Stability of canine factor VIII activity and von Willebrand factor antigen concentration in vitro

The in vitro stability of canine factor VIII activity, von Willebrand factor antigen concentration and the ratio of these two factors was studied. Samples were stored for up to 48 hours, either as plasma or as whole blood, at 4 degrees, 20 degrees and 37 degrees C. Factor VIII activity was generally stable in both plasma and whole blood samples for up to 48 hours at 4 degrees or 20 degrees C. The concentration of von Willebrand factor antigen was more stable in samples stored as plasma than whole blood, and for a shorter time than factor VIII activity. Consequently, the stability of the ratio of these two factors was relatively poor in vitro.     

The estimation of factor VIII levels in horse, cattle, sheep and pig plasma by the use of synthetic chromogenic substrate

Factor VIII level in horse, cattle, sheep and pig plasma was estimated by the use of synthetic chromogenic substrate S-2222 (benzoyl-isoleucyl-glutamyl-glycyl-arginyl-p-nitronilide). The highest level of this factor was stated in pig, the lowest one in sheep plasma.     

Stability of canine factor VIII: coagulant activity in vitro.

A pilot study was undertaken to assess the stability of canine factor VIII:coagulant (FVIII:C) activity over three days, under various storage conditions (plasma at 4, 20 and 37 degrees C, whole blood at 4 and 20 degrees C). Blood collected from normal and hemophiliac dogs was used. Both plasma and whole blood samples appeared to be stable for up to 48 h at 4 and 20 degrees C. A subsequent study evaluated FVIII:C stability at 4 and 20 degrees C when stored as whole blood only. Samples were tested at 0, 24 and 48 h after collection. At 4 degree C there was a significant decline at 24 h (p less than 0.05), from 110% to 97% (mean values). Although the mean value was further decreased at 48 h (89%) this was not significant (p greater than 0.05). No significant change in FVIII:C activity was observed in whole blood stored at 20 degrees C for 24 or 48 h (110% and 107% respectively). These results suggest that canine whole blood samples collected into sodium citrate stored at 20 degrees C are adequate for routine FVIII:C assay for up to 48 h after collection.     

Changes in factor VIII activity and von Willebrand factor antigen concentration with age in dogs.

The factor VIII activity of 38 German shepherd puppies, 6-12 weeks old, submitted for diagnosis of haemophilia A was measured. Eight of these puppies had values higher than would be expected for haemophiliacs, but less than the reference range for adult dogs. A further sequential study of 21 puppies (6-26 weeks of age) indicated that the factor VIII activity of puppies is generally less than that of adult dogs until about 14 weeks of age. Changes in the concentration of von Willebrand factor antigen in the puppies were irregular. These variations are probably not sufficient to interfere with accurate diagnosis of haemophilia A in most affected young dogs, but may interfere with the detection of heterozygotes in young bitches.     

Evaluation of a chromogenic assay to measure the factor Xa inhibitory activity of unfractionated heparin in canine plasma

BACKGROUND: Unfractionated heparin (UFH) has a complex pharmacologic profile that necessitates patient monitoring to prevent inadequate anticoagulation or overdosage and hemorrhage. Factor Xa inhibitory assays (to measure anti-Xa activity) are used to adjust UFH dosage and define safe and effective regimens for specific thrombotic disorders in humans. OBJECTIVE: In this study, the accuracy, linearity, and clinical utility of a chromogenic assay were assessed for monitoring UFH anti-Xa activity in canine plasma samples. METHODS: A commercial assay (Rotachrom Heparin, Diagnostica Stago, Parsippany, NJ, USA) was used to measure anti-Xa activity in canine plasma samples spiked with different concentrations of UFH. Background absorbance and assay linearity were compared for canine and human plasmas. Percentage recovery of UFH anti-Xa activity and intra- and interassay imprecisions were investigated by multiple measurements of canine plasma to which known amounts of UFH were added. The spiked plasma samples also were used to determine the heparin sensitivity of an activated partial thromboplastin time (aPTT) test. RESULTS: Canine plasma samples were assayed at a higher dilution than were human plasma samples (3:8 versus 4:8) to eliminate higher background anti-Xa activity in canine plasma. Using this modification, the recovery of anti-Xa activity in canine plasma was linear (R2 > .9) at concentrations of 0 - 0.75 U/mL UFH. Intra- and interassay imprecisions for plasma samples containing 0.5 U/mL UFH were <10%, whereas samples containing 0.25 U/mL UFH had imprecisions of 13% and 24%, respectively. The anti-Xa activity range of 0.5 - 0.75 U/mL caused prolongation of aPTTs to 1.5 - 2.5 times the assay mean. CONCLUSION: Plasma anti-Xa activity of dogs treated with UFH can be accurately monitored using this commercially available chromogenic assay.     

Effect of acepromazine, xylazine and thiopentone on factor VIII activity and von Willebrand factor antigen concentration in dogs

The effect of acepromazine maleate, xylazine and thiopentone on the packed cell volume, plasma protein content, factor VIII activity and von Willebrand factor antigen concentration of blood was studied in normal dogs. The same variables were measured in dogs with haemophilia A given acepromazine maleate and thiopentone. Both the packed cell volume and plasma protein content decreased after the administration of either acepromazine maleate or xylazine. Values were not changed further after administration of thiopentone. Changes in the haemostatic variables measured were generally small. Consequently, blood samples collected from dogs under the influence of premedicant doses of acepromazine maleate or xylazine, and when subsequently anaesthetised with thiopentone, are adequate for the assay of factor VIII activity and von Willebrand factor antigen concentration for establishing an animal's haemophilia A and von Willebrand's disease status.     

DDAVP-induced increases in coagulation factor VIII and von Willebrand factor in the plasma of conscious dogs

Infusion of the vasopressin analogue DDAVP into five normal dogs at doses of 0.1-2.0 micrograms DDAVP per kg body weight induced dose-dependent increases in the plasma content of coagulation factor VIII and von Willebrand factor. Plasma concentrations of von Willebrand factor (determined antigenically as factor VIII-related antigen and functionally as coagglutinin cofactor activity) and coagulation factor VIII were measured immediately before and at 10, 30, and 120 min after 10-min intravenous infusions of DDAVP. The greatest increases in coagulation factor VIII were produced with the 2.0 micrograms/kg dose. Ten minutes after infusion the mean increase in coagulation factor VIII was 32 units/dl (concentrations of all indices were reported relative to concentrations in a standard canine plasma pool, arbitrarily assigned a concentration of 100 units/dl) and this increase did not change significantly throughout the duration of the experiment. At 10 min post-infusion, the mean factor VIII-related antigen concentration increased 81 units/dl (dose = 2.0 micrograms/kg) and did not change significantly for the duration of the experiment. The maximum mean increase in coagglutinin cofactor activity, 141 units/dl, occurred 10 min after infusion (dose = 1.0 microgram/kg). Coagglutinin cofactor activity decreased significantly from peak activity by 120 min post-infusion.     

The effects of desmopressin on plasma factor VIII/von Willebrand factor activity in dogs with von Willebrand''s disease.

Eight unanesthetized normal dogs and seven dogs with von Willebrand's disease (vWD) were given desmopressin (0.6 micrograms/kg, IV) in order to determine the effects of this drug on plasma Factor VIII/vWF activity. Seven of the normal dogs and four of the vWD dogs were administered an equal volume of saline (control infusion) on another occasion. The other three vWD dogs underwent major surgery after treatment with desmopressin. Plasma FVIII coagulant activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), and FVIII-ristocetin co-factor activity (FVIII:RC) were quantitated before infusion and at 60 minutes postinfusion. Activities were expressed as a percentage of the activity of a pooled canine plasma (12 dogs) arbitrarily designated as having 100% FVIII:C, vWF:Ag, and FVIII:RC activity. Plasma FVIII:C activity increased by 28% in the normal dogs and by 37% in the dogs with vWD. Plasma vWF:Ag increased more than twofold in normal dogs after desmopressin treatment. In the vWD dogs the average increase was also twofold, however there was much greater variability between dogs with increases ranging from 1.2 fold to 2.4 fold. Plasma FVIII:RC activity almost doubled in normal dogs, however like vWF:Ag, the increases in vWD dogs were more variable. One vWD dog had no increase in FVIII:RC while in the remaining six dogs FVIII:RC increases ranged from 1.8 to 2.9 fold. The results of this study indicate that a single intravenous dose of desmopressin (0.6 micrograms/kg) causes a significant elevation in plasma vWF:Ag and FVIII:RC activity and a much lesser increase in FVIII:C activity in normal unanesthetized dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of desmopressin on plasma factor VIII and von Willebrand factor concentrations in Greyhounds

Objective To determine the effect of 1-Deamino-8-D-argi-nine vasopressin on plasma concentrations of von Willebrand factor and factor VIII in Greyhound blood donors, and to compare the response of 1-Deamino-8-D-arginine vaso-pressin injection on plasma concentrations of von Willebrand factor between groups with different resting plasma concentrations of von Willebrand factor.
Animals Fifteen Greyhound blood donors were used. Dogs were grouped into three categories depending on their von Willebrand factor concentrations.
Procedure Desmopressin was administered subcuta-neously at 1 mg/kg to all dogs. Plasma von Willebrand factor and factor VIII concentrations were measured before and 10, 20, 30, 45, 60, 90 and 120 min after desmopressin injection.
Results The von Willebrand factor and factor VIII concentrations in all dogs increased significantly and remained higher than base-line throughout the 2 h period.
Conclusion Desmopressin is useful in increasing von Willebrand factor concentrations in Greyhound blood donors, including those with low resting concentrations.     

哺乳动物体细胞核移植方法研究进展

1997年克隆绵羊Dolly的诞生，揭开了哺乳动物体细胞核移植的序幕。多年来该技术已应用于多种哺乳动物，并成功获得体细胞克隆绵羊、小鼠、牛、山羊、猪、猫、免和骡等。同时，核移植技术本身也得到改进，从显微注射为主的传统方法逐步向手工操作方向发展，在克隆效率相当的情况下，后者实现了方法的简单化和生产力的提高，因而备受关注。当然这些新方法只是在个别物种中获得成功，其广泛应用还有待于进一步的研究和证实。     

Determination of the enzymatic activity of trypsin and chymotrypsin using denatured 125I-labeled albumin

The method of the determination of trypsin and chymotrypsin activity by means of modified 125I-labelled albumin is described. The radioalbumin, denatured with an alkaline solution of urea, is about 20 times more sensitive to enzymatic hydrolysis than native radioalbumin. Owing to the higher sensitivity of the proposed procedure, as compared with other available techniques, this method enables accurate determination of lower enzyme concentrations than it has been possible until now. The method is suitable for the measurement of the rate of the proteolysis of raw enzymatic extracts as well as for exact kinetic measurements of purified enzymes.     

Determination of the activity of luteinizing hormone in PMSG preparations using testosterone synthesis in vitro

The activity of luteinizing hormone (LH) in serum gonadotropin (PMSG) was studied by a bioassay in vitro, using the cells of mouse testes prepared by the combination of enzymatic and mechanical diffociation. The activity of individual preparations was calculated according to the amount of produced testosterone measured by radioimmunoassay. Comparison with the 2nd international standard of PMSG (WHO) showed that in the PMSG substandard (Dessau) the activity of LH was twice lower, and in three charges of the commercial PMSG preparation (Bioveta, Ivanovice in Haná) four-and-a-half times lower than declared. No great variability of LH activity was observed among the charges of the commercial PMSG preparation.     

Assay for endogenous heparin in plasma of livestock using a synthetic chromogenic substrate

The levels of endogenous heparin in the plasmas of horses, cows, sheep and pigs were determined with the use of synthetic chromogenic substrate benzoyl-isoleucyl-glutamyl-glycyl-arginyl-p-nitroanilide (S-2222). The lowest heparin concentrations were stated in cattle plasma, the highest ones in the plasma of pigs.     

Haemophilia A (factor VIII deficiency) in a litter of Weimaraners

Inherited coagulopathies are reported in a number of dog breeds. However, to date, there is no report of Weimaraners suffering factor VIII deficiency (haemophilia A). We report the discovery of haemophilia A in both males from a single litter of Weimaraners. Haemophilia A in human beings often results from a de novo stochastic mutation. We found no evidence using currently available screening tests of haemophilia A in relatives as far back as three generations making a stochastic mutation possible in this litter.     

A combined deficiency of factor VIII and contact activation defect in a family of cats

The coagulation parameters of a litter of kittens born to an obligate carrier of haemophilia A (classical haemophilia, factor VIII deficiency) are described. Three of four kittens were found to have an intrinsic coagulation defect, but only one was haemophilic. Factor XII deficiency was confirmed in one female, the other female and the dam being carriers of the defect. A confirmed haemophilic male from a previous litter was also found to be a factor XII deficient carrier.     

Comparison of factor VIII:C and factor IX sensitivity of different commercial APTT reagents for canine plasma

In the present study, six commercial reagents for the determination of the activated partial thromboplastin time (APTT) were compared with respect to their factor VIII:C and factor IX sensitivity for measurements of canine plasma. For this purpose, plasma with different levels of factor VIII:C or factor XI activity (100, 80, 70, 60, 50, 40, 30, 20, 15, and 10% [factor VIII:C: additionally 5%]) was prepared by mixing pool plasma with plasma of dogs with haemophilia A or B. Double measurements of three different sample mixtures were carried out for each activity level. The sensitivity of the reagents was measured first based on the ratios of the coagulation time to the 100% values. In addition, the single factor activity (F VIII:C/IX(X0.975)), whose accompanying APTT corresponded to the upper limit of the reference range (97.5%-quantile, n = 50) of the respective reagent, was determined graphically. The APTT reflected a decrease of factor IX activity generally more sensitive than a reduction of factor IX activity of an identical degree. Based on ratios distant differences respecting factor VIII:C and factor IX sensitivity were found between different reagents using two way analysis of variance (p < 0.05). Significant differences between various reagents were also found with respect to the F VIII:C(X0.975) and the F IX(X0.975). These corresponded to values between 27 and 50% or 32 and 64%, respectively, dependent on the reagent. As a result, the more sensitive reagents fulfilled the demands on the sensitivity of APTT in humans. Based on the latter criterion the highest sensitivity for both factors was found for the same reagent (Pathromtin) consisting of kaolin as a contact activator and human placental phospholipid. Respecting all proofed reagents, however, no relation was found between contact activator and single factor sensitivity.     

Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays.