Factor VIII coagulant antigen in hemophilic plasma: a comparison of five alloantibodies

Five independent alloantibodies directed to factor VIII coagulant antigen (VIII:CAg) were assessed against normal, von Willebrand's disease, and severe hemophilia A plasmas. Immunoradiometric assays (IRMAs) were developed for each antibody, one of which had arisen "spontaneously" and four in transfused hemophiliacs. The correlations between assays were very high for normal, vWd, and both CRM+ and CRM- hemophiliacs. This suggests that IRMAs maybe developed from almost any reasonably high titered alloantibody and used with confidence in diagnosing CRM- hemophilia A in utero by fetoscopy.     

Pattern of factor VIII inhibitors in patients with hemophilia A in the north east of Iran

This survey was conducted to evaluate coagulation factor VIII:C inhibitors among 102 hemophilia A patients from different cities of Khorasan province in north east of Iran in order to identify and characterize the pattern of inhibitor formation in these patients population. For this purpose, we randomly obtained plasma samples of 102 hemophilia A patients (44 patients with severe, 28 patients with intermediate and 30 patients with mild hemophilia A) and studied them using two tests: the APTT mix and Bethesda test were performed. In the whole group 20 patients (19.6%) factor VIII inhibitors were detected. These were in 11 patients with severe, five patients with intermediate and four patients with mild hemophilia A. None of patients with hemophilia A had previously been studied for the presence of an inhibitor, so there was no existing history of inhibitor evaluation.     

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.     

A randomized placebo-controlled double-blind study of danazol in hemophilia A.

A randomized double-blind placebo-controlled crossover trial of danazol was carried out in 19 cases of hemophilia A. Danazol was given for 3 months at a dose of 150 mg/day to patients under 15 years of age, and 300 mg/day to older patients. The basal factor VIII:C level was 8.3 +/- 5.6% (mean +/- SD), and after 3 months of danazol treatment was 15.3 +/- 11.0% (p = 0.02). Six patients (basal factor VIII:C 2-22%) showed a 1.36- to 2.87-fold elevation of factor VIII:C levels after danazol. 0/2, 1/4 and 5/13 cases of severe, moderate and mild disease, respectively, responded. Decreases in the number of bleeding episodes and cryoprecipitate requirement were seen in the responders. No adverse reactions to danazol were encountered. Danazol appears to raise the factor VIII:C levels in selected cases of hemophilia A.     

Prevalence of antibodies against parvovirus B19 in Norwegians with congenital coagulation factor defects treated with plasma products from small donor pools.

The seroprevalence of antibodies against parvovirus B19 in 308 Norwegians with coagulation factor defects of different types and severities was assessed by an IgG antibody capture radioimmunoassay (GACRIA). The overall seroprevalence was 62%. The seroprevalence among subjects with different types of coagulation factor defects was related to the type and severity of the coagulation factor defect: severe hemophilia A 64%, moderate and mild hemophilia A 58%, severe hemophilia B 88%, moderate and mild hemophilia B 73%, and von Willebrand's disease 52%. The prevalence of parvovirus B19 antibodies among household contacts and blood donors was 49% and 42% respectively. This study confirms that replacement therapy with coagulation factors is accompanied by an increased risk for acquiring parvovirus B19 infection. However, the prevalence of parvovirus B19 antibodies among Norwegian hemophiliacs is well below the prevalence reported from other countries and probably reflects the small numbers of donors in plasma pools used for the preparation of coagulation factor concentrates.     

Assay of factor VIII antigen (VIII:CAg) in 294 Haemophilia A patients by a new commercial ELISA using monoclonal antibodies

Monoclonal antibodies (MoAbs 833 and D4H1) directed against human factor VIII (FVIII) have been produced on a large scale to measure VIII:CAg by two-site ELISA (Asserachrom VIII:CAg, Diagnostica Stago). F(ab’)₂ from MoAb 833 were used for coating and bound VIII:CAg was revealed with MoAb D4H1 coupled to peroxidase. Control plasma (100 VIII:CAg U dL^?1 by comparing with the International Standard) was used as reference. The assay sensitivity was 0.1 U dL^?1 VIII:CAg. No apparent effect of the plasma proteins was observed provided plasma dilution was 5. Thus this ELISA allowed us to estimate VIII:CAg levels of 0.5 U dL^?1 in plasma. Levels of VIII:CAg were similar to those of VIII:C (correlation coefficient r= 0.87) in plasma from normal individuals (32 cases) and in patients with von Willebrand disease of various types (30 cases). Among 294 patients with Haemophilia A (HA), 161 had severe HA (VIII:C < 1 U dL^?1). Among those patients, 124 were cross-reacting material (CRM) negative with undetectable VIII:CAg and 37 were CRM+ (VIII:CAg 1– 31 U dL^?1). In 42 patients with moderate HA (VIII:C 1– 5 U dL^?1), 33 were CRM reduced (VIII:CAg 0.5–8 U dL^?1) and nine were CRM+ with a VIII:CAg/VIII:C ratio of 6–91 (mean 34.3). In mild HA (91 cases with VIII:C 6 U dL^?1), 29 patients were classified as CRM+ (VIII:C 6–57 U dL^?1, VIII:CAg 17–130 U dL^?1 and VIII:CAg/VIII:C ratio 1.8–13.7 (mean 4.51)). In 62 CRM reduced patients there was a linear correlation between VIII:C (6–39 U dL^?1) and VIII:CAg (2–36 U dL^?1) levels (r= 0.88). In conclusion, this sensitive assay allows us to distinguish the quantitative CRM reduced and negative from the qualitative (CRM+) abnormalities in Haemophilia A.     

Analysis of factor VIII coagulant antigen in normal, thrombin-treated, and hemophilic plasma.

The relationship between Factor VIII coagulant antigen (VIII:CAg) and Factor VIII-associated von Willebrand factor (VIII:vWF), and the effect of thrombin on VIII:CAg have been determined in plasma by using complexes of VIII:CAg and 125I-labeled human anti-VIII:CAg-Fab. Antibody-treated plasma samples were electrophoresed on NaDodSO4/polyacrylamide agarose gels and analyzed by autoradiography. The major VIII:CAg-125I-labeled Fab complex that persisted in NaDodSO4 had Mr 3.2 x 10(5). This Mr value was confirmed by column chromatography and sucrose density centrifugation and is presumed to reflect a free VIII:CAg of Mr 2.7 x 10(5). Minor bands were also present on autoradiograms of normal plasma corresponding to Mr values of 2.5, 1.85, and 1.7 x 10(5) (free VIII:CAg related proteins with Mr values of 2.0, 1.35, and 1.2 x 10(5), respectively). None of the VIII:CAg bands was present in plasma samples from five patients with severe hemophilia A. No radioactivity was associated with VIII:vWF multimers on NaDodSO4 gels. Thrombin treatment of normal plasma eliminated the radioactive band at 3.2 x 10(5) and increased the intensity of a band of Mr 1.7 x 10(5). Generation of this presumed VIII:CAg fragment of Mr is approximately equal to 1.2 x 10(5) coincided with a thrombin-induced increase in Factor VIII coagulant activity. These data demonstrate that the form of VIII:CAg detected in normal plasma is not covalently linked to VIII:vWF multimers and is absent in plasma from five hemophilia A patients. Thrombin-induced proteolysis of VIII:CAg can be detected in microliter quantities of normal plasma.     

Use of porcine factor VIII in the treatment of patients with acquired hemophilia

Data have been collected from 47 centers in Europe and North America on the treatment with porcine factor VIII concentrate of 74 acute bleeding episodes in 65 patients with acquired hemophilia. The median initial anti-human factor VIII auto-antibody inhibitor level was 38 Bethesda unit (BU)/mL (range 1.2 to 1,024) whereas that against porcine was 1 BU/mL (range 0 to 15). The mean initial dose of porcine factor VIII infused was 84 IU/kg, which increased the plasma factor VIII:C activity by 0.85 IU/mL. Therapy was continued for a mean of 8.5 days during which time the average number of infusions was 11. Objective clinical responses were rated as good or excellent in 78% of recipients. Side effects were uncommon; only one patient experienced a severe anaphylactic reaction necessitating the discontinuation of porcine FVIII therapy. After therapy, no increase in the median level of anti- human FVIII or anti-porcine antibody was noted in the group as a whole, although 13 patients showed individual increases in either anti-human or anti-porcine antibody levels or both of more than 10 BU/mL. Of the 7 patients who subsequently rebled, 5 were successfully re-treated and 2 did not respond to further porcine factor VIII treatment. Porcine factor VIII is safe and clinically effective treatment for bleeding episodes associated with acquired hemophilia and should be considered as first-line therapy for patients whose acquired anti-factor VIII:C antibody cross-reacts with porcine factor VIII:C at low levels.     

Aminoglycoside suppression of nonsense mutations in severe hemophilia

Aminoglycoside antibiotics exhibit their bactericidal effect by interfering with normal ribosomal activity. In this pilot study, we have evaluated the effect of the aminoglycoside antibiotic gentamicin on the factor VIII (FVIII) and IX levels of severe hemophiliacs with known nonsense mutations. Five patients were enrolled and each patient was given 3 consecutive days of gentamicin at a dose of 7 mg/kg intravenously every 24 hours. Two patients (patient no. 1: hemophilia A, Ser1395Stop; and patient no. 5: hemophilia B, Arg333Stop) showed a decrease in their activated partial thromboplastin time (aPTT), an increase in their FVIII (0.016 IU/mL, 1.6%) or FIX (0.02 IU/mL, 2%) levels, and an increase in thrombin generation. The remaining 3 patients (patient no. 2: hemophilia B, Arg252Stop; patient no. 3: hemophilia A, Arg2116Stop; and patient no. 4: hemophilia A, Arg427Stop) showed no response in the aPTTs or factor levels, but one (patient no. 2: hemophilia B, Arg252Stop) showed an increase in the factor IX antigen level (2%-5.5%) that persisted throughout the period of the study and was concordant with an increase in thrombin generation. Gentamicin is unlikely to be an effective treatment for severe hemophilia due to its potential toxicities and the minimal response documented in this report. This study, however, does provide a proof of principle, suggesting that ribosomal interference with a less toxic agent may be a potential therapeutic mechanism for severe hemophilia patients with nonsense mutations.     

Liver transplantation in hemophilia A

Four patients with hemophilia A have undergone liver transplantation in our institution, three successfully. The first was a 21-year-old man with chronic active hepatitis (CAH) in whom the effects of previous abdominal operations prevented the satisfactory technical insertion of the new liver. He died intraoperatively. The second patient was a 15- year-old boy with CAH who began to synthesize factor VIII coagulant activity (F VIII:C) within 18 hours of successful liver transplantation and has continued to do so for almost 2 years (F VIII:C range 0.89 to 3.20 U/mL). The first 2 months of his postoperative course were complicated by infections, but since that time he has done well and has returned to school. The third patient was a 48-year-old man with portal fibrosis and severe ascites. He synthesized F VIII:C (range 0.96 to 1.50 U/mL) within six hours after reestablishment of circulation through the new liver. His postoperative course was complicated by numerous infections, and he died with sepsis and an acquired immunodeficiency-like syndrome 4 months after transplantation. The fourth patient was a 47-year-old mild hemophiliac with CAH who produced adequate factor VIII:C levels following transplantation (range 0.79 to 2.80 U/mL). These patients demonstrate that liver transplantation in hemophiliacs with end-stage liver disease may be lifesaving and results in correction of the F VIII:C deficiency and associated hemorrhagic tendency.     

Factor VIII:C and VIII:CAg Response in Patients with Haemophilia A and von Willebrand's Disease after Administration of Different Factor VIII Concentrates or Plasma

S ummary . Factor VIII procoagulant activity (VIII:C) and factor VIII procoagulant antigen (VIII:CAg) were studied in seven patients with haemophilia A after administration of three different factor VIII concentrates or plasma. The in vivo recovery of VIII:CAg was less than that of VIII:C and the disappearance rate of VIII:CAg was much higher either when concentrates or plasma were given. The half-life of VIII:C was thus about 12 h but of VIII:CAg only about 3 h or less. Six patients with von Willebrand's disease were studied after administration of AHF- Kabi. In contrast to haemophilia A the discrepancy between VIII:C and VIII:CAg disappearance rates was not present in von Willebrand's disease, since both VIII:C and VIII:CAg showed a typical progressive increase. We conclude that factor VIII:C given to haemophilia patients does not behave like native VIII:C, not even when fresh plasma is used. Patients with von Willebrand's disease are capable of forming a normal VIII:C when appropriately stimulated.     

Simplified immunoradiometric assay for factor VIII coagulant antigen

A simplified, non-competitive, solid phase immunoradiometric assay has been developed for the quantitation of factor VIII coagulant antigen (VIII:CAg)--the antigenic counterpart of FVIII coagulant activity (VIII:C). Both homologous and heterologous antibodies to human factor VIII (FVIII) were used in this assay. Initially, FVIII in a test sample was attached to immobilized, human IgG obtained from a polytransfused haemophilia A patient with a high titre antibody to VIII:C. The bound FVIII was then detected using rabbit 125I-IgG specific for human FVIII. The concentration of VIII:CAg correlated well with VIII:C levels in the plasma from normal donors (r = 0.84, n - 15). Homozygote von Willebrand's disease patients had undetectable levels of VIII:CAg in their plasma. Patients with severe haemophilia A (VIII:C less than 0.01 u/ml) could be divided into groups on the basis of the VIII:CAg levels, i.e. those having undetectable VIII:CAg and other with measurable VIII:CAg. VIII:CAg detected in normal serum was less than 0.002 u/ml. In this assay the use of human antibody to FVIII is considerably decreased compared to other methods for VIII:CAg, and the time-consuming steps to immunopurify human anti-FVIII antibody are eliminated.     

Poor response to danazol in hemophilia

We gave danazol (600 mg/day orally for 14 days) to eight adults with mild or moderate hemophilia A, one with severe hemophilia A, and one with moderate hemophilia B. In the patient with severe hemophilia A, the levels of factor VIII two to four days after an infusion of factor VIII concentrate were higher than expected, suggesting a prolonged half- life. In one patient with mild hemophilia A, a questionable slight increase in factor VIII was noted at the end of the study. No change was seen in factor levels of other subjects. Therapy was terminated early, at eight days, in a patient who developed severe muscle cramps, and at ten days in a patient with a severe rash. Another patient developed hepatic dysfunction three days after completing the 14-day trial. In this trial, the side effects of danazol outweighed its meager and questionable benefits.     

A female hemophilia A combined with hereditary coagulation factor XII deficiency: a case report.

A 2-year-old Japanese girl with easy bruising and arthropathy was demonstrated to have severe hemophilia A (Factor VIII activity: less than 0.01 U/ml). She had normal 46XX karyotype. Her brother also had hemophilia A, and her mother and grandmother seem to be hemophiliac carriers. Additionally, activated partial thromboplastin time (APTT) of the patient was disproportionately prolonged and there were reduced levels of coagulation factor XII in the patients and members of the maternal trait which are compatible with heterozygous factor XII deficiency. Her father had both normal factor VIII and factor XII levels. Southern blotting analysis of genomic DNA from the propositus and family members with factor VIII and factor XII DNA probes revealed no gross alterations. This patient represents a female hemophilia A combined with heterozygous factor XII deficiency. Nonrandom inactivation of a normal X-chromosome (extreme lyonization) may be the basis for the expression of hemophilia A in this female patient.     

Current Status of Swedish Hemophiliacs

ABSTRACT. A comprehensive survey of the Swedish hemophilic population (hemophilia A and B) was undertaken in 1980. The total number of known hemophiliacs was 564. The ratio of hemophilia A to B was 4:1. The severe form constituted about 30% of each type. The incidence has remained fairly constant since 1957 and was 1.67 per year per 10000 males born. The prevalence was 6.8 per 100000 inhabitants. There was an increase in the prevalence in all counties but the increase varied considerably, 19–383%. The overall increase of hemophilia was 123%. Among mild cases the increase was 245%, whereas it was only 48% among the severe hemophiliacs. This skewness was attributed to better laboratory diagnoses and increased life expectancy. The proportion of mild cases increased from 35% in 1960 to 54% at the time of the present investigation. The situation was the reverse for the severe cases, a decrease from 45% to 30%. The mean and median ages among moderate and mild hemophiliacs deviated only little from the corresponding figure in the total population. Among severe cases, however, mean and median age were 8–13 years less than in the normal population.     

Degradation of factor VIII coagulant antigen by proteolytic enzymes

S ummary . The factors responsible for the lability of factor VIII coagulant activity (VIII:C) and factor VIII coagulant antigen (VIII:CAg) are poorly understood. In this study the VIII:C and VIII:CAg are studied after incubation with plasmin, trypsin or α-chymotrypsin. Both isolated human VIII:CAg and VIII:CAg associated with factor VIII-related antigen (VIII R:Ag) are evaluated. The antigenic sites of the VIII:CAg are somewhat more stable to the action of these enzymes than the functional activity, although both follow a generally parallel degradation. A biphasic decay curve is seen in the initial time points. No stabilization of the functional or antigenic reactivity is observed in the presence of the VIII R:Ag.
Lower concentrations of each enzyme cause an initial rise in the factor VIII:C in the presence of VIII R:Ag, but not in the isolated VIII:CAg. Higher concentrations of α-chymotrypsin cause activation of VIII:C and a slight decrease in the VIII:CAg values in both preparations. These enzymes may play a modulating role in the coagulation cascade through the activation and degradation of VIII:C and VIII:CAg.     

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.     

The effect of liver disease on factors V, VIII and protein C

The components of the factor VIII complex were estimated by immuno- and bioassays in 85 patients with liver disease. The plasma concentrations of the antigens were elevated in 65% (VIII:CAg) and in 76% (VIIIR:Ag) of patients while the biological activities were elevated in only 14% (VIII:C) and 15% (VIII:RiCof). There was no correlation with C-reactive protein, used as a measure of an acute phase reaction (X2 = 0.7; P = 0.1); or with severity of liver disease as judged by prothrombin ratio (P = 1.0) but highest values were observed in patients with cholestatic liver disease. Following parenteral vitamin K there was a significant fall in both the biological activity of VIIIC (36%) and of VIII:CAg (38%) in 13 vitamin K deficient patients (P less than 0.001) but no change in 23 vitamin K replete patients or in the VIIIR:Ag levels in either group. Factor V levels were lower in patients with parenchymal liver disease (0.54 +/- 0.1 units/ml, mean +/- SEM, n = 12; normal range 0.5-1.5 units/ml) than in patients with extrahepatic cholestasis who were vitamin K deficient (1.2 +/- 0.1 units/ml, P less than 0.0001). The levels of protein C antigen, the vitamin K dependent protease which inactivates factors VIII:C and V, was at the lower end of the range in both groups (0.7 +/- 0.1, mean +/- SEM, n = 18, normal range 0.74-1.4 units/ml). There was no significant change in either protein C antigen or factor V following vitamin K. The discrepancy between the biological activity of factor VIII and the antigen levels could represent accumulation of partially degraded factor VIII or production of a hypoactive form. There is no evidence that the reduction in VIIIC and VIII:CAg following vitamin K was mediated by protein C.     

Severe hemophilia and physiologic inhibitors of coagulation.

Patients with hemophilia demonstrate quite variable clinical phenotype even in cases with the same level of deficient factor or the same molecular abnormality. Different interacting factors including congenital and acquired alterations of coagulation inhibitors can modulate both clinical expression and severity of hemophilia. In this study, plasma levels of factor VIII (FVIII), factor IX (FIX) as well as protein C (PC), protein S (PS), and antithrombin (AT) plasma levels were measured in 80 patients with severe hemophilia A and B. Patients were divided into two groups according to the risk of bleeding: the first group (n = 32) with mild bleeding (< 2 bleeds/year), and the second group (n = 48) with severe bleeding (> or = 2 bleeds/year). Both hemophilia groups showed significantly decreased PC plasma levels compared to levels in healthy control subjects (the first group: p < 0.0001 and second group: p < 0.01). The difference in PC plasma levels between the first and second hemophilia group was significant (p < 0.05). Moreover, there was positive correlation between age and the functional PC in both hemophilia groups. Our results suggest that decreased PC plasma levels can testify to a slightly protective effect of the PC pathway on the severity and frequency of bleeding in patients with severe hemophilia A and B.     

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.