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.