D. E. G. Austen

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

Summary. 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.

Factor VIII of Small Molecular Weight and its Aggregation

Summary. Work on the disaggregation of factor VIII into sub‐units has been extended and it is shown that factor VIII of small molecular weight may be aggregated to a high molecular weight species probably identical to the nod unreacted factor. In addition some practical modifications were introduced to improve yield and reproducibility. The reaction of dithiothreitol and high concentrations of saline were compared using both human and bovine factor VIII. Small factor VIII which was produced had a molecular weight of 230 000 and the act of disaggregation illustrated a clear difference in behaviour between active factor VIII and factor VIII related antigen. These results may be considered to oppose the hypothesis that factor VIII related antigen is the protein carrier of factor VIII.

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.

Identification of sources of inter-laboratory variation in factor VIII assay.

Summary. A repeated finding of national and international collaborative studies of standard factor VIII preparations has been that systematic differences exist between laboratories in their measurement of the relative activities of the same pairs of factor VIII preparations.

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.

The Chromatographic Separation of Factor VIII on Aminohexyl Sepharose

Summary. 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 and Characterization of the Factor in Porcine and Bovine Plasma which Aggregates Human Platelets

Summary. 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.

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.

Clotting factor IX levels in C/EBPα knockout mice

Previous studies have indicated that C/EBPα 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/ebpα gene are significantly deficient in factor IX transcription.