Summary: Through the use of DEAE‐cellulose under the conditions reported in this paper which were particularly selective for the adsorption of clotting factors, the chromatographic behaviour of Factors I, II, V, VII, VIII, IX and X was studied using different buffer systems. Human plasma, supernatant of Fraction I of Cohn and human Factor‐VIII concentrates were used as starting materials. As some of the chromatographic systems used do not perceptibly modify the physical and chemical properties of the plasma or its derivatives, they allow the techniques to be included in a general scheme of routine plasma fractionation. The conclusions drawn from the chromatographic behaviour of the factors studied have led to the preparation of a concentrate of Factors II, IX and X for clinical use, a concentrate of Factor VII and an artificial substrate for the assay of Factor VII.

THE treatment of haeinophilic bleeding often requires the use of concentrated Factor VIII.Casillas, Simoiictti and Pavlovsky (1959) compared the available methods of separating Factor VIII from liurnan plasma and found that the technique of Blomback, Blombsck and Nilsson (1959) was the most satisfactory. The preparation made by this process (called FI-0)is still contaminated with many other proteins, notably fibrinogen, which hinder its concentration and the better characterization of the active component.

Bio-resin thrombin preparations were found to contain three weak precipitinogens. The clotting activity was not demonstrably associated with the precipitinogenic systems. Further, work was done on methods for the purification of citrate resin thrombin, and its clotting activity is also not associated with a precipitinogenic system. The N-terminal amino acid of both bio-resin thrombin and citrate resin thrombin was found to be glutamic acid. The two preparations were found to be homogeneous upon ultracentrifugal examination and could not be differentiated on the basis of sedimentation constants. Since "citrate" activation and "bio" activation produce eventually similar thrombin material, the autocatalytic activation of prothrombin in 25% sodium citrate solution can be used as an ideal model of prothrombin activation. The prothrombin first dissociates to form a derivative that does not form thrombin in the two-stage analytical reagents. Then a second alteration occurs in which the derivative again may form thrombin in the two-stage analytical reagents. Then thrombin activity appears as esterase activity, then as clotting activity. Later the clotting activity may be lost and finally also the esterase activity. The original prothrombin is a precipitinogen while the active thrombin is not.

The PVP precipitating properties for human and bovine factor VIII and fibrinogen were studied and a new technique for the fractionation of factor VIII and fibrinogen was developed. The precipitation was performed at different temperatures and different PVP concentrations and the best conditions for the technique were chosen. The technique consists of two steps: (a) precipitation of factor VIII from undiluted plasma with PVP and (b) washing of the precipitate with a glycine--saline solution. The final concentrate contains 90% of the factor VIII and 20% of the fibrinogen of the original plasma.

Dear Sir, Factor VIII is composed of two different antigenic moieties with different activities: Antigen 1 (Al), associated to the coagulant factor (VIII C) and antigen 2 (A2), associated to the von Willebrand factor (VIIIVWF) in human F.VIII and to the platelet aggregating factor (PAF) in bovine F.VIII. We have already described molecular varieties lacking VIII C and keeping 100% of VIII VWF or PAF (1). We are now describing a simple technique in order to obtain a molecular variety having only VIII C. An ultrasonication desintegrator of the type used by bacteriologists was used for the ultrasonication of human and bovine samples. During ultrasonication (20 Kc; 60 W) a rapid total inactivation of VIII VWF (assayed as ristocetin cofactor) or PAF (Fig. 1) occurs in 30 sec. In highly purified human or bovine factor VIII the VIII C begins to inactivate after 2 min, whereas in medium purity F. VIII it is more stable in function of time, and much more stable in the case of plasmas.

SummaryThis publication describes the results obtained by treatment of haemophiliacs with factor VIII preparations isolated from Cohn fraction I by use of tannic acid, FI-O-Ta.The authors stress the rapidity of the disappearance of factor VIII after injection. Transfusions are generally well tolerated. One reaction of the pyrogen type has been observed and also a case of activation of the fibrinolytic system.A second purification method by means of chromatography on DEAE-cellulose is described.

Summary. The adsorption on tricalcium phosphate and on barium sulphate of factor VIII from bovine and human plasma and from human factor VIII concentrates was studied. A technique for the preparation of a concentrate of bovine factor VIII by direct adsorption of plasma with tricalcium phosphate and then filtration on agarose gel has been developed. The use of EACA, glucose and glycine markedly increase the yield and make possible the lyophilization of highly purified factor VIII. The technique can be used on a preparative scale using 10–30 litres of bovine plasma.

Highly purified bovine factor VIII prepared according to our technique and having a specific activity of 500 U/mg protein has been studied. The chemical analysis of the preparation revealed it to be composed of amino acids, lipids (8–10%) and carbohydrates (7 %). The lipid moiety can be removed by chromatography. Different mechanisms of inactivation of bovine factor VIII and the possible molecular changes induced by the inactivating agents were studied. EDTA and EGTA provoke a weakening of the bonds linking the structural elements of the molecule, which allows for the separation of two different components of the molecule by gel filtration. Upon treatment with thrombin, the carbohydrate content of bovine factor VIII decreases without any apparent degradation of the protein moiety of the molecule. The fact that precipitating and neutralizing antisera against factor VIII were obtained, shows that the molecules modified by EDTA and thrombin still have the antigenic properties of factor VIII. The inhibitor developed by hemophiliacs transfused with human factor VIII is bound to bovine factor VIII, forming a complex which is stable upon agarose gel filtration. Immunological studies of the purified complex reveal that it is composed of bovine factor VIII and human γ-globulin. Bovine factor VIII in the complex retains some antigenic determinants which bind rabbit anti-serum against bovine factor VIII, as shown by neutralization of the antiserum and by precipitation studies.