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PubMed1977

Factor VIII coagulant activity and factor VIII-related antigen released from isolated perfused human spleens.

Eight human spleens were perfused for up to 65 h at normothermia and the coagulant Factor VIII activity measured in the perfusate. In addition, in three experiments Factor VIII-related antigen was determined in the perfusate. Although the spleens were pathologically enlarged and the normal structure involved by different diseases, all spleens released Factor VIII coagulant activity and Factor VIII-related antigen. On average the total amount of Factor VIII coagulant activity released was equivalent to that of 3.5 l of human plasma.

Antigens↗
PubMed1989

Intersubunit fluorescence energy transfer in human factor VIII.

Human factor VIII circulates as a series of active heterodimers composed of a light chain (83 kDa) linked by divalent metal ion(s) to a variable sized heavy chain (93-210 kDa). Purified factor VIII subunits were modified with sulfhydryl-specific fluorophores. Probe selection was based upon the limited number of free cysteine residues in each subunit. Levels of probe incorporation suggested the presence of a single reactive cysteine residue per subunit. Amino-terminal sequence analysis of fluorescent tryptic peptides derived from the modified subunits indicated fluorophore attachment sites at Cys528 of the heavy chain (A2 domain) and Cys1858 of the light chain (A3 domain). Subunit reassociation was measured by fluorescence energy transfer using light chain modified with N-[1-pyrenyl] maleimide (fluorescence donor) and heavy chain modified with 7-diethylamino-3-[4'-maleimidophenyl]-4-methylcoumarin (fluorescence acceptor). Donor fluorescence quenching paralleled the formation of factor VIII clotting activity, and both effects were saturable with respect to added heavy chain. Based upon the degree of donor quenching, a distance of 20 A was calculated separating the two fluorophores. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A3 domain of light chain in the factor VIII heterodimer.

Amino Acid Sequence↗
PubMed2000

Porcine factor VIII provides clinical benefit to patients with high levels of inhibitors to human and porcine factor VIII.

The development of an inhibitor against Factor VIII is an important complication of haemophilia and occurs in approximately 31% of patients [1]. Despite various approaches to their management, the presence of these inhibitors remains a major cause of morbidity and mortality. Inhibitors may be low level [<5 Bethesda Units (BU)] or high level [>10 BU]. Low-level inhibitors usually remain low and do not respond to administered factor VIII with a rise of the inhibitor titre; high-level inhibitors, in contrast, are characterized by a rapid rise in titre following treatment with factor VIII. Modalities of treatment of acute bleeding episodes in patients with inhibitors include 'overcoming' the inhibitor with very large doses of factor VIII, 'bypassing' the inhibitor blockade with products such as activated prothrombin complex concentrates or recombinant factor VIIa, 'removing' the inhibitor by plasmapheresis or immunoabsorption and 'repressing' it with immunosuppressive drugs and immune tolerance induction. An alternative approach is to use a porcine factor VIII concentrate which does not cross-react with the human factor VIII inhibitor. Following treatment with porcine factor VIII, functional factor VIII can be detected and the therapeutic levels correlate with cessation of bleeding. The use of porcine factor VIII may, however, result in the development of antiporcine factor VIII antibodies, limiting its use. Experience in patients with high-titre inhibitors indicates that porcine factor VIII therapy could be used in the presence of factor VIII inhibitors [2-11], including inhibitors to porcine factor VIII [5,7], and that haemostasis may be obtained in the absence of detectable levels of circulating factor VIII [7,11].

Animals↗
PubMed1997

Factor VIII inhibitors in previously treated haemophilia A patients with a double virus-inactivated plasma derived factor VIII concentrate.

Antibodies to factor VIII (inhibitors) are usually produced at the beginning of treatment with factor VIII and are rare in multitransfused patients. Such antibodies are deemed to be patient-related, as supported by the description of a number of associated risk factors. However, a second category of inhibitors has recently been identified, namely antibodies occurring in multitransfused patients as a result of exposure to a particular factor VIII concentrate. A first outbreak of product-related inhibitors was recently described. The present paper describes the second well-documented occurrence of such inhibitors. Eight out of 140 multitransfused patients with severe haemophilia A developed an inhibitor to factor VIII shortly after changing treatment to a double-virus inactivated plasma-derived factor VIII concentrate. In addition to solvent-detergent treatment, this concentrate was pasteurised at 63 degrees C for 10 hours. Exposure to the pasteurised product before inhibitor detection ranged from 9 to 45 days. Inhibitor titers varied between 2.2 and 60 Bethesda Units and recovery of transfused factor VIII ranged from 0.21 to 0.68 (expressed as i.u./dl factor VIII rise per i.u./kg administered). In contrast to usual inhibitors in haemophilia A patients, these product-related inhibitors showed complex inhibition kinetics. They were found specific for the factor VIII light chain. The inhibitors gradually declined when exposure to the pasteurised product was stopped, despite further treatment with other factor VIII concentrates. The present data stress the importance of carefully monitored clinical studies, both in previously treated and previously untreated patients, before introduction of a new or modified clotting factor concentrate.

Adolescent↗
PubMed1989

Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments.

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3'-ends by selective restriction-enzyme digestion and used as templates for 'run-off' mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

Antibodies, Monoclonal↗
PubMed1992

Pharmacokinetics of two pasteurized factor VIII concentrates by different and multicenter assays of factor VIII activity.

We assessed the pharmacokinetic characteristics of a new high-purity pasteurized FVIII concentrate in comparison with an intermediate purity pasteurized concentrate, produced by the same manufacturer. The study was designed as a cross-over single-dose pharmacokinetic investigation in 8 non-bleeding patients with severe hemophilia A. All patients were given 25 IU/kg of each of the two concentrates, with an interval of at least one week between the two administrations. Decay curves were assessed by collecting 10 serial blood samples over 36 hours following the end of infusion. The concentration of Factor VIII in blood samples was determined in triplicate in three different laboratories using each of the following assay methods: a one-stage clotting assay, a two-stage clotting assay, and a two-stage chromogenic-peptide substrate assay. All pharmacokinetic parameters were calculated by model-independent methods. The two products were found to differ significantly both in the clearance, which was on average 13.8% lower for Haemate P, and in the in-vivo recovery, which was 11.7% lower for Factor VIII:C P on the average. In comparison with previous pharmacokinetic data obtained from other heated Factor VIII concentrates, the clearance of Haemate P was found to be significantly slower, while the half-life of both products was longer. No differences were observed in the Vd-area. These findings indicate that the purification procedures to which both products are subjected do not increase the in-vivo rate of plasma disappearance of Factor VIII.

Adolescent↗
PubMed1999

Spontaneous inhibitors of factor VIII: kinetics of inactivation of human and porcine factor VIII.

Autoantibodies to factor VIII (FVIII)(spontaneous inhibitors) often inactivate FVIII in a complex fashion (type II inhibitors) as compared with alloantibodies (hemophilic inhibitors), which usually demonstrate second-order reaction kinetics (type I inhibitors). The infusion of porcine FVIII in patients with spontaneous inhibitors may give rise to anti-porcine FVIII antibodies. The purpose of this study was to determine whether these were type I or type II inhibitors. Plasma from 8 patients with spontaneous inhibitors and 6 patients with hemophilia with inhibitors were studied. Equal volumes of patient plasma and either pooled normal human plasma or porcine FVIII (Hyate-C) were incubated at 37 degrees C for 90 minutes. Aliquots were removed immediately after mixing and at 30-minute intervals and assayed for FVIII by using a two-stage method. The values for residual FVIII were log-transformed and plotted against the time of incubation, and the resultant curves were analyzed for goodness of fit (coefficient of determination, r2) by using linear and exponential equations. The values were examined by paired t tests; P values were two-tailed. Values are expressed as mean +/- SD. The titers of spontaneous inhibitors against human FVIII ranged from 2.6 to 416 Bethesda Units (BU), and those against porcine FVIII ranged from 0.7 to 47 BU. Samples were diluted so that the FVIII levels in the mixtures before incubation were similar: human, 0.44 U/ml; porcine, 0.47 U/ml; P = not significant. Four of the 8 patients with spontaneous inhibitors inactivated human FVIII in a fashion consistent with complex kinetics; their r2 values with the linear equation were less than 0.90. All r2 values improved when the exponential equation was used (linear, 0.90 +/- 0.08; exponential, 0.92 +/- 0.06; P = .007). In contrast, r2 values with porcine FVIII were the same (0.94) with either the linear or the exponential equation (P = not significant). r2 for the 6 hemophilic inhibitors showed no significant difference between the linear and exponential equations; the values were 0.99 with human FVIII and 0.95 with porcine FVIII. In non-hemophilic patients, antibodies developing to porcine FVIII have kinetics of inhibition that are second order (type I), even though antibodies to human FVIII in these same patients may have complex (type II) kinetics.

Animals↗
PubMed1986

Characterization of four monoclonal antibodies to factor VIII coagulant protein and their use in immunopurification of factor VIII.

Four monoclonal anti-VIII:C antibodies were obtained from the fusion of the splenocytes of one Balb/C mouse with a specific activity ranging from 2.3 to 45,000 U/mg when purified from ascitic fluid. Only one antibody was able to inhibit completely Factor VIII:C in normal plasma. The four antibodies could bind Factor VIII:CAg in plasma and commercial concentrate both in liquid and solid phase, and were suitable for immunopurification of Factor VIII:C. Three antibodies competed with polyclonal anti-VIII:CAg Fab' in a liquid phase IRMA, and all of them were able to displace their own binding to Factor VIII:CAg. Competition studies between monoclonal antibodies for the binding to Factor VIII:CAg were performed and showed the recognition of different epitopes and various functional impact. These studies indicate that at least one antibody, with the lowest anti-VIII:C titer clearly recognizes a different epitope of VIII:C than those recognized by the others. Affinity constants ranged from 10(9) to 10(10) l/mole.

Animals↗
PubMed1996

In vitro factor VIII recovery during the delivery of ultrapure factor VIII concentrate by continuous infusion.

Factor VIII (FVIII) replacement by continuous infusion has been advocated as a cost-effective method for maintaining stable plasma levels of FVIII in the hemophilia A patients during surgery or life-threatening hemorrhage. Continuous delivery of monoclonal or recombinant FVIII concentrates to our pediatric patients using a traditional delivery system (dilution in normal saline of 2-10 U/ml infused at a rate of 20 ml/hr) has frequently yielded higher than expected factor usage to achieve desired levels and unexpected variability in plasma levels under presumed steady-state conditions. To determine if diminished in vitro FVIII recovery was responsible for these observations, a study of four ultrapure concentrates during 8 hr of in vitro continuous delivery was performed using four delivery systems. When reconstituted concentrate was added to normal saline in polyvinylchloride bags at a concentration of 10 U/ml (method IA), monoclonal products showed a stable recovery of 84-109% of time 0 levels. Recombinant product recovery dropped to 57-76% of time 0 levels before reapproximating the time 0 level at 2 hr. The addition of 10 mg/ml human albumin to the bags (method IB) did not improve recoveries. When reconstituted concentrate was delivered undiluted (method IIA), the early drop in recombinant recovery was eliminated; stable recovery of 78-117% of time 0 level was achieved with all products. In using method IA, a large discrepancy was seen between the actual time 0 recoveries and those expected based on vial assays, most striking for recombinant products (49-57% of expected. Method IIA allowed 75-90% recovery; addition of 20 mg/ml albumin of reconstituted but undiluted concentrate (method IIB) maximized recovery at 85-98% of expected.

Albumins↗
PubMed1997

[Prostate carcinoma associated spontaneous factor VIII:C inhibitor hemophilia. Successful therapy of severe hemorrhagic complication with porcine factor VIII in a 75-year-old patient].

Inhibitors of factor VIII are a rare condition in non-hemophiliacs, but they are frequently responsible for life threatening hemorrhage. Acquired factor VIII:C inhibitors represent the spontaneous development of autoantibodies that partially or completely neutralize the plasma coagulant activity of the clotting factor. The autoantibodies can arise in diverse clinical settings, in older adults they are frequently associated with immunologic disorders or malignancies. We report of a 75-year-old man with acquired factor VIII:C inhibitor associated with adenocarcinoma of the prostate and a successful treatment of a severe bleeding complication with porcine factor VIII. A 75-year-old man was admitted because of a hematoma of his right cheek and an isolated prolonged aPTT. Acquired factor VIII:C inhibitor was identified as the cause and immuno-suppressive therapy was begun. In the clinical course severe hemorrhaging occurred and was successfully treated with porcine factor VIII (Hyate:C). The initially high inhibitor titer of 32 Bethesda Units (BU) disappeared. As the cause of acquired factor VIII:C inhibitor a newly diagnosed adenocarcinoma of the prostate is likely. After complete remission of acquired factor VIII:C inhibitor radiation therapy was begun. Six months after severe hemorrhaging the patient was clinically stable and PSA levels were normal. This case demonstrates the necessity of a precise diagnosis and therapy regimen of this coagulopathy based on clinical and laboratory data. In the absence of hemorrhage immuno-suppressive therapy with corticosteroids is indicated, in a patient with severe bleeding and high inhibitor titer (> or = 5 BU) porcine factor VIII should be administered.

Adenocarcinoma↗
PubMed2004

Management of haemophilic patients with inhibitors in major orthopaedic surgery by immunadsorption, substitution of factor VIII and recombinant factor VIIa (NovoSeven): a single centre experience.

Inhibitors of factor VIII or FIX in haemophilic patients are a common and serious complication associated with an increased risk of life-threatening bleeding during elective surgery. Substitution therapy fails to be effective, therefore an alternative treatment is needed. We have performed six major elective orthopaedic interventions in four patients with haemophilia A and inhibitors. A preoperative immunadsorbant therapy with Therasorb to eliminate inhibitors was successful in four cases, but during FVIII substitution inhibitors increased on day 4 to day 6 after surgery, leading to decreasing FVIII levels. Therefore, therapy was changed to recombinant FVIIa (rFVIIa; NovoSeven). Two interventions had to be covered with sole rFVIIa therapy as immunadsorbant therapy failed to be effective in one case and the need for acute intervention did not allow pretreatment in the other. We did not see increased bleeding during or after surgery when compared to our experience with non-inhibitor haemophilic patients. In conclusion, a preoperative decrease of inhibitors from immunadsorbant therapy, perioperative substitution of FVIII and changing treatment to rFVIIa when inhibitors are increased, is a safe and economic therapy for guaranteeing haemostasis in major elective orthopaedic surgery. On the contrary, sole therapy with rFVIIa allows immediate surgical intervention without a long hospital stay prior to surgery and a need for laboratory monitoring of inhibitor titres and FVIII levels. Our findings support data previously published.

Adult↗
PubMed2003

Factor VIII and von Willebrand factor changes during normal pregnancy and puerperium.

Gestation is a challenge to haemostasis and it is associated with significant haemostatic changes. Several studies have evaluated von Willebrand factor in normal pregnancy, but none considered the personal history of bleeding. We studied a group of healthy non-bleeding women (184 pregnant, 64 puerperium, 37 non-pregnant) to evaluate normal ranges and their relationship to blood group and parity. The von Willebrand factor increased markedly from non-pregnant values up to the end of early puerperium (P < 0.0001), while factor VIII only showed a slight increase. Factor VIII and von Willebrand factor activity remained within the normal range for non-pregnant women. The return to non-pregnant factor levels occurred in late puerperium, later than previously reported. Only factor VIII was significantly lower in the O blood group (P = 0.035). As regards parity, there were no differences in factor VIII, von Willebrand factor antigen and von Willebrand factor ristocetin cofactor between primigravidae and multigravidae for any period studied (P = 0.888, 0.999, and 0.237, respectively). Our results provide reference ranges that may help to design a study in von Willebrand factor disease in pregnancy.

Adolescent↗