Universal leucodepletion is being introduced in the U.K. to reduce a theoretical risk of Creutzfeldt-Jakob disease (CJD) transmission. If CJD infectivity is associated with leucocytes, any cell fragmentation associated with filtration could reduce the potential benefit. Four types each of whole blood, red cell and platelet leucodepletion filters were assessed after holding of blood units for at least 4 h at 22 degrees C. In all cases the mean residual leucocyte content was <1 000 000 per unit, with only two individual filtered whole blood units having a leucocyte content exceeding this. Evidence of leucocyte fragmentation during filtration was sought but not found by assay of soluble elastase, beta-thromboglobulin and normal prion protein, as well as by isotopic labelling of leucocyte external membrane. These preliminary studies indicate that it was possible to prepare leucodepleted blood components by filtration at room temperature, and that this appeared not to be associated with overt cell fragmentation. Definitive demonstration that fragmentation does not occur requires the development of improved general (non-specific) assays for cell membrane fragments.

Antithrombin III (ATIII) and heparin cofactor II (HCII) are currently thought to be the most important protein mediators of the anticoagulant and antithrombotic activities of glycosamino-glycans. A simple, quantitative method for assessing the affinity of a protein for a sulphated polymer in the liquid phase, based on competition with immobilised heparin, has been developed. Using this technique, the binding of ATIII and HCII to a wide range of glycosaminoglycans and other sulphated polymers have been compared, and the contributions to binding of size, degree of sulphation and backbone structure of the polymers analysed. In the presence of the high protein concentrations found in plasma, unfractionated heparin inhibited the binding of ATIII to immobilised heparin with a Ki of 1 x 10-6. Binding was destroyed by N-desulphation. 1 Results with a range of low molecular weight (LMW) heparins and heparan sulphates are consistent with the view that they all contain the ATIII-binding sequence, but at a lower molar ratio than heparin. Highly sulphated synthetic polymers such as dextran sulphate bound ATIII by a different mechanism, which was molecular weight-dependent. The affinity of HCII for heparins increased markedly with heparin chain length. Binding was largely, but not entirely, mediated by sulphate residues. HCII bound to heparan and dermatan sulphates with lower affinities than to heparin, and to synthetic sulphated polymers with similar or higher affinities. Pentosan polysulphate (SP54) bound HCII as effectively as did heparin. Binding of HCII to dextran sulphate was highly dependent on molecular weight. The affinity of HCII for a sulphated polymer appears to depend both on its chain length and density of sulphation. Thus the profiles of binding of ATIII and HCII to glycosaminoglycans and other sulphated polymers are quite different. This technique is useful both for investigating the interactions of existing therapeutic anticoagulants and assessing new products.

Heating of blood products to reduce viral infectivity is now a standard practice. Such treatment may also modify the constituent proteins, reducing their activity or altering their structure with potentially harmful consequences for the recipient. Partially denatured proteins frequently form aggregates, which are often immunogenic and could precipitate immune complex formation, allergic reactions and kidney damage. In addition they may contribute to the development of AIDS after HIV infection by inducing a persistent state of T-cell activation. Protein aggregate formation in factor VIII and factor IX (II + X) concentrates has been investigated by fast protein liquid chromatography (FPLC), which proved to be a rapid, convenient method for this purpose. Freeze-drying alone resulted in aggregate formation in intermediate purity FVIII concentrates, but not in FIX concentrates. However, aggregates were detected after heating the FIX concentrate at 80°C for 72h in the dry state. Dry heating of intermediate purity FVIII concentrates to 68°C for 24h also increased the content of protein aggregates, which contained fibrinogen and fibronectin but little IgG. In this product, the aggregate content after heating correlated with total protein concentration. A higher purity FVIII concentrate selectively depleted in fibrinogen and fibronectin also contained protein aggregates after freeze-drying, but heating this product at 80°C for 72h resulted in a relatively small increase in aggregate content. Haemophiliacs receiving regular injections of heated concentrates are constantly exposed to protein aggregates. They should be monitored for any harmful effects, and manufacturers should aim to reduce the aggregate content of their products.

Unfractionated heparin, pentosan polysulphate (SP54) and the low molecular weight heparins CY216 and CY222 were injected subcutaneously at a minimum of weekly intervals into 5 healthy volunteers. The dose was 75 mg in all cases. Concentrations of administered glycosaminoglycan in serial plasma samples and voidings of urine were measured using a competitive binding assay, and biological activity was assessed in plasma using APTT and anti-Xa clotting assays. There was wide individual variation in the absorption of unfractionated heparin as indicated both by the maximal plasma concentrations reached 2–3 h after injection and by the area under the concentration vs. time curve. The efficiency of absorption increased and the individual variation decreased with decreasing molecular weight of the administered glycosaminoglycan. Urinary excretion correlated with plasma concentration, and recovery in the urine also increased with decreasing molecular weight. Similar patterns of uptake and clearance were indicated by the APTT and competitive binding assays, but anti-Xa clotting activity could be detected in the plasma after clearance of the administered glycosaminoglycan.

The vascular tree of cadaver legs was washed out with a detergent - glucose mixture and the soluble activator precipitated with 8%PEG 6,000. The redissolved precipitate was chromatogranhed on 4% agarose where it exhibited an apparent molecular weight of 8 × 105 and was well resolved from lipids, factor VIII antigen, albumin and haemoglobin. The active material eluted in the void volume of G-200 Sephadex when using 0.15 M NaCl eluant, but was dissociated and retarded by re-chromatography in 1 M NaCl or lysine. The dissociated, purified enzyme had an apparent molecular weight of 56,000 and was a serine protease. The enzymic activity of the carrier complex was stable for several days at 4°C, but the dissociated enzyme was rapidly inactivated at 4°C.