Clotting factor IX levels in C/EBP alpha knockout mice.

Previous studies have indicated that C/EBP alpha is involved in the regulation of factor IX and mutations of a C/EBP recognition element in the factor IX promoter result in haemophilia B. We now report that mice homozygous for the deletion of the c/ebp alpha gene are significantly deficient in factor IX transcription.

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.

Protein engineering of the hydrophobic domain of human factor IX.

Vitamin K-dependent plasma proteins contain a highly conserved hydrophobic domain located between the gamma-carboxyglutamic acid (Gla) domain and the first epidermal growth factor (EGF)-like domain. Here we have used protein engineering of the hydrophobic domain in human factor IX to investigate its function in intact factor IX. Mutant proteins were generated by site-directed mutagenesis and in vitro expression in Madin-Darby canine kidney (MDCK) cells. All of our mutants, including one with a deletion of the entire hydrophobic domain, were activated by factor XIa, showing that this domain is not required for factor IX activation. The results with the mutant Phe41-->Val suggest that the hydrophobic domain interacts with the adjacent EGF-like domain. Our data for the Phe41-->Asp mutant is consistent with, but cannot prove, a role for this residue in the maintenance of a phospholipid-binding structure required for factor IX function.

Subcutaneous injection of factor IX for the treatment of haemophilia B.

Haemophilia B patients are normally treated, either prophylactically or in response to bleeding episodes, by frequent intravenous injections of factor IX purified from blood donors. Here we show in model animal experiments that purified human factor IX, when injected subcutaneously, is rapidly (in 3-11 h) and reasonably efficiently (30-40% of an equivalent intravenous dose) transported at least partly by the lymphatic drainage of the skin into the bloodstream, mostly in a biologically active form. This suggests that patients could be treated prophylactically by subcutaneous rather than intravenous injection, where the short delay in raising plasma factor IX to haemostatic levels would be clinically acceptable. More generally, our studies emphasize that the subcutaneous route of injection should be useful for other therapeutic proteins, including other clotting factors, which have to be delivered to the bloodstream, as long as their half-life is at least a few hours allowing time for transport into the general circulation.

Production of factor VIII deficient plasma by immunodepletion using three monoclonal antibodies.

Factor VIII deficient plasma was made from pooled, HIV antibody and hepatitis B antigen screened, normal human plasma by cryoprecipitation and immuno-depletion, using three different monoclonal antibodies bound to Sepharose columns, in series. These monoclonal antibodies are specific respectively for von Willebrand factor, factor VIII heavy chain and factor VIII light chain. The immunodepleted plasma contained less than 0.002 u/ml factor VIII coagulation activity (VIII:C) less than 0.0001 u/ml von Willebrand factor antigen and 1-2 g/l fibrinogen, while the levels of other clotting factors were unchanged. This immunodepleted plasma was compared with commercial factor VIII deficient plasma obtained from a severe haemophilia A patient as substrate in the one-stage factor VIII assay. Plasmas obtained from 20 normal subjects and 28 patients with von Willebrand's disease or haemophilia A were assayed for VIII:C using the two substrates. The results were very highly correlated (r = 0.96). The columns have high capacity and can be regenerated at least 10 times. Large-scale production of a substrate for factor VIII assays free of virus contamination is now feasible.

Expression of active human clotting factor IX from recombinant DNA clones in mammalian cells.

Haemophilia B, or Christmas disease, is an inherited X-chromosome-linked bleeding disorder caused by a defect in clotting factor IX and occurs in about 1 in 30,000 males in the United Kingdom. Injection of factor IX concentrate obtained from blood donors allows most patients to be successfully managed. However, because of impurities in the factor IX concentrate presently in use, this treatment involves some risk of infection by blood-borne viruses such as non-A, non-B hepatitis and the virus causing acquired immune deficiency syndrome (AIDS). Because of the recent concern about the increasing incidence of AIDS amongst haemophiliacs, a factor IX preparation derived from a source other than blood is desirable. Here, we report that after introduction of human factor IX DNA clones into a rat hepatoma cell line using recombinant DNA methods, we were able to isolate small amounts of biologically active human factor IX.

Partial separation of blood clotting factors, albumin, and IgG by continuous free film electrophoresis.

Blood plasma and factor VIII concentrate have been fractionated by continuous free film electrophoresis in an apparatus which was designed to overcome the problems of overheating usually inherent in this technique. Results show that complete separation of plasma proteins is unlikely to be obtained by a single passage through this apparatus. However, a useful degree of fractionation may be obtained with certain proteins, for example fibrinogen and IgG. It should be possible to concentrate blood clotting factors if the electrophoresis is used in combination with a conventional separation procedure such as adsorption on aluminium hydroxide. The advantage of the electrophoretic technique is that it is a continuous process using the minimum of extraneous chemicals. The disadvantage is that it introduces a 40-fold dilution into the fractionation.

Factor VIII fractionation on aminohexyl Sepharose with possible reduction in hepatitis B antigen.

The method of factor VIII purification by chromatography on aminohexyl Sepharose has been extended so that up to 100 ml of intermediate purity concentrate can be processed on an 8.6 ml column. The product has a specific activity of 1.8 International Units of factor VIII per mg of protein. Most of the fibrinogen is removed and antibodies to blood group substances A and B are not detectable by haemagglutination techniques. Hepatitis B antigen has been reduced to one sixteenth of that in the starting material, in preliminary experiments. The process has the added advantage that it concentrates the factor VIII relative to the starting material.

A comparison of the Bethesda and New Oxford methods of factor VIII antibody assay.

A collaborative trial has been carried out under the auspices of the International Committee on Thrombosis and Haemostasis to compare the Bethesda and New Oxford methods of antibody assay. It was found that errors between laboratories were much greater than those with laboratories and each laboratory had a bias whereby it always rated samples high or low with respect to the other laboratories. However there was excellent agreement in the order in which laboratories ranked antibody samples and if a standard antibody sample could be provided there would be a significant improvement in numerical agreement between laboratories. On average, for this exercise, a result for a given sample in Bethesda units was 1.21 times the result in New Oxford units although it must be stressed that this ratio could vary from sample to sample.

The chromatographic separation of factor VIII on aminohexyl sepharose.

A new method of factor VIII purification has been devised which involves chromatographic separation on aminohexyl-substituted agarose. Relatively large volumes of starting material can be processed compared to the volume of agarose employed and satisfactory yields are obtained. Factor-VIII-clotting activity is separated from the other related substances and resultant products appear to be stable. While separation of clotting activity occurs with relative ease, the antigen and ristocetin co-factor are hardly segregated, if at all. This provides a little more information about the interrelation of these substances. Results suggest that ion-exchange is involved in the mechanism of separation but additional hydrophobic or steric effects cannot be ruled out.

Assay of ristocetin co-factor using fixed platelets and a platelet counting technique.

A new assay of ristocetin co-factor has been developed which is based on platelet counting using a Coulter Counter. A modified method of washing and fixing platelets has also been devised to provide a suitable platelet preparation which can be used in this assay. These fixed platelets have consistently proved to be satisfactory and have given high levels of maximum percentage aggregation. They have been stored for periods up to 6 months without significant deterioration. The method of platelet counting is simple in operation and sensitive to relatively low levels of platelet aggregation. It is precise and seems to offer distinct advantages over existing methods.

Assay and characterization of the factor in porcine and bovine plasma which aggregates human platelets.

A sensitive and objective bioassay has been developed to measure the factor present in porcine or bovine plasma which aggregates human blood platelets. The technique is based on direct measurement of platelet aggregation using an electronic particle counter. Using this assay it has been established that platelet aggregating activity is present in both bovine and porcine plasma and in therapeutic concentrates of factor VIII prepared from these materials. Platelet aggregating activity was found to be a function of the factor-VIII-related antigen concentration and independent of factor-VIII coagulant activity. The activity is absent in the plasma from pigs with von Willebrand's disease. It is concluded that factor-VIII-related antigen and the platelet aggregating factor are closely related and may represent one protein species.

Detection of carriers of haemophilia: a 'blind' study.

A 'blind' study has been made to try to find out if it is possible to diagnose carriers of haemophilia. A group of 34 obligatory carriers of haemophilia were compared with 34 normal women. Levels of factor VIII activity, factor VIII-related antigen, factor V and ratio of factor VIII activity to factor VIII-related antigen were measured. In the carrier group the mean level of factor VIII activity and the mean level of the ratio of activity to antigen were each approximately half of those found in the normal women. The mean level of factor V was the same in both groups of women. By setting the lower limit of normal at the lowest level of the different factors found in the normal women, 12 out of 34 (35%) carriers could be distinguished on the basis of their factor VIII level alone; 24 out of 34 (71%) could be detected on the basis of the ratio of factor VIII activity to factor VIII related antigen and 25 out of 34 (73%) could be detected if both factor VIII activity and the ratio were taken into account. It is concluded that consideration of both the level of factor VIII activity and the ratio of factor VIII activity to factor VIII-related antigen is of some value in detecting carriers of haemophilia. The number of carriers detected (73%) in the present study is not as high as that found by other workers.