K. W. E. Denson

Antithrombin III, Antifactor Xa and Heparin

Methods have been devised for the measurement of antithrombin and of the natural inhibitor of activated factor X.

The mode of action of antibodies which destroy factor VIII. II. Antibodies which give complex concentration graphs.

Summary. Antibodies which destroy factor VIII may follow second‐order kinetics (Biggs et al, 1972) or a more complex type of reaction may occur. The present communication concerns the complex reactions and evidence is presented which supports the hypothesis that these antibodies may react with factor VIII to form a complex with some factor‐VIII activity.

An investigation of three patients with Christmas disease due to an abnormal type of factor ix

Three patients with Christmas disease whose plasma was shown to have a prolonged one-stage prothrombin time with ox brain thromboplastin have been investigated. These patients have an inhibitor for the reaction between factor X, factor VII, and ox brain extract. The abnormal constituent responsible for this inhibitor appears to be factor IX whuch is functionally inactive but antigenically indistinguishable from normal factor IX. It is proposed that patients might be classified into haemophilia B+ for patients with this defect (Christmas disease+) and haemophilia B− (Christmas disease−) for patients who have classical Christmas disease.

The Fate of Prothrombin and Factors VIII, IX and X Transfused to Patients Deficient in these Factors

The treatment of patients with coagulation defects consists of an attempt temporarily to replace in their blood the factor they lack. This is achieved by giving whole plasma or concentrates of the appropriate factor prepared from human or animal blood. There is now no reasonable doubt that this treatment is effective. With increasing precision of the methods used to assay the different factors it is becoming possible to relate the blood level achieved in the patient to the dose of material administered and to study the rate of disappearance of the factor from the patients circulation. Accumulation of this information for all treated patients should improve treatment. If bleeding is related to the concentration of a particular factor in the patients blood, it is clearly important to be able to predict what concentration is likely to be reached after a given dose and to predict how long it is likely to be maintained. If the response of patients to therapeutic material prepared by different methods is not the same, this again is important because allowances must be made for the variations in calculating the effective dose.

The Mode of Action of Antibodies which Destroy Factor VIII.: I. ANTIBODIES WHICH HAVE SECOND-ORDER CONCENTRATION GRAPHS

Summary. In 1959 Biggs & Bidwell proposed the hypothesis that the reaction between antibody and factor VIII could be interpreted as a second‐order reaction and that in antibody excess the amount of antibody present could be measured using a linear calibration graph relating the logarithm of residual factor VIII to antibody concentration after incubation for a fixed period of time. This hypothesis has been re‐examined because antibody is seldom present in excess in most routine test systems and in view of a new and attractive hypothesis put forward by Pool & Miller (1972). All the evidence seems to suggest that the original hypothesis of Biggs & Bidwell is adequate for assessing the potency of antibodies of the type occurring in many haemophilic patients.

The Interaction of Factors VIII and IX

It has been shown by Macfarlane, Biggs, Ash and Denson (1) and Biggs, Macfarlane, Denson and Ash (2) that factors viii and ix, react in the presence of calcium chloride and traces of thrombin to form factor x activator (factor viiza) (figure 1). The curves shown in figure 1 were obtained using a one-stage method for following the development of factor x activator activity, and a two-stage assay method to follow the destruction of factor viii when mixtures of factor viii, ix a, thrombin, and calcium chloride were pre-incubated. It can be seen that coincident with the disappearance or consumption of factor viii in figure 1, there is development of an activator of factor x. It appeared that factor viii was a substrate in this reaction, and that formation of the factor x activator was catalysed by both activated factor ix (factor ixa) and thrombin. The effect of thrombin was unmistakebly clearcut. There appeared to be little doubt that even traces of thrombin had a powerful effect on factor viii alone, at first activating it and subsequently destroying it. Acceleration in the rate of formation of the factor x activator by different concentrations of thrombin is shown in figure 2. The overall reaction was depicted by the following equations In these experiments, the observed activity, which was attributed to the formation of viii a, could equally well have been due to contaminating amounts of factor x in the various preparations of factor ix used.

THE MODE OF ACTION OF A COAGULATION INHIBITOR IN THE BLOOD OF TWO PATIENTS WITH DISSEMINATED LUPUS ERYTHEMATOSUS (DLE).

SOME patients with disseminated lupus erythematosus (DLE) have in their blood a coagulation inhibitor which will lengthen the clotting time of normal blood. Some of these patients also have a low level of prothrombin as measured by the two-stage method. The two defects do not seem to be causally connected. Several authors have noted that the defects in all of the recorded cases have features in common which suggest a common origin and mode of action for the inhibitor. The patients have a defrnite but usually slight lengthening of the one-stage prothrombin time; if, in this test, the brain extract is much diluted, the difference between the clotting times of the normal and the patients plasma is exaggerated. The thromboplastin generation test is characteristically normal (in 10 of 11 cases). This result is generally taken to imply normality of the factors involved in the formation of intrinsic prothrombin activator. The explanations so far advanced for the mode of action of the inhibitor have seemed incomplete. They do not define a sufficiently specific activity and when some specific mechanism has been proposed the degree of abnormality has seemed inadequate to account for the defect. The present investigation is concerned with the mode of action of the inhibitor. The experimental methods used in this work and some of the deductions made are based on certain assumptions about the theory of blood coagulation, the evidence for which has been given in detail by Biggs and Macfarlane (1962). For the convenience of readers, the part of this theory relevant to this work is outhed below:

Experiments with Factor VIII Separated from Fibrinogen by Electrophoresis in Free Buffer Film

Approaches to an understanding of the chemistry of Factor VIII (antihaemophilic factor) have been few and necessarily indirect since Factor VIII occurs in plasma in only trace amounts, possibly less than 1 mg./l., is very labile, and the biological assay methods are subject to large errors. There have been repeated suggestions that the actual active Factor‐VIII molecule might be rather small and that in plasma it is associated with another protein (Thelin and Wagner, 1961; Surgenor, 1964). Methods of purification of Factor VIII are essentially modifications of methods applicable to fibrinogen. The highest purification of Factor VIII is probably that of Michael and Tunnah (1963) but the protein of even their best preparation is at least 50 per cent fibrinogen. Methods directed towards removing fibrinogen specifically by conversion to fibrin or heating result in a loss of Factor‐VIII activity. It seemed possible, therefore, that if there is an association between Factor VIII and another protein, the partner might be fibrinogen.