Cecil Hougie

A simple assay method for factor X (Stuart-Prower factor).

Conclusion A new assay method for factor × is described. Human plasma is treated with bentonite resulting in a substrate rich in prothrombin but deficient in factors VII and X. Factor V and fibrinogen are also depleted but these factors are replaced by addition of Al(OH)3-treated plasma. Russell viper venom is used to compensate for the factor VII deficiency. The method is a simple and reproducible one. The results obtained compare well with the original method using plasma from a patient with a congenital deficiency of factor X.

Reactions of Stuart factor and factor VII with brain and factor V.

Summary Previous work showing that factor V, serum, brain and calcium react together to form an active prothrombin conversion factor (“extrinsic thromboplastin”) is confirmed. The active components in serum in respect of this reaction include both factor VII and SF but not PTC. SF appears to influence yield while factor VII primarily determines rate.

Effect of Russell's Viper Venom (Stypven) on Stuart Clotting Defect

Summary It is shown that Russells viper venom (Stypven) does not correct the prolonged one-stage prothrombin time of patient with a congenital hemorrhagic diathesis previously thought to be deficient in factor VII. The factor deficient in this patient which is referred to as the Stuart factor, is readily distinguishable from both the Prower factor and factor VII since the prolonged prothrombin times of plasma deficient in either of these two factors are completely corrected by Stypven. The finding that Stypven does not correct the clotting defect of Stuart factor deficient plasma implies that certain modified one-stage “specific” assay methods for prothrombin using Stypven as a source of factor VII are not, in fact, specific, and measure both prothrombin and Stuart factor.

Evidence that factor VIII and the ristocetin aggregating factor (VIIIRist) are separate molecular entities.

The most highly purified preparations of human factor VIII have at least three clearly defined properties. They shorten the clotting time of hemophilic plasma, an activity which will for convenience be referred to as VIIIcoag; they give a precipitin reaction with a rabbit antibody prepared against the preparation, a property referred to as the factor VIII related-antigen (VIIIR-Ag); and in the presence of the antibiotic, Ristocetin, they correct the defective aggregation of platelets found in von Willebrands disease (1-4), an activity which will be referred to as VIIIRist. There are two prevailing concepts: (a) that each property represents a function of the same molecule, (b) that there is more than one molecule, each with one or more properties (5). The purpose of the experiments described below was to determine whether VIIIcoag, VIIIRist, and VIIIR-Ag reside on the same or on different molecules. The addition of a naturally occurring human factor VIII antibody known to be of the IgG class neutralizes VIIIcoag without affecting the expression of VIIIRist or VIIIR-Ag (3). Rabbit antibodies, on the other hand, prepared against highly purified factor VIII preparations neutralize VIIIRist and VIIIR-Ag (3), but their VIIIcoag neutralizing activity may be relatively low (6, 7). These findings are not inconsistent with the one-molecule hypothesis as the antibody could neutralize one side chain carrying an active site without affecting others. If the one-molecule hypothesis is correct, following the neutralization of VIIIcoag or VIIIRist by the appropriate antibody, the addition of a second precipitating antibody directed against the IgG class should precipitate both VIIIcoag and VIIIRist activities bound to the first antibody; as the factor VIII molecule would carry all the active sites, the supernatant will be devoid of all the remaining properties of the molecule.

Anticoagulant Action of Protamine Sulphate.

Summary Protamine sulphate in low concentrations affects both rate and yield of blood thromboplastin but has no effect on formed thromboplastin. Relatively higher concentrations inhibit a reaction between blood thromboplastin, prothrombin and calcium.

The Passovoy Defect: Further Characterization of a Hereditary Hemorrhagic Diathesis

We studied a coagulation abnormality present in 12 members of five kindreds who bruised easily and bled excessively after minor trauma. Their activated partial thromboplastin times were between 32 and 39 seconds (normal, 22.8 to 28.8 seconds). Prothrombin times, thrombin times, platelet-function tests and the levels of factors XII, XI, IX, VIII, prekallikrein and high-molecular-weight kininogen were normal. Within these kindreds inheritance of prolonged partial thromboplastin times followed an autosomal and probably dominant pattern. The prolonged thromboplastin times were corrected by normal plasma and by normal plasma adsorbed with celite, but there was no mutual correction between plasmas of the patients. These subjects shared a common defect in the intrinsic pathway of coagulation that we designate by the probands surname, Passovoy.

Pathogenesis of haemophilia; a critical analysis of the evidence for the Bridge anticoagulant.

UNTIL recently there were two hypotheses for the pathogenesis of haemophilia. The ‘anticephalin-excess’ hypothesis postulates that the bleeding tendency in haemophdia results from an excess of a circulating dubitor, ‘anticephalm‘. This substance is believed to be present in normal blood at a lower titre acting to prevent intravascular clotting; an important corollary of this hypothesis is that there is a f d complement of coagulation factors in haemophilic blood (Tocantins, 1951). In a critical and exhaustive study, Graham and Barrow (1957) showed that the original interpretation of the experimental data believed to support h s hypothesis is probably incorrect, and that these experiments can be explained on a basis other than the presence of ‘anticephalin’. The second hypothesis is that haemophdia is due merely to a deficiency of a coagulation factor, annhaemophilic factor (AHF). This second hypothesis constitutes the orthodox view. A third theory has now been advanced by Nour-Eldin and Wilkinson (1958a) after a study of 10g patients with haemophdia. These workers found that in all these patients the incomplete plasma thromboplastin formed in the absence of AHF clotted normal substrate plasma in a shorter time than haeniophilic plasma. The addition of AHF to the haeniophilic substrate plasma did not materially affect the results. They deduced from this that diminished concentration of AHF in the substrate was not the cause of its prolonged clotting time and attributed this prolongation to the presence of an anticoagulant which counteracts the action of incomplete plasma thromboplastin. They also showed that the incomplete thromboplastin formed on using haemophilic plasma absorbed with aluminium hydroxide (Al(OH),) and diluted I in 5 was inore than that produced on using the same plasma undiluted and interpreted h s finding as further evidence supporting the existence of an idubitor in haemophilia. From the results of further experiments they concluded that the anticoagulant has no destructive action on AHF and inhibits a reaction between Christmas factor, AHF and platelets and not merely a reaction between Christmas factor and AHF. The circulating anticoagulant, therefore, differed from any other previously described, in particular those found complicating haemophilia (Hougie, 1955; Lewis, Ferguson and Arends, 1956). Nour-Eldin and Wilkinson named h s new inhibitor Bridge anticoagulant after the first patient in whose plasma it was detected. They believe that haemophilla is characterized by both the presence of the Bridge anticoagulant and a deficiency of AHF. These workers performed similar experiments on fourteen patients with Christmas disease and believe that this condition is also characterized by the presence of Bridge anticoagulant in addition to a deficiency of Christmas factor. The dual hypothesis theory is an attractive one since it could explain the relative mildness of clinical manifestations in those patients with von Willebrand’s disease who have the same low level of AHF as haemophdiacs with moderately severe c h c a l manifestations. This difference in clinical severity obviously cannot be due to AHF deficiency alone, since t h i s is * This work was supported by grant H-3536 from the National Heart Institute, N.I.H., Public Health Service, U.S.A. t Postal address: Box 32, University Hospital, Charlottesville. Virginia, U.S.A.