The type of factor VIII deficient plasma used influences the performance of the Nijmegen modification of the Bethesda assay for factor VIII inhibitors.

We have investigated the influence of the type of factor VIII deficient plasma used on the assay results of the Nijmegen modification of the Bethesda method for factor VIII inhibitors. Immuno depleted factor VIII deficient plasmas, lacking besides factor VIII also von Willebrand factor, gave decreased inhibitor titres compared to assay results with factor VIII deficient plasmas containing von Willebrand factor suggesting the need of the latter in the test system for the stability of factor VIII:C. Moreover the performance of the assay with immuno depleted plasma was contaminated in a certain type of this plasma by the presence of a factor VIII:C inhibitor. Chemically depleted factor VIII deficient plasma appeared to give falsely elevated titres when used in combination with other types of deficient plasmas as substrate plasma in the factor VIII:C assay due to the presence of activated factor Va in the preparation. Suggestions are described with respect to the observed limitations in order to obtain reliable results.     

An effect of predilution on VIII:C determination by the one-stage assay

Factor VIII concentrates are usually prediluted in Owrens buffer, before use of the one-stage assay for VIII:C determinations. We found that predilution of high purity Factor VIII concentrates in fresh VIII:C deficient plasma gave VIII:C estimates about 3 times higher than predilution in Owrens buffer. Predilution in reconstituted lyophilized VIII:C deficient plasma gave VIII:C estimates about 1.6 times higher. Likewise the VIII:C in a plasma sample can be estimated to e.g. 0.03, 0.06 or 0.10 IU/ml depending on whether the reference curve is established by predilution of the reference plasma in fresh VIII:C deficient plasma, in reconstituted lyophilized VIII:C deficient plasma or in Owrens buffer, respectively. It is shown that the major part of the differences between the effects of the various prediluents on the VIII:C determinations, can be accounted for by differences in the content of Factor V, Fibrinogen, and vitamin K dependent coagulation factors in the assay mixtures.     

Standardization of Factor VIII-III. Establishment of a stable reference plasma for Factor VIII-related activities

An international collaborative study has been carried out to establish a reference plasma for Factor VIII-related activities. The freeze-dried reference plasma, coded 80/511, was assayed against fresh normal plasma, local standards and another freeze-dried plasma. There was good agreement between laboratories for the comparison of the two freeze-dried plasmas, but wide variation in the comparison of plasma 80/511 with fresh normal plasma and local standards, indicating the differences in Factor VIII content of local pooled plasmas. There were no significant differences between the one-stage and two-stage assays of VIII:C, or between electroimmunoassay (EIA) and immuno-radiometric (IRMA) assays of VIII:Ag. However, in VIII R:RCoF (ristocetin co-factor) assays, the aggregometry methods gave lower values than the macroscopic and counting methods for the comparison of freeze-dried against fresh normal plasmas. From the combined results of assays against each laboratory's fresh normal plasma, potencies were assigned to plasma 80/511 as follows: VIII:C 0.73 VIIIR:Ag 0.87 International Units VIIIR:RCoF 0.80 per ampoule VIIIC:Ag 0.95. Results from accelerated degradation studies indicated that losses of each VIII-related activity in plasma 80/511, when stored at -20 degrees C, should be less than 0.01% per year, indicating its suitability to serve as a long-term reference preparation. Plasma 80/511 has been established by the WHO Expert Committee on Biological Standardization as the 1st International Reference Preparation for Factor VIII-Related Activities in Plasma.     

Coagulation activities of plasma microparticles

An evaluation of the effect of plasma microparticles (MP) on in vitro coagulation has been undertaken using platelet rich (PRP), platelet poor (PPP) and platelet free (PFP) plasmas prepared by differential centrifugation. MP provide coagulant material which shortens the activated partial thromboplastin time (APTT) and dilute simplastin time (DSTT) which is different from that contributed by commercial phospholipid preparations. The amount of platelet factor three (PF3) available in plasma is directly correlated with the centrifugal force used in its preparation and is present in large amounts in the MP pellet remaining after preparation of PFP. Factor VIII (F.VIII:C) and von Willebrand factor (vWf) were associated with the MP fraction but could be separated from MP on sucrose gradients. The effect of MP on the APTT was independent of the F.VIII:C/vWf and was not solely due to their PF3 content. Plasma prepared for routine coagulation assays contains MP which contribute to the APTT and DSTT and should be considered in their assessment. High speed centrifugation of plasma reduces the F.VIII:C/vWf:Ag/RCoF levels and this may contribute to losses of these proteins during preparative procedures utilising high speed centrifugation.     

Recombinant factor VIIa does not induce hypercoagulability in vitro

Recombinant factor VIIa (rVIIa) has been reported to be clinically effective and safe in haemophilic patients with inhibitor antibodies. Compared to activated prothrombin complex concentrates the risk of thrombotic complications seems to be very low after rVIIa administration. Determination of free thrombin generation has been shown to identify hypercoagulability. Therefore, free thrombin and prothrombinase activity (Xa generation) were assessed after extrinsic activation of rVIIa supplemented factor VIII and factor IX deficient plasma. Free thrombin generation was also determined after supplementation of (activated) prothrombin complex concentrates. Addition of 150 U rVIIa/ml shortened the clotting times markedly in control, factor VIII, and factor IX deficient plasma. In contrast, free thrombin and Xa generation were not different in the absence or presence of 150 U rVIIa/ml. Addition of (activated) prothrombin complex concentrates resulted in a marked increase of free thrombin generation in all investigated plasmas. Although in vitro studies cannot reflect specific clinical circumstances our results support the notion that rVIIa does not induce a hypercoagulable state as sporadically observed after administration of (activated) prothrombin complex concentrates.     

An effect of predilution on potency assays of factor VIII concentrates

The effect of prediluent on potency assays for Factor VIII concentrates was evaluated systematically using a well defined one stage Factor VIII assay. It was demonstrated that potency values were higher when Factor VIII deficient plasma was used as the prediluent compared to identical assays performed when barbital buffered saline solution was used as prediluent. Prediluent is therefore an important variable to evaluate in establishing the validity of any proposed Factor VIII potency assay. Since the goal of Factor VIII potency measurement is to predict therapeutic effect, the use of Factor VIII deficient plasma is the more satisfactory alternative where a difference exists, since this diluent approximates best the conditions which occur when a Factor VIII concentrate is injected into the bloodstream of a patient with hemophilia A.     

Contact factors are responsible for the thrombogenicity of prothrombin complex.

In order to determine the factors responsible for the thrombogenicity of prothrombin complex (PC), preparations of PC were prepared from normal plasma and from plasmas deficient in Factor XII, Factor XI, prekallikrein and high molecular weight kininogen. PC isolated from normal or Factor XI deficient plasma was thrombogenic unless the plasma was treated with kaolin prior to the isolation procedure. PC isolated from plasmas deficient in Factor XII, prekallikrein, or high molecular weight kininogen were not thrombogenic even when the kaolin adsorption step was omitted. These results indicate that Factor XIIa activation of prekallikrein is the initial reaction responsible for the thrombogenicity of PC.     

Novel factor V C2-domain mutation (R2074H) in two families with factor V deficiency and bleeding

The molecular basis of Factor V deficiency has been defined in few patients only. We report a homozygous nucleotide change (G6395A) in two Tunisian probands with Factor V deficiency and bleeding episodes. This substitution results in the replacement of an arginine (R) by a histidine (H) in amino acid position 2074, located in the Factor V C2-domain. Mutations in this protein domain have not previously been described. Several lines of evidence support that this sequence variant is indeed disease causing: 1) Crystal structures of Factor V and molecular C2-domain modeling studies of H2074 suggest that the conserved R2074 is required for correct folding; 2) Structure-function studies of selective Factor V mutants (R2074A) demonstrate the importance of R2074 for structural stability of the Factor V C2-domain and for cofactor activity (1); 3) In Factor VIII, point mutations in codon 2209, which corresponds to position 2074 in Factor V, cause hemophilia A.     

The formation of a thrombin-like material (TLM) following stimulation of leukocytes

When thrombin, tissue thromboplastin or Russell's viper venom was added to a suspension of either lymphocytes or neutrophils containing normal plasma, aggregation of these cells ensued. The aggregate formed one gelatinous mass which was readily separable from the cell free supernatant, an aliquot of which caused platelet aggregation. This leukocyte derived platelet aggregatory substance had characteristics similar to thrombin but not AGEPC. When plasma deficient in Factor V was substituted for normal plasma, the platelet stimulatory substance was not produced. Substitution with plasma deficient in Factor VII, VIII, IX, X or XI was without effect. Thrombin clotting time measurements indicated a generation of activity, relative to thrombin, of about 3.0 U/5 X 10(6) cells.     

Freeze-dried whole plasma: evaluating sucrose, trehalose, sorbitol, mannitol and glycine as stabilizers

Several groups report stability results for freeze-dried whole plasma intended for use as a transfusion product [Hellstern P, Sachse H, Schwinn H, Oberfrank K. Manufacture and in vitro characterization of a solvent/detergent-treated human plasma. Vox Sang 1992;63:178-185; Trobisch H. Results of a quality-control study of lyophilized pooled plasmas which have been virally inactivated using a solvent detergent method (modified Horowitz procedure). Beitr Infusionsther 1991;28:92-109; Hugler P, Trobish H, Neuman H, Moller, Sirtl C, Derdak M, Laubenthal H. Quality control of three different conventional fresh-frozen plasma preparations and one new virus-inactivated lyophilized pooled plasma preparation. Klin Wochenschr 1991;69:157-161; Krutvacho T, Chuansumrit A, Isarangkura P, Pintadit P, Hathirat P, Chiewsilp P. Response of hemophilia with bleeding to fresh dry plasma. Southeast Asian J Trop Med Public Health 1993;24:169-173; Chuansumrit A, Krasaesub S, Angchaisuksiri P, Hathirat P, Isarangkura P. Survival analysis of patients with haemophilia at the International Haemophilia Training Centre, Bangkok, Thailand. Haemophilia 2004;10:542-549]. Plasma coagulation properties are substantially impaired in these freeze-dried plasmas, while pH levels are close to alkaline. In this work, plasma supplemented with 60mM sucrose, trehalose, mannitol, sorbitol or glycine was freeze-dried. The samples were subjected to forced degradation at 40 degrees C for 10 days in order to quickly evaluate the effectiveness of the different stabilizers. Initial PT, APTT and TT values were 14.4+/-0. 5s, 31.4+/-1.5s and 18.3+/-0.6s, respectively. At the end of the degradation period, PT, APTT and TT were substantially prolonged, and were 19.1+/-0. 5s, 43.1+/-0.6s and 26.1+/-1.0s, respectively. In the presence of glycine, at the end of the degradation period, PT, APTT and TT values remained close to the initial values and were 15.5+/-0. 4s, 35.7+/-0.9s and 19.4+/-0.2s, respectively. Percent activities of the coagulation factors V, VII, VIII, IX, X and the coagulation inhibitors protein C, protein S and antithrombin III were recorded. Factors V and VIII were most prone to degradation. Factor V and VIII activities, in control plasma, were approx. 44+/-3.5% and 58+/-2.3%, at the end of storage. In contrast, much higher factor V and VIII activities were maintained in the lyophilized glycine-supplemented plasma: approx. 60+/-3.5% and 74+/-7.0%, correspondingly. The most stable protein was protein C, which showed no signs of degradation under the testing conditions of this study. All tested stabilizers provided protection. Glycine, however, outperformed all tested polyols, providing superior preservation of plasma clotting properties. Thermograms of 60mM glycine in water and 60mM glycine in plasma show that, in the presence of plasma, glycine does not crystallize. The process of freeze-drying caused a complete loss of plasma pCO(2) (gas) and a substantial increase in plasma pH. Citric acid was found to be a suitable pH adjuster for lyophilized/rehydrated plasma.     

Regulation of bovine activated protein C by protein S: the role of the cofactor protein in species specificity

The anticoagulant activity of bovine activated Protein C was observed to exhibit species specificity when tested in the plasmas from cow, dog, rabbit, and human. The relative effectiveness in descending order was cow, dog, human, and rabbit. When bovine Protein S was added to each of these plasmas, the species specificity of bovine activated Protein C was lost. No activated Protein C cofactor activity could be detected in either rabbit or human plasma. Bovine activated Protein C enhanced the rate of inactivation of both rabbit and human Factor Va in serum. The rate of activated Protein C catalyzed inactivation of rabbit and human Factor Va could be enhanced by the addition of bovine Protein S. These results indicated that the species specificity of the anticoagulant activity of bovine activated Protein C is mediated by a cofactorenzyme interaction rather than an enzyme-substrate interaction. These results further demonstrated that the anticoagulant activity of activated Protein C is due, in part, to the inactivation of Factor Va.     

Activation of human plasma prekallikrein: Influence of activators,activation time and temperature and inhibitors

Plasma prekallikrein (PKK) was activated with Cephotest and dextran sulphate (DS) under various conditions and the resulting kallikrein (KK) assayed on a chromogenic peptide substrate for this enzyme. Cephotest: plasma ratios of 30:1 and DS concentrations of between 0.001 and 0.01% (equal volumes plasma and DS) gave maximal activities when activation proceeded at 0°C. However, enzyme activity was also dependent on incubation time and temperature and activation times of 3 minutes (Cephotest) and 15 minutes (DS) at 0°C gave highest activities. Incubation at 37°C gave progressively lower KK yields with Cephotest and very little KK activity was detected with DS at this temperature. These results were shown to be due to progressive inhibition of plasma KK at 37°C. With C¹-esterase inhibitor deficient plasma higher KK activities were obtained with both activators at 0°C and the loss of activity on incubation at 37°C was markedly reduced. The addition of pure C¹-esterase inhibitor to the deficient plasma prior to activation resulted in a normal activity and normal inhibition profile. Gel filtration studies on normal and C¹-esterase deficient plasmas after activation at 0°C with Cephotest revealed that both free KK and KK-α₂ macroglobulin complexes were present in the activated plasmas. In the deficient plasma both activities were higher indicating that in normal plasma some KK is immediately bound by α₂-macroglobulin and C¹-esterase inhibitor even when activation takes place at 0°C.     

Rapid isolation and purification of antibody to factor VIII by protein A

A simple and reproducible method has been developed for rapidly isolating and purifying antibodies to Factor VIII from the plasma of both haemophilic and non-haemophilic patients. The one-step separation of these immunoglobulins makes use of the high affinity binding of protein A with the Fc region of the human lgG1, 2 and 4 subclasses but not with the lgG3 subclass. Small protein A-Sepharose CL-4B gel columns are used to isolate the lgG subclasses which contain the anti-Factor VIII activity from as little as 0.5 ml of plasma. The yield of the antibody is 70–80%. When the purification procedure is combined with a solid phase agarose gel assay for antibody to Factor VIII, a large number of samples can be tested whilst only small amounts of patient's plasma are required. The plasma of a patient with antibody to Factor V was also fractioned by protein A gel with similar results.     

The Factor V (Leiden) test: evaluation of an assay based on dilute Russell Viper Venom Time for the detection of the Factor V Leiden mutation

In the present study a new clotting assay for the detection of an increased resistance of coagulation factor V against degradation by activated protein C (Factor V Leiden mutation, FVLM) was evaluated. The Factor V (Leiden) Test (Gradipore, North Ryde NSW, Australia) is based on the dilute Russell Viper Venom Time (DRVVT), which is prolonged when the plasma sample is preincubated with dilute whole Agkistrodon contortrix contortrix venom for activation of protein C (PC). In contrast to the DRVVT based global assay, Protein C Pathway Test (Gradipore, North Ryde NSW, Australia) this new assay is expected to be more specific for FVLM because of optimized amounts of the venom. The test result is expressed as the ratio between the DRVVT with and without addition of the venom. The following precision values were found: intraassay coefficient of variation (CV): 5.53% (n=20) in the normal range, 4.30% (n=20) in the pathological range; interassay CV: 6.90% (n=10) and 7.64% (n=10), respectively. A normal range (5th to 95th percentile) of 2.12 to 3.08 was calculated from 50 healthy controls. A ratio below 2.12 was found in all samples from patients with FVLM (n=21), in 9 of 12 patients with PC, in 0 of 6 with protein S (PS), and in 0 of 4 with antithrombin (AT) deficiency. There was, however, a good discrimination between carriers of the FVLM (highest ratio 1.44) and patients deficient in PC (lowest ratio 1.59), in particular when samples were prediluted with factor V deficient plasma FVDP (1.16 vs. 1.96, respectively). Predilution of samples with FVDP caused a clear discrimination between controls and patients deficient in PC, PS, AT, and FVLM-positive individuals and also in patients on oral anticoagulant treatment. Our data show that the Factor V (Leiden) Test discriminates well between carriers of the FVLM and healthy controls or patients deficient in PC, PS, and AT. Individuals presenting values between the lower cutoff of controls and the range in which FVLM-positive individuals are found are highly suspicious for protein C deficiency.     

Protein C activity levels in endotoxin-induced disseminated intravascular coagulation in a dog model

The levels of protein C (PC) and other coagulation factors were monitored during endotoxin-induced disseminated intravascular coagulation (DIC) in the dog. Initial evaluation of the effectiveness of intradermal administration of bolus endotoxin quantities into the dog, demonstrated induction of DIC in the canine, without the severe side effects associated with bacterial sepsis. Quantitative determination of canine plasma protein C levels were performed using a multiple step amidolytic assay, that included a specific precipitation of the vitamin K-dependent proteins from citrated plasma, followed by thrombin activation (and neutralization) and subsequent measurement of the activated protein C (APC) by chromogen hydrolysis. This investigation demonstrated, that over a twenty-four hour interval, intradermal administration of endotoxin produces a gradual decrease in the PC activity levels, concomitant with a significant reduction in the Factor V, Factor VIII and fibrinogen levels and platelet count, and a prolongation of the Prothrombin Time and Partial Thromboplastin Time. During the first 6 hours, protein C levels fell below the pre-levels and remained significantly lower in the surviving dogs. Thus, this endotoxin-induced DIC animal model permits evaluation of various hemostatic parameters, yet diminishes the severe clinical findings associated with DIC.     

Absence of blocking antibody in non-inhibitor haemophilic plasma.

This study examined the hypothesis that non-inhibitor haemophilic plasma contains antibodies which are specific for sites other than the active procoagulatn site on factor VIII, and that some of them might be sufficiently close to the active site that pre-incubation of such plasma with factor VIII would block the subsequent binding of inhibitor antibody. Among the 26 non-inhibitor plasmas examined, none was found to contain such blocking antibody. This result does not eliminate the possibility that antibody is present in such non-inhibitor plasmas which is neither specific for the active enzyme site of factor VIII nor capable of blocking the binding of antibody which does have that specificity.     

Approaches to the measurement of the in vivo response to factor VIII concentrate infusion

The in vivo response to the infusion of 12 lots of Factor VIII Concentrate was determined in a five Centre collaborative study. The principal aim of the study was to compare the responses obtained, after infusion into a single haemophiliac, of several lots of Factor VIII Concentrate made by a newly licensed process with those obtained after the infusion of several lots of Factor VIII Concentrate made by a previous process. Factor VIII Concentrates made by two other manufacturers were also evaluated. There was considerable inter-laboratory variation in the results obtained for the Factor VIII:C content of both the Factor VIII Concentrates and the ex vivo plasma samples. However, on pooling the data, the mean in vivo recovery was not statistically different from the expected response based on the potency assigned by the manufacturers. These data indicate that the results from a single laboratory may not be reliable in ascertaining either the in vitro or the in vivo potency of a particular lot of Factor VIII Concentrate and suggest that in the absence of a Factor VIII inhibitor, the in vitro and in vivo potency of a particular lot of Factor VIII Concentrate should be ascertained by several laboratories and the results pooled. Such pooled information then can be used to predict reliably the in vivo recovery of factor VIII: C activity without resorting to a formal in vivo response study.     

Treatment of hereditary protein C deficiency with stanozolol

Five type I protein C deficient male patients received 5 mg stanozolol b.i.d. during 4 weeks. After four weeks of treatment plasma protein C activity increased from 0.42 to 0.74 U/ml and protein C antigen from 0.49 to 0.75 U/ml. This approximately 1.6 fold increase in plasma protein C was accompanied by an increase in factor II antigen (1.5 fold), factor V activity (1.6 fold), factor X antigen (1.1 fold), antithrombin III antigen (1.3 fold) and heparin cofactor II antigen (1.5 fold), while the concentration of factor VII, factor VIII, and factor IX activity, and of protein S antigen remained unchanged. Prothrombin fragment F1+2, measured in two patients, increased 1.3 fold. In addition to its effect on procoagulant and anticoagulant factors stanozolol had profibrinolytic effects, reflected in an increase in tPA activity and in the concentration of plasminogen. These data indicate that in type I protein C deficient patients stanozolol increases the concentrations of both procoagulant and anticoagulant factors and favours fibrinolysis. The efficacy of stanozolol in preventing thrombotic disease in type I protein C deficient patients, however, remains to be established. During the four weeks of stanozolol treatment no thrombotic manifestations were observed in the protein C deficient patients.     

Clinical application of a chromogenic substrate method for determination of factor VIII activity

A chromogenic substrate kit for the determination of factor VIII activity (COATEST Factor VIII) has been evaluated in five different laboratories, one of them using a semi-automated procedure. This chromogenic method was compared to one-stage clotting assays for factor VIII determination in plasmas from healthy subjects, carriers of hemophilia A, severe, mild and moderate hemophilia A as well as von Willebrand's patients. In all these cases, a high correlation between these two methods was obtained (r = 0.96-0.99, n = 385) with a good agreement of the assigned potencies at all levels of factor VIII. A good correlation (r = 0.94) was also obtained for the levels of factor VIII after infusion of concentrates in six severe hemophiliacs or after administration of DDAVP to von Willebrand's patients. The chromogenic method is insensitive to preactivation of factor VIII by thrombin, thus yielding valid potency assignments also in these situations. The precision was higher with the chromogenic method than with the one-stage clotting assays (C.V. = 2-5% vs 4-15%). Altogether, the new chromogenic substrate method has proven itself suitable for determination of factor VIII in plasma and concentrates.