Specific factor VIII-related antigen fragmentation: an in vivo and in vitro phenomenon

Factor VIII concentrates have been shown to have reduced ability to correct the bleeding time defect in von Willebrand's disease and to have abnormal mobility of their VIIIR:Ag on crossed immunoelectrophoresis. This report concerns the partial characterization of a fragment of VIIIR:Ag that lacks some of the normal antigenic determinants present on VIIIR:Ag. It is present in commercial factor VIII concentrates, but not in cryoprecipitate, and is recovered from the plasma of hemophilic patients who have been infused with these concentrates. The fragment is also produced during disseminated intravascular coagulation. Although it has a similar mobility on SDS agarose electrophoresis to the smallest multimer of VIIIR:Ag, they are not immunologically identical.     

Acquired von Willebrand's disease in association with Wilm's tumor: regression following treatment

A 9-yr-old female presented with a Wilm's tumor and a coagulopathy consistent with von Willebrand's disease. Factor VIII procoagulant activity (VIII C), factor VIII related antigen (VIIIR:Ag), and von Willebrand factor activity (VIII:vWf) were decreased. There was no evidence for a circulating inhibitor of the factor VIII molecular complex. von Willebrand's antigen II (vW AgII), which is deficient in hereditary von Willebrand's disease, was decreased below detectable levels in this patient. The coagulation studies, VIIIR:Ag, and vW AgII levels returned to normal following therapy of the Wilm's tumor. Wilm's tumor must be included as one of the malignancies associated with acquired von Willebrand's disease. Immunofluorescent studies of the tumor specimen showed normal endothelial staining of VIIIR:Ag by semiquantitative techniques and a lack of specific tumor adsorption of VIIIR:Ag The presence of normal amounts of tissue VIIIR:Ag has not previously been demonstrated in acquired von Willebrand's disease. Since we failed to demonstrate an inhibitor in the plasma in this patient, the etiology of the acquired von Willebrand's disease in this patient appears to differ from other cases of acquired von Willebrand's disease. The finding that vW AgII is decreased in this patient, similar to that reported in hereditary von Willebrand's disease, supports the close association of vW AgII to VIIIR:Ag, even though they are immunologically and biochemically distinct.     

Intranasal desmopressin (DDAVP) by spray in mild hemophilia A and von Willebrand's disease type I

Summary Desmopressin acetate (1-desamino-8-Darginine vasopressin, DDAVP) has mostly been given by the parenteral route for the treatment of mild hemophilia A and von Willebrand's disease type I. In the present study the hemostatic effects of desmopressin acetate administered intranasally by spray in a dose of 300µg and intravenously 0.3–0.4µg/kg were assessed and compared in 8 patients with hemophilia A and 22 patients with von Willebrand's disease type I. A bioequivalent response to intravenous and intranasal desmopressin acetate was found in Factor VIII coagulant activity (VIII:C) in the hemophilia patients. In the von Willebrand patients, an equivalent shortening of the bleeding time was seen after the two modes of administration, even though intravenous injection gave a higher increase in plasma levels of VIII:C and vWF:Ag. In five patients with von Willebrand's disease the duration of the spray effect on VIII:C and vWF:Ag was followed for 24 h. After 12 h the mean level of VIII : C was 1.4, and of vWF:Ag 1.5, times the basal level. The findings suggest that the spray can be recommended for home or prophylactic treatment of patients with mild hemophilia A and von Willebrand's disease.     

Factor VIII complex in progressive systemic sclerosis

Factor VIII complex and its related activities (Coagulant, Antigen and Ristocetin Cofactor) have been investigated in 23 patients with Progressive Systemic Sclerosis (PSS) divided into two groups: acrosclerosis and diffuse sclerosis. All Factor VIII-related activities were higher in PSS patients than in normal subjects. No difference in F. VIII-related Antigen (F. VIIIR:Ag), F. VIII-related Ristocetin Cofactor (F. VIIIR:Co) and F. VIII Coagulant activity (F. VIII:C) was found comparing the patient groups. F. VIII:C was increased significantly less than F. VIIIR:Ag and F. VIIIR:Co in both patient groups. Some hypotheses about the pathogenesis of this increase are discussed.     

A variant of von Willebrand's disease with abnormal expression of factor VIII procoagulant activity

Reports on variants of von Willebrand's disease are numerous, but many of these are based on tests that will show marked fluctuations with time and tests that might not be similar in affected family members. This report describes 8 patients with a new variant of von Willebrand';s disease in which there is a normal APTT, slightly reduced one-stage factor VIII:C assay (VIII:C-1), and a drastically reduced two- stage factor VIII:C assay (VIII:C-2). The VIII:C in this variant is more readily adsorbed to AI(OH)3. This variability in VIII:C assays and excessive adsorption to AI(OH)3 are corrected by the addition of either hemophilic plasma or hemophilic factor-VIII-related antigen. This variant is stable with restudy on multiple occasions and is inherited in a stable fashion in three generations of one family. The multimeric structure of the VIIIR:Ag appears normal, although the concentration is moderately reduced. The differences in functional activity, the adsorption to AI(OH)3, and the differences between functional and antigenic (VIII:C Ag) assays of VIII:C support that this is a functional abnormality of type I von Willebrand's disease.     

Management of cesarean section under replacement therapy with factor VIII concentrates in a pregnant case with congenital combined deficiency of factor V and factor VIII

The congenital combined deficiency of Factor V and Factor VIII, a rare bleeding disorder, was identified in a 25-year-old woman. She was admitted to our hospital with a complaint of genital bleeding. Her prothrombin time and activated partial thromboplastin time were prolonged. She had low levels of Factor V coagulant activity (F. V:C) 14%, and Factor VIII coagulant activity (F. VIII:C), 12%, and normal levels of von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (Rcof) and Protein C antigen. Her Protein C inhibitor level was slightly low. Her Rcof, vWF:Ag and F. VIII:C were elevated following administration of 1-deamino-8-D-arginine-vasopressin (DDAVP), but her F. V:C remained unchanged. Four years later, her F. VIII:C rose to 70% during the course of her pregnancy, but her F. V:C value remained low. It was expected that the vaginal delivery would be possible at the termination of pregnancy. Premature rupture of the membranes and an anomaly of rotation appeared in the course of delivery, however, and cesarean section was accomplished without excess bleeding under replacement therapy with Factor VIII concentrates. These findings suggested that DDAVP and Factor VIII concentrates were useful for management of her delivery. However the mechanisms of the rise of plasma F. VIII:C during pregnancy in a case with congenital combined deficiency of Factor V and Factor VIII are unclear.     

Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand's disease

S ummary . Factor VIII was purified from cryoprecipitate by ion exchange chromatography on solid phase polyelectrolyte E-5 (PE-E5). The product was highly purified (3.5 u VIII: C/mg protein) compared to conventional concentrate (0.3 u VIII: C/mg protein) with low fibrinogen, low isoagglutinin titre, and a ratio of factor VIII coagulant activity (VIII: C) to factor VIII related antigen (VIIIR: Ag) of 16:1.
Trial infusions of this material (PE VIII) were given to three patients with severe haemophilia A and one patient with homozygous von Willebrand's disease. These patients also each received separate infusions of intermediate purity concentrate (IPC) for comparison. There were no adverse effects. The mean half life of VIII: C after PE VIII infusion in the haemophiliacs was 10.9 h and after IPC was 12.1 h, a statistically insignificant difference. The survival of factor VIII coagulant antigen (VIII:CAg) was similar to that of VIII:C. In contrast, the half life of VIII:C and of VIII:CAg was very short after infusion of PE VIII in the patient with von Willebrand's disease (2.4 h). IPC when infused in this patient produced a typical secondary rise of VIII:C. Two bleeding episodes in severe haemophiliacs were satisfactorily treated with PE VIII. PE-E5 deserves further study as a means of preparing clinical concentrates of factor VIII.     

Measurement of factor VIII procoagulant antigen in normal subjects and in hemophilia A patients by an immunoradiometric assay and by an enzyme-linked immunosorbent assay

Factor VIII coagulant antigen (FVIII:Ag) and FVIII coagulant activity (FVIII:C) were measured in 102 healthy individuals, in 5 hemophilia A carriers and in 21 hemophilia A patients before and after infusion of heat-treated high-purity FVIII concentrates. Factor VIII:Ag was determined by a solid-phase micro enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and by a conventional solid-phase immunoradiometric assay (IRMA). Factor VIII:C was assessed using a one-stage assay. The micro ELISA was decidedly more precise than the IRMA. There was a close correlation between the results obtained by the three assays in the plasma of healthy subjects and in hemophilia A carriers. After transfusion of FVIII concentrates to hemophilia A patients, the FVIII:Ag recoveries were significantly lower than the FVIII:C recoveries and the biological half-life of FVIII:Ag was significantly shorter than for FVIII:C. The calculated half-life of FVIII:C was longer than in any previous study.     

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.     

Serial studies in von Willebrand's disease: variability versus "variants"

The variability of laboratory findings in von Willebrand's disease (vWd) was evaluated by performing serial studies of bleeding time (BT), factor VIII coagulant activity (VIII:C), factor-VIII-related antigen (VIIIR:Ag) and ristocetin cofactor (VIIIR:Rcof) in 50 individuals from 25 families with this disorder. The types of results were characterized from 1 to 16 based on the possible combinations of findings using these four tests. The only patients observed to have consistently abnormal results of all four tests were three individuals with homozygous vWd. Individuals with autosomal dominant vWd were found to have a variety of results and all 16 possible types were observed. Although a consistent pattern was present within some families, others with equivalent history of bleeding demonstrated widely variable types of results. The results within some families, others with equivalent history of bleeding demonstrated widely variable types of results. The results of serial studies of the same tests in 10 normal individuals indicated relative stability, with nearly all values within the usual range of normal, but some independent variation of factor-VIII-related activities was observed. These studies indicate that: (1) the results of BT, VIII:C, VIIIR:Ag, and VIIIR:Rcof vary considerably from time to time in many individuals with vWd, (2) a classification of "variants" of vWd based solely on such studies may be inappropriate, particularly if the tests are not repeated, and (3) repeated testing may be required to establish the diagnosis of vWd in some individuals.     

Phage display technology: a tool to explore the diversity of inhibitors to blood coagulation factor VIII

Hemophilia A is a X-linked bleeding disorder that is caused by the functional absence of blood coagulation factor VIII. The bleeding tendency in hemophilia A patients can be corrected by the administration of plasma-derived or recombinant factor VIII concentrates. A serious complication in hemophilia care is the development of factor VIII neutralizing antibodies (inhibitors) that arise as a consequence of factor VIII replacement therapy. The majority of factor VIII inhibitors are directed toward epitopes located within the A2, A3, and C2 domains of factor VIII. In this article, we summarize current knowledge on the epitope specificity of factor VIII inhibitors. In addition, we will discuss recent information on the molecular characteristics of human anti-factor VIII antibodies generated by phage display technology.     

In vitro characterization of commercial factor VIII concentrates: Long-term follow-up

Summary Fifty batches of Factor VIII concentrates from 12 producers were characterized in a long-term follow-up. The following parameters were measured: Factor VIII:C, Factor VIIIR:AG, Factor VIII:Rcof, specific activity (U Factor VIII:C/mg protein), fibrinogen, IgG, IgM, IgA, isoagglutinins, Hb_s-AG, heparin-like activity, thrombin-like activity, antithrombin III, Factor VIII-stability at room temperature, and the rate of complete dissolution of the lyophilizate. In most preparations there was an unacceptable batch-to-batch variation of both Factor VIII complex and contaminating proteins, which exceeded the inter-assay coefficient of variation of the applied test systems. Nevertheless, different brands could be recognized by their typical protein pattern. The results obtained suggest that the standardization of Factor VIII concentrates of unknown composition is still accompanied by considerable risks.     

Use of a Simple Visual Assay of Willebrand Factor for Diagnosis and Carrier Identification

A visual assay of factor VIII-related Willebrand factor (VIIIR:WF) is described which utilizes formaldehyde-fixed platelets, end points being read in microflocculation tiles. Four dilutions of a sample can be assessed simultaneously, and the correlation with aggregometric assays is high (r = 0.91). Measurement error is 8.0% for a single assay in triplicate and less than 5% if an assay is repeated three times. The method has been used for 2 years by the coagulation genetics group at Chapel Hill for diagnosing subjects with von Willebrand's disease and assigning genotypes to members of families transmitting this disorder. Its utility in classifying known carriers of haemophilia A has also been examined, both in conjunction with assays of VIII:C and in a three-way test with assays of VIII:C and VIIIR:Ag. As predicted by the Lyon hypothesis, the rate of false negative diagnosis was higher than false positive diagnosis, but the overall rate of misclassification on single plasma samples was 7/51 = 13.7%. The error rate was the same whether discrimination was based upon assays of VIII:C vs. VIIIR:Ag, VIII:C vs. VIIIR:WF, or VIII:C vs. VIIIR:Ag vs VIIIR:WF, the same individuals being misclassified by each method. The observed rate of misclassification was well within the rates reported by others and very similar to our previous experience. We have concluded that this method of assaying VIIIR:WF is highly useful for diagnosing vWd, detecting inhibitors to VIIIR:WF, and examining large numbers of column fractions. It is a useful supplement, although it cannot yet substitute for, assays of VIIIR:Ag in detecting carriers of haemophilia A.     

Alterations of Factor VIII von Willebrand Factor in Clinical Conditions Associated with an Increase in its Plasma Concentration

Factor VIII-related antigen (VIIIR:Ag) was consistently higher than factor-VIII procoagulant activity (VIII:C) in 57 patients with clinical conditions characterized by acute-phase reactions. Two different methods for measuring VIII:C (one- and two-stage assays) and VIIIR:Ag (electroimmunodiffusion and immunoradiometric assay) gave concordant results in the majority of cases. In 43% of plasma samples, crossed immunoelectrophoresis in agarose gel was characterized by the appearance of an additional, fast-moving precipitin peak which was immunologically identical with the major, slower-moving VIIIR:Ag peak. The fast-moving peak was detected in all the patients with clinical conditions typically associated with increased plasma proteolysis (DIC, acute pancreatitis, during thrombolytic therapy). It was present in a smaller proportion of cases with liver and renal failure and malignancies and in the post-operative period. The additional VIIIR:Ag peak is thought to be the result of in vivo factor VIII/von Willebrand factor fragmentation by proteolytic enzymes.     

Influence of sex, blood group, secretor character, smoking habits, acetylsalicylic acid, oral contraceptives, fasting and general health state on blood coagulation variables in randomly selected young adults

Blood samples were drawn from 129 randomly selected young adults. Intake of acetylsalicylic acid (ASA), contraceptive drugs, smoking habits and health state were registered. Males had significantly higher systolic blood pressure, shorter bleeding time and lower VIII:C. Smoking was only correlated to some variables assessed in the female group. Users of oral contraceptives smoked more, had a shorter bleeding time and higher fibrinogen levels. Factor VIIIR;Ag was elevated only in female smokers with blood group non-O. Non-secretors had shorter bleeding times and a tendency towards higher VIIIR:Ag.     

Preclinical animal models for hemophilia gene therapy: predictive value and limitations

Hemophilia A and B are excellent candidate disorders for the application of somatic cell gene therapy. One of the major advantages in the preclinical development of hemophilia gene therapy strategies has been the availability of several animal models for both hemophilia A and B. These models recapitulate many of the phenotypic aspects of human hemophilia and have proven to be very informative in exploring the efficacy and safety of gene therapy. Considerable progress has been made in the design of gene therapy protocols, and over the last 5 years it has been shown that long-term phenotypic correction, with sustained therapeutic levels of factor VIII (FVIII) and factor IX (FIX), can be attained in FVIII- and FIX-deficient mice and dogs using various viral vector-mediated gene therapy approaches. These animal models also have elucidated potential complications of gene therapy protocols, including acute vector-associated toxicities and the induction of neutralizing antibodies to the FVIII and FIX transgene products. Nevertheless, although the preclinical paradigm of hemophilic mouse followed by hemophilic dog studies has proven to be extremely helpful in evaluating the efficacy and safety of potential clinical gene therapy protocols, several limitations to these animal models still exist. This review presents a summary of the animal models available for hemophilia gene therapy, and highlights the various strengths and weaknesses of these models.     

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.     

Distribution of plasma fibronectin (cold-insoluble globulin) and components of the factor VIII complex after heparin-induced precipitation of plasma

Factor VIII procoagulant (VIII:C) activity, factor VIII coagulant antigen (VIII:CAg), von Willebrand ristocetin cofactor (VIIIR:RC) activity, factor VIII-related antigen (VIIIR:Ag), and plasma fibronectin (CIg; cold-insoluble globulin) were measured in the heparin precipitable fraction (HPF) and heparin supernatant fraction (HS) of normal human plasma. Following heparin induced precipitation, most measurable VIII:C activity (77% +/- 24%) was recovered in the HS. Although there was little VIII:C activity (less than 1%) in the HPF, 20% +/- 6.5% VIII:CAg was present as well as CIg (81% +/- 5.6%). VIIIR:RC activity (72% +/- 12%), and VIIIR:Ag (34 +/- 5.2%). As assessed by Na dodecyl SO4 glyoxyl agarose electrophoresis, the multimeric forms of plasma VIIIR:Ag could be resolved into a series of bands. Larger multimers tended to precipitate with the HPF whereas the smaller multimers tended to remain supernatant. Plasma from a subject with congenital afibrinogenemia was also studied. Although the afibrinogenemic HPF contained CIg, neither VIIIR:RC activity nor VIIIR:Ag was precipitated. However, both were present in the HPF from afibrinogenemic plasma to which fibrinogen had been added, suggesting that they are incorporated in this precipitate because of an affinity for fibrinogen. The ability of heparin to induce precipitation of CIg while leaving most VIII:C activity in the supernatant plasma may be useful in the preparation of procoagulant-rich plasma subfractions, since VIII:C can subsequently be recovered in good yield by cryoprecipitation.     

Severe factor VIII and factor IX deficiency in females.

A survey of 11 hemophilia centers produced data concerning 28 females with extremely low levels of factor VIII or IX coagulant activity. Ten of the 28 have hemophilia A, six have hemophilia B, and 12 have severe von Willebrand's disease. The 16 females who have severe factor VIII or factor IX deficiency as an isolated defect exemplify several of the possible genetic explanations for the occurrence of hemophilia in females. All 16 bruise excessively, and several have had recurrent hemarthroses. Three of these girls, ages five, 10 and 23 years, have evidence of chronic hemophilic arthropathy. The 12 females with severe von Willebrand's disease are either homozygous for von Willebrand's disease or severely affected heterozygotes. All 12 have mucous membrane bleeding. In addition, five of the 12 have recurrent hemarthroses and three have evidence of chronic joint disease. However, the major problem in the adult females with von Willebrand's disease has been extreme menorrhagia. One of the seven adults underwent irradiation sterilization and another had a hysterectomy because of menorrhagia. The others have been managed with anovulatory drugs or plasma infusions and EACA. Despite menorrhagia, five pregnancies and deliveries have been uneventful in three of these women.     

Carrier detection in hemophilia A: a cooperative international study. II. The efficacy of a universal discriminant

Factor VIII (F.VIII) and von Willebrand factor (VWF):Ag data collected by eight laboratories on a total of 336 obligatory carriers of hemophilia A and 137 normal women were used to answer several questions concerning the construction of linear discriminants for carrier detection. It was found: that a "universal" linear discriminant can be constructed which is suitable for use in all laboratories and is nearly as effective as laboratory-specific discriminants; that inclusion of age and ABO blood type data improved the efficacy of these discriminants; that substitution of alternative assays for F.VIII and VWF:Ag did not generally improve the efficacy of the discriminants over that obtained using the bioassay for F.VIII:C and Laurell's immunoassay for VWF:Ag; that linear discriminants were far more effective than discriminants based on the F.VIII:C/VWF:Ag ratio. A step-wise procedure is given which any laboratory may follow in using the universal discriminant for carrier detection.