Characteristics of the Factor VIII Protein and Factor XIII in Various Factor VIII Concentrates

The in vitro properties of 5 factor VIII preparations (AHF-Kabi, Hemofil Hyland, AHF-Profilate Abbott, Kryobulin Immuno and Factorate High Purity Armour) and an ordinary cryoprecipitate were studied with reference to factor VIII clotting activity (VIII:C), factor VIII clotting antigen (VIILCAg), factor VIII related antigen (VIIIR:Ag) (EI, IRMA, CIE), ristocetin cofactor activity (VIIIR:RCF), fibrinogen and factor XIII. All the preparations with the exception of Factorate had higher levels of VIII:CAg than VIII:C indicating inactivation of the biological activity of VIII:C during the procedure. AHF-Kabi (fraction I-0) and the cryoprecipitate, the only preparations capable of normalising the defect in patients with von Willebrand's disease, showed the same level of VIIIR:Ag determined by EI and by IRMA, while all the other preparations (i.e. cryoprecipitates purified further in different ways) had considerably lower levels of VIIIR:Ag determined by IRMA than by EL Based on these in vitro techniques it seems to be possible to predict which preparations can be used successfully in patients with von Willebrand's disease, while no such conclusions can be made from VIIIR:RCF determinations. EI yielded similar concentrations of factor XIII a subunit in all the preparations tested. 3 functional assays showed high factor XIII activities in AHF-Kabi but low or no activities in the others. Thus, considerable differences were found of the in vitro properties of the proteins in 5 factor VIII concentrates and a cryoprecipitate. The action of proteases and the techniques used in the purification procedure are probably of crucial importance for the properties of the various factors.     

Discrepant in vivo recovery of factor VIII:C following infusion of two different monoclonal factor VIII preparations

Background: We experienced decreased recovery of factor VIII:C after commercial monoclonal factor VIII infusions. We report the recovery data obtained from infusing two brands of monoclonal factor VIII.Methods: Factor VIII:C activity was measured before and after an infusion of one of two monoclonal factor VIII preparations. The increments were calculated and expressed as a function of units of factor VIII administered per kilogram.Results: Monoclate increments averaged 2.3% (range: 1.6–3.2%). Antihemophilic factor (human) method M (AHFM) yielded an average increment of 1.4% (range: 1–1.7%).Conclusions: The two monoclonal factor VIII preparations used are not equivalent in activity. In switching brands, one must recalculate the prescribed dose based on the institution's experience in recovery of VIII:C from the particular brand to be prescribed. Cost calculations and comparisons should be based on the recovered factor VIII:C increments rather the unit purchase price.     

Fatal Iron Intoxication with Multiple Coagulation Defects and Degradation of Factor VIII and Factor XIII

A severe iron intoxication in a 15-year-old girl resulted in profound damage to vital organs and multiple clotting defects, but no haemorrhagic diathesis. Investigation revealed not only impaired synthesis of coagulation factors but also abnormal proteo-lysis by plasmin as well as by other proteolytic enzymes liberated from leucocytes and damaged tissue cells affecting especially factor VIII and factor XIII.     

Factor VIII, ABO blood group and the incidence of ischaemic heart disease

Summary. Relations of factor VIII activity, FVIIIC, and von Willebrand factor antigen (vWFAg), with ischaemic heart disease (IHD) were examined in 1393 men aged between 40 and 64 years at entry to the Northwick Park Heart Study (NPHS) who experienced 178 first major episodes of IHD during an average follow-up period of 16.1 years. After allowing for the large factor VIII differences between the main ABO blood groups, FVIIIC was probably associated with IHD incidence, possibly more strongly with fatal than non-fatal episodes. Thus, an increase of 1 standard deviation in FVIIIC raised the risk of fatal IHD by about 28%. vWFAg was also significantly associated with fatal events. The observed relation of FVIIIC with IHD incidence probably underestimates the true strength of the association because of the considerable within-person and laboratory variability in factor VIII measurements. FVIIIC and vWFAg were strongly correlated ( r = 0.57) and in statistical terms there may be little to choose between them in long-term studies of IHD. Taking account of evidence that haemophiliacs seem to experience less IHD than expected, high factor VIII levels may contribute to the incidence of IHD by increasing thrombogenic potential. The incidence of IHD was significantly higher in those of blood group AB than in those of groups O, A or B, particularly for fatal events. There was no evidence that the FVIIIC and vWFAg associations with IHD are determined by ABO group. The factor VIII and ABO blood group effects therefore appeared to be independent. Group AB may be a genetic marker of characteristics influencing other indices of IHD risk such as short stature, NPHS men (though not women) of group AB being about 2 cm shorter than those of other groups.     

Potency and mass of factor VIII in FVIII products

Summary.  Several factor (F) VIII products of different origin and structure are being used for haemophilia A treatment worldwide. The assessment of FVIII concentration in these products is done using activity assays, which are dependent upon the assay and its modifications. To evaluate FVIII products for potency and for FVIII concentration and specific activity, three activity-based assays [activated partial thromboplastin time (APTT), intrinsic FXase and synthetic coagulation proteome] and two immunoassays (ELISA and western blotting) were used in this study with albumin-free full-length recombinant (r) FVIII as a standard. In all activity assays, products A and B (both contain full-length rFVIII) at 1 U mL⁻¹ showed potency similar to that of the 0.7 n m (1 U mL⁻¹) rFVIII standard. Product E (contains truncated rFVIII) was less potent in the APTT (83% of standard) and product C (contains plasma FVIII) was less potent in FXase assays (66%). The ELISA immunoassay revealed that the specific activity of FVIII proteins in products A–C and E varied over a wide range (3900–13 200 U mg⁻¹) and was higher for most lots when compared with the standard (5000 U mg⁻¹), whereas the specific activity of product D (contains plasma FVIII) was lower than expected (3200–4800 U mg⁻¹). (i) FVIII potency estimated in different assays gives dissimilar results; (ii) the specific activity of FVIII in various FVIII products is different and inconsistent. Thus, the administration of an equal FVIII potency in units means the administration of different amounts of FVIII protein, which may partly explain apparent discrepancies in product performance.     

Factor VIII inhibitors: in vivo decrease of inhibitory activity during calcium infusion

The effect of parenteral calcium on altering the activity of factor VIII inhibitors has been studied in three patients, two nonhemophiliacs and one hemophiliac. The patients were studied during both intravenous calcium infusion, factor VIII replacement, and combinations thereof. When compared to factor VIII replacement alone, a greater diminution of inhibitory activity was noted whenever calcium was infused prior to and during factor VIII replacement. This was demonstrated by both conventional coagulation assays and an agarose gel method. These observations could have therapeutic implications and may aid in further understanding the relationship of calcium to factor VIII and its inhibitor.     

Factor VIII Concentrate Prepared from DDAVP Stimulated Blood Donor Plasma

Human fraction 1-0 (AHF-Kabi) was prepared from plasma from blood donors who had received an i.v. injection of DDAVP (0.2 μg per kg b.w.) and tranexamic acid (0.01 g per kg b.w.) 15 min before collection of the blood. The factor VIII preparation from such plasma contained twice as much VIII:C, VIIIR:Ag, and VIIIR:RFC as normal fraction 1-0. Normal fraction 1-0 and DDAVP fraction 1-0 were given to 2 patients with severe haemophilia A. The in vivo response of the DDAVP fraction 1-0 corresponded to the in vitro values. No differences in survival time were seen. Hence, it is possible to produce factor VIII concentrates with at least double the yield by increasing the factor VIII level in blood donors by i.v. injection of DDAVP.     

Activation profiles of factor VIII in concentrates reflect one-stage/chromogenic potency discrepancies

We have investigated the possibility that differences in the profile of factor VIII (FVIII) activation, by thrombin, may help to explain the one-stage/chromogenic potency discrepancies in two therapeutic concentrates. A Method M concentrate and a recombinant B-domain-deleted (B-DD) concentrate were found to have one-stage/chromogenic ratios of approximately 1.15 and 0.70, respectively, relative to the World Health Organization (WHO) 6th International Standard (IS) FVIII concentrate, whether pre-diluted in FVIII-deficient plasma or buffer (+/- von Willebrand factor, VWF). The activation of FVIII, by thrombin, was followed in a buffer medium (+/- VWF) and all three concentrates showed similar times to reach peak FVIII coagulation (FVIII:C) activity. However, despite the use of equivalent amounts of FVIII:C for all three concentrates, the B-DD concentrate reached a higher peak level and maintained higher FVIII:C compared with the WHO 6th IS throughout the incubation period, whereas the Method M concentrate reached a lower peak level and maintained lower FVIII:C throughout the incubation period. We propose that the higher levels of FVIII:C found with the B-DD concentrate and the lower levels with the Method M concentrate, following activation, may be reflected in the potencies obtained by the chromogenic method and may be consistent with one-stage/chromogenic ratios of < 1.0 and > 1.0 respectively.     

Anticoagulant thrombins

Thrombin plays both procoagulant and anticoagulant roles in the blood coagulation cascade. This dual role is influenced allosterically by the binding of Na⁺ near the primary specificity pocket of the enzyme. Recent findings demonstrate that it is possible to engineer recombinant thrombins that have practically lost anticoagulant activity but retain their anticoagulant properties. These anticoagulant thrombins bear substitutions in or around the Na⁺ binding site, provide important clues on structure–function relations, and offer a promising alternative to current anticoagulant therapies. In addition, they demonstrate the importance of Na⁺ as a coagulation factor and broaden our understanding of the function and regulation of all vitamin K–dependent clotting enzymes.     

Factor VIII Inhibitor Postpartum

Acquired factor VIII deficiency in women postpartum due to a factor VIII inhibitor is rare and the etiology is unknown. In this study a case report and a review of the literature are given. The haemorrhagic diathesis resembles classic haemophilia, with the exception that ecchymoses and tissue bleeding occur more frequently. The potency of the inhibitor may vary from weak to strong and the inactivation of factor VIII coagulant activity (factor VIII-C) by the inhibitor is of a non-linear type. Severe bleeding has been fatal in a few cases, but factor VIII concentrate substitution has usually been successful without anamnestic response of inhibitor activity. There is no convincing evidence that immunosuppression is effective, also because the natural history of the disease is characterised by a spontaneous disappearance of the factor VIII-C inhibitor. Treatment of bleeding symptoms with factor VIII concentrate should therefore not be reserved for life threatening haemorrhages only.     

Factor VIII inhibitor in insulin-dependent diabetes mellitus

Acquired factor VIII inhibitor was found in a 69-year-old white male with insulin-dependent diabetes mellitus. He presented with left lower abdominal pain and hematoma after a fall. Preoperative hemostasis studies were normal except for prolonged aPTT. Prolonged aPTT was not corrected by 1:1 mixture with normal fresh plasma and incubation showed further prolongation with time. Factor VIII:c was 3.5%. The inhibitor titer was 7.5 Bethesda units. The possible mechanism causing antibody to factor VIII was postulated to be an autoimmune process and/or increased immunogenicity owing to glycosylation of factor VIII coagulant protein.     

Molecular defects in coagulation Factor VIII and their impact on Factor VIII function

Molecular defects in Factor VIII (FVIII), such as haemophilia A-related mutations or denaturative conformational changes, may affect the stability of FVIII as well as its interactions with physiological activators, von Willebrand Factor, phospholipid, or conformationally sensitive antibodies. We summarize the contemporary assays which allow identification of impaired functional interactions of FVIII that cause a reduction or loss of its cofactor activity and/or increased immunogenicity. These assays can potentially be used for detection of molecular defects in FVIII and elucidation of the function impaired by these defects.     

The Release of Plasminogen Activator and Factor VIII after Injection of DDAVP in Healthy Volunteers and in Patients with von Willebrand's Disease

Increases in plasma concentrations of VIII:C, VIII:CAg, VIIIR:Ag and plasminogen activator (PA) were observed in 50 healthy volunteers given i.v. injections of DDAVP (desaminocys¹-8-D-arg-vasopressin). The PA activity reached its maximum immediately after the injection, VIII:C, VIII:CAg and VIIIR:Ag after 3040 min. However, a positive correlation was found when the PA and VIII:C responses in each of the normals were analysed. DDAVP was also administered to 3 patients with severe von Willebrand's disease. 2 of the patients displayed no changes in VIII:C, VIII:CAg, VIIIR:Ag or VIIIR:RCF and there was no increase in PA. The third patient responded with an increase in VIII:C and to a minor degree in VIII:CAg. This patient developed fibrinolytic activity, but in the lower normal range. In 3 other patients with mild von Willebrand's disease DDAVP caused increases in VIII:C, VIII:CAg, VIIIR:Ag and PA. We feel that the combined data may support the concept that one and the same target cell is involved in the DDAVP mediated release of factor VIII related activities and PA.     

Factor VIII and transmissible spongiform encephalopathy: the case for safety

Haemophilia A is the most common inherited bleeding disorder, caused by a deficiency in coagulation factor VIII (FVIII). Current treatment of haemophilia A is based on repeated infusions of plasma-derived FVIII concentrate or of recombinant FVIII, which may be exposed to plasma-derived material of human or animal origin used in its tissue culture production process. We review epidemiological and experimental studies relevant to blood infectivity in the transmissible spongiform encephalopathies (TSEs, or 'prion' diseases), and evaluate the hypothetical risk of TSE transmission through treatment with plasma-derived or recombinant FVIII.     

Human parvovirus PARV4 in clotting factor VIII concentrates

BACKGROUND AND OBJECTIVES: Parvoviruses are small non-enveloped DNA viruses, relatively resistant to virus inactivation procedures. The recently identified human parvovirus PARV4, including a related genotype 2 virus (also termed PARV5), has been found to be a contaminant of pooled plasma used in the manufacture of plasma-derived products. This report describes an investigation to determine whether PARV4 is present in clotting factor concentrates. MATERIALS AND METHODS: Factor VIII concentrates manufactured in the past 30-35 years were screened for PARV4 and human parvovirus B19 (B19V) sequences. Viral loads in products testing positive for PARV4 were quantified using a consensus TaqMan assay designed to a highly conserved region. DNA sequence analysis was performed to confirm the genotypes present. RESULTS: From a total of 175 lots of factor VIII concentrate, 28 of these contained PARV4 sequences, and in two lots both genotypes 1 and 2 were found to be present. The highest viral loads observed exceeded 10(5) copies per ml. The majority of factor VIII concentrates testing positive for PARV4 were manufactured in the 1970s and 1980s. Human B19V was also a frequent contaminant of these products. CONCLUSIONS: PARV4 was detected in 16% of factor VIII concentrates, particularly in older batches from the 1970s and 1980s. The significance in terms of the viral safety and potential transmission to recipients of these products is not yet known.     

Factor VIII inhibitors in haemophiliacs: a single-centre experience over 34 years, 1964-97

A retrospective study of the natural history of factor VIII inhibitors in haemophilia A patients experienced in a single comprehensive haemophilia centre over three decades is reported. 431 haemophilia A patients of all severities have been followed-up for a total of 5626 patient-years. The frequency of inhibitors was 10% in the severe haemophilia A patients and 37% occurred in children <10 years. The majority of the patients received several products before developing the inhibitors. 59% of patients had <50 exposure days and 48% were high responders (>5 BU). An 8-year (1987-95) inhibitor-free period was seen during which all previously untreated patients were treated with an intermediate-purity factor VIII concentrate. A moderate haemophiliac with a missense mutation that has not been described in association with inhibitor is reported. Six HIV-positive patients preserved their antibody response to factor VIII even at the advanced stage of their disease.