Roger E. Benson

Ristocetin cofactor activity of purified canine factor VIII: inhibition by plasma proteins.

Abstract Canine plasma does not support the ristocetin-induced agglutination of fixed washed human platelets (FWP) under conditions that readily produce agglutination with human plasma. Purified canine factor VIII (FVIII) resembles purified human FVIII as it supported a pronounced ristocetin-induced agglutination of FWP in a system essentially free of extraneous proteins. Susceptibilities of these ristocetin cofactor activities (RCoF) to inhibition by plasmas or bovine serum albumin (BSA) were compared. Canine plasma, canine or human von Willebrands disease (VWD) plasmas, and BSA completely inhibited the RCoF of purified canine FVIII at concentrations that had minimal effects on this activity of purified human FVIII.

The Value of Age‐ and Sex‐Matched Controls for Coagulation Studies

Summary. Several coagulation parameters and plasminogen levels were studied in 80 normal people divided into eight paired and sex‐matched age groups: prepubertal children, postpuberal children, young adults, and adults over 50. The data indicate that factor‐VII and ‐IX activities increase with age, with a cluster of lower activity for children and higher activity for adults. Factor‐VIII levels appear to decrease with age, although this effect could be due to greater anxiety in the pre‐ and postpubertal children at the time of venipuncture. The adults showed no significant change in factor‐VIII activity with age, but partial data indicate that factor‐VIII levels are higher in adults with blood group A than those with blood group O. The age‐related changes in factor‐VII, ‐IX, and possibly ‐VIII activities did not vary between sexes. By contrast, plasminogen increased strikingly with age in males and decreased with age in females. With fibrinogen, a similar effect was found for adults, though not for the entire population. These findings indicate the importance of using appropriate age‐ and sex‐matched controls for coagulation and plasminogen assays, especially in patients with mild inherited or acquired coagulation disorders.

Binding of Low-Molecular-Weight Canine Factor VIII Coagulant from von Willebrand Plasma to Canine Factor VIII-Related Antigen

Summary. Plasmas having no detectable factor VIII‐related antigen but moderate factor VIII coagulant were obtained from two unrelated dogs homozygous for von Willebrands disease and with the severe clinical expression of the disease. When these plasmas were gel‐filtered in a buffer at physiologic ionic strength, the factor VIII coagulant eluted in the bed volume as a single well‐defined peak. Addition of protease inhibitors, including diisopropylfluorophosphate, did not change the elution pattern. Each plasma was then combined individually with plasmas from six different mutants of canine haemophilia, all of which had normal factor VIII‐related antigen but no detectable factor VIII coagulant. The factor VIII coagulant elution profile of these combined plasma resembled that of normal canine plasma. Slightly over half of the recovered factor VIII coagulant coeluted in the void volume with the factor VIII‐related antigen; the rest eluted as a second, distinct peak of lower molecular weight. These results demonstrated that part of the factor VIII coagulant of the von Willebrand plasmas had bound to the factor VIII‐related antigen of the haemophilic plasmas. This finding supports the theory that factor VIII exists as a macromolecular complex of nonidentical components in normal citrated plasma.

Factor VIII-related antigen in canine hemophilia and von Willebrand's disease: Variation when measured with different agaroses☆

The source of agarose used in the Laurell electroimmunoassay was found to produce variations in the factor VIII-related antigen levels of plasmas from dogs with von Willebrands disease and hemophilia A. Similar artifactual variations in antigen were not observed for normal canine plasmas.

Physical Relationship between Canine Factor VIII Coagulant Activity and Factor VIII-Related Antigen

SummaryTwo of three insolubilized anti-canine FVIII systems removed similar amounts of VIII-C and VIII-RA from canine plasma. These data suggest that VIII-C and VIII-RA form part of one circulating complex.The authors thank Dr. W. P. Webster, Chapel Hill, N.C., for providing the canine hemophilia A plasma and Joanne E. Kull for her assistance in collecting canine plasma samples and immunizing the rabbits.Summary Two of three insolubilized anti-canine FVIII systems removed similar amounts of VIII-C and VIII-RA from canine plasma. These data suggest that VIII-C and VIII-RA form part of one circulating complex. The authors thank Dr. W. P. Webster, Chapel Hill, N.C., for providing the canine hemophilia A plasma and Joanne E. Kull for her assistance in collecting canine plasma samples and immunizing the rabbits.

Partial Characterization of the Factor VIII‐Stimulating Material of Rabbit Liver Perfusates: Relationships to Rabbit Plasma Factor VIII

Previous studies in our laboratory showed that effluent from perfused rabbit livers stimulates the production of factor‐VIII activity in perfused rabbit spleens. When concentrated supernatants of the hepatic perfusates were chromatographed on Sephadex G‐200, BioGel A‐15M and Sepharose 4B, the fractions with the greatest factor VIII‐stimulating capacity eluted in the void volume (V°). Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the Sephadex G‐200 V° fraction revealed components with a wide range of molecular weights. Rabbit factor VIII was isolated from ethanol‐concentrated, citrated rabbit plasma by Sepharose 4B filtration. The factor‐VIII activity appeared in the V° when eluted with physiological saline but was retarded on columns eluted in 0.8 M sodium chloride. Further analysis with SDS gels demonstrated that the Sepharose‐purified factor‐VIII preparation entered the polyacrylamide gels only after reduction with 2‐mercaptoethanol. Goat antirabbit factor VIII antiserum prepared against the gel‐purified antigen contained factor VIII‐neutralizing activity, was apparently monospecific by double‐diffusion and produced faint, single peaks by electro‐immunodiffusion. Antibody neutralization studies with the antiserum showed that the concentrated supernatants of hepatic perfusates contain fourfold greater factor VIII‐neutralizing antigen than procoagulant activity, whereas control perfusates, lacking factor VIII‐stimulating capacity, had negligible antibody‐blocking effect. The presence in hepatic perfusates of both high‐molecular‐weight factor VIII‐stimulating material and large amounts of factor VIII‐neutralizing antigen lacking procoagulant activity suggests that they are interrelated.

A practical technique for preparation of antiserum to canine factor VIII-related antigen

Identification and measurement of canine factor VIII-related antigen (VIIIR:Ag) has many clinical and research applications, including differential diagnosis of hemophilia A and von Willebrands disease, use as a marker for specific cell types, and elucidation of the structure of this factor VIII component. We have developed a practical method for producing antibodies to canine VIIIR:Ag that uses 2% agarose filtration for purification and identifies the antigen by correlation with the elution of the peak A280 fraction in the void volume. Antisera for electroimmunoassay (EIA) can be produced in less than four weeks from simple starting materials and with commonly available laboratory equipment. This technique would be useful for either clinical veterinary or comparative research laboratories.

Assessing the specificity of anti-canine-factor VIII-related antigen

Antibodies to canine factor VIII-related antigen should be monospecific to accurately study this proteins location in tissue and its structure. Because no uniformly accepted standard for monospecificity exists for anti-factor VIII-related antigen, we have compared the sensitivities of three common evaluation techniques--immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis. Unabsorbed rabbit anti-canine factor VIII-related antigen appeared monospecific against whole plasma by all three methods; however, multiple specificities were recognized against a plasma cryoconcentrate. These results demonstrate the insensitivity of the techniques under certain conditions, which can lead to erroneous interpretation of data.