of homologous recombination between gene A located on the 22. introne of the F8C gene and one of its telomeric copies located about 500 kb from 5'end of the factor F VIIIC gene. This study presents the results of this mutation screening in 84 nonrelated patients with hemophilia A. Inversion was identified in 22 (50%) of 44 patients with severe form and in 1 (from 13) with moderate form of the disease. Distal type of inversion was more frequent (82.6%) than proximal one. The identification of iversions enabled direct DNA diagnosis in 50% of patients with severe form of the disease and will be successfully used in the prenatal diagnosis and carrier testing, mainly in families with sporadic occurrence of the disease. (Tab. 1, Fig. 2, Ref. 18.)     

Factor VIII inhibitors in mild and moderate-severity haemophilia A

Inhibitors are an uncommon complication of mild haemophilia, occurring in 3-13% of patients and usually arising during adulthood. The risk of inhibitor development in this group appears to be associated with relatively few high-risk factor VIII genotypes clustered in the A2 and C2 domains, especially the Arg593-Cys and the Trp2229-Cys mutations. Kindreds with these mutations have an inhibitor incidence of up to 40%. These mutations may induce a stable conformational change in the factor VIII molecule rendering it antigenically distinct from wild-type factor VIII. Inhibitors in mild haemophilia usually cross-react with endogenous factor VIII reducing the basal VIIIC to < 0.01 IU/ml, and causing spontaneous bleeding. This bleeding is sometimes severe and life-threatening, two-thirds of patients developing a pattern of soft tissue, gastrointestinal (GI) and urinogenital bleeding reminiscent of acquired haemophilia. Bleeding has been treated with human and porcine factor VIII, bypass therapy and DDAVP. Recombinant factor VIIa and DDAVP have the advantage that they do not induce an anamnestic rise in inhibitor titre. About 60% of these inhibitors disappear in the remainder over a median of 9 months. Few of these inhibitors recur, suggesting that most such patients have become tolerant. The inhibitors persist long-term and remain troublesome in about 40% of patients. The limited data available on immune tolerance induction in this group indicate a generally poor response to this approach. Two of nine achieved tolerance, with a partial response in a further four. Inhibitors are an uncommon but life-threatening complication of haemophilia. This complication should be considered when selecting the treatment modality for patients with a family history of inhibitors, and DDAVP used whenever possible.     

Preparation of cryoprecipitated factor VIII concentrates

Factors affecting the yield of factor VIII in cryoprecipitate have been investigated in the context of a blood component program. Both in vitro and in vivo measurements were used to assess the effects of critical variables on the yield of factor VIII activity. Variables such as anticoagulant, plastic bag, mixing during collection, and platelet contamination had no significant effect on yield of factor VIII activity in cryoprecipitate. Among the most critical factors affecting yield were storage time of whole blood and procedures for freezing, thawing, and reconstitution. The following procedures were found to assure a 60 per cent recovery of factor VIII in cryoprecipitate: 1)processing of whole blood within six hours of collection; 2)use of a technique to freeze plasma within 30 minutes either in a -70 C ethanol bath or -85 C freezer; 3)rapid thawing (1 1/2 hour or less) in a 4 C circulating water bath; 4)centrifugation at 4,500 X g for 10 minutes at 4 C followed by draining of the supernatant in a 4 C cold room; 5) storage of the precipitate at -20 C until ready for use; 6) thawing in a 37 C water bath for at least 15 minutes followed by addition of 20 ml of 0.15 M saline for a 20 minute period at room temperature, and gentle mixing before pooling units for transfusion. The recovery of factor VIII in cryoprecipitate appears to be limited to about 65 per cent by its solubility in plasma at 4 C. Therefore, further effort to increase the amount available for treatment should involve improving the supply of plasma for its preparation and decreasing the cost of processing.     

Continuous arterial infusion of protease inhibitor with supplementary therapy for the patients with severe acute pancreatitis--clinical effect of arterial injection of ulinastatin

We treated five patients with severe acute pancreatitis by continuous arterial infusion (CAI) of protease inhibitor, nafamostat mesilate. Arterial injection (AI) of ulinastatin was performed in four cases and AI of antibiotics (IPM/CS) was done in one case, as supplemental therapies of CAI. Abdominal pain disappeared in 7.9 hours on the average, abdominal tenderness disappeared in 5.0 days and laboratory data lately recovered. All five cases treated by these therapies were cured without hemodialysis or surgical treatment in acute phase. AI of ulinastatin through arterial infusion catheter is pharmacokinetically more effective, because it yields a relatively high concentration of the drug at the acting site when compared with that of intravenous injection. Furthermore ulinastatin inhibits different types of protease from nafamostat mesilate. Therefore the clinical effect of CAI of nafamostat mesilate is enhanced by the combined therapy with AI of ulinastatin. It is also suggested that arterial injection of ulinastatin might be effective for the control of abdominal pain and that arterial injection of antibiotics might have an advantage on prevention of infectious pancreatic necrosis.