The Survival of Factor Viii (Antihemophilic Globulin) and Factor Ix (Plasma Thromboplastin Component) in Normal Humans*

Abstract

There is much evidence favoring the concept of coagulation as a continuous process in the normal subject. In order to learn more about the dynamics of this process, we have been studying the survival of physiological amounts of the various clotting factors in the normal subject. With the development of isotope labeling, it has for the first time become possible to carry out such studies. We (1-3) and others (1-10) have applied this technique to studies of platelet and fibrinogen turnover. These studies were possible because both platelets and fibrinogen are present in large amounts and can be separated in relatively pure form. Isotope techniques have not yet been applied to a study of the other proteins involved in coagulation because they are present in relatively trace amounts and cannot yet be isolated in sufficiently pure form.In order to overcome these problems, we have developed a technique of in vivo labeling, separating, and counting two of the clotting proteins, Factor VIII (antihemophilic globulin) as an example of a protein consumed during coagulation and Factor IX (plasma thromboplastin component, Christmas factor) as an example of a clotting protein not consumed during coagulation. Three mc of S"-DL-methionine was inj ected intravenously into suitable humans. Subj ects were selected who had no evidence of liver disease or of bleeding tendency. The labeled amino acid was used by the recipient in the formation of plasma proteins. The more rapid the turnover of the protein, the higher the specific activity would be. Twenty-four hours later, the subject was phlebotomized. ACD solution was used as anticoagulant, and 500 ml of blood was obtained with as little trauma as possible. The blood was immediately infused into the normal human recipients in whom the survival of the labeled protein was to be followed.Separating labeled proteins. This was accomplished by immunological methods.A partially purified Factor VIII preparation was made from human plasma by the method of Spaet and Kinsell (11). This mixture of proteins was made into a 10% solution with distilled water and mixed in a 1:1 suspension with Freund adjuvant.2 The mixture was injected intramuscularly twice a week for a period of 2 months into a group of twelve rabbits. After a 10-day rest period, the rabbits each received an iv injection of 3 ml of an alum-precipitated preparation made from a 5% solution of the original partially purified Factor VIII preparation. Seven days later, the rabbits were bled into 0.1 vol of 0.1 M sodium oxalate, the plasmas were separated and pooled, and 0.1 vol of 0.1% aqueous merthiolate was added.Blood was drawn from a patient with hemophilia whose Factor VIII level when measured by the partial thromboplastin technique (12) was less than 1%. This blood was drawn into 0.1 vol of 0.1 M sodium oxalate, and 0.1 vol of 0.1%So aqueous merthiolate was added.

Studies on the Response of Patients with Classic Hemophilia to Transfusion with Concentrates of Antihemophilic Factor

“…Relatively few observations were possible in four of the patients; in these, the first posttransfusion specimen was obtained between 15 and 30 min after completion of the infusions and the half-life was calculated from the peak values observed. In patient D, a specimen was obtained immediately following administration of the concentrate and a second 25 min later; in this patient, a biphasic decline in antigen levels was observed as has been suggested by other studies (9). The half-life in this patient therefore was calculated from the determinations made 25 min after completion of treatment (that is, at the time when the first postinfusion specimens were obtained from the other patients).…”

“…Only one previous study with which we are familiar employed an immunological technique to determine the halflife of infused AHF. Information not available at the time of that study now raises questions as to the specificity of the antibody employed, but nevertheless Adelson and his colleagues, using such a technique, also reported a half-life of AHF considerably longer than that 12 hr usually observed when clot-promoting activity assays alone are employed (9).…”

Immunologic differentiation of classic hemophilia (factor VIII deficiency) and von Willebrand's disease

“…0. 18 A multiple comparion test (37) was used. Our hypotheses are by no means satisfying, but it is interesting to note that in another situation, Fabry's disease, the transfusion of normal plasma is followed by a rise in the concentration of an enzyme, ceramide trihexosidase, present in diminished amounts in this disorder (12).…”

“…Using physicochemical methods, Hershgold, Silverman, Davison, and Janszen (13) and Barrow and Graham (14) reported the isolation of material similar to AHF from hemophilic plasma. Heterologous antisera directed against relatively crude AHF preparations have been prepared by several investigators (15)(16)(17)(18)(19)(20). Utilizing such heterologous antisera, Shanberge and Gore (16), Berglund (17), and Piper and Schreier (19) demonstrated that the plasma of hemophiliacs neutralized the capacity of the antisera to inactivate AHF.…”

Molecular Immunology of Factor VIII

“…More extensive studies have since yielded information which, though in itially contradictory, now appears to support these original conclusions. Several investigators utilizing heterologous antiserum either were not able to dem onstrate these Factor VIII antigens in hemophilic plasma (8)(9)(10) or could find them only in a small minority of cases, usually those retainin g some procoagulant activity (11,12). Additional studies with antibodies having specificity for apparently differ ent intramolecular sites have demonstrated antigenic expressions of Factor VIII in almost all hemophilic plasmas, including those without detectable Factor VIII procoagulant activity and lacking functional site-associated antigens (13)(14)(15).…”