Factor VIII Activity in Haemophilic Plasma unmasked by Synthetic Organic Anions

HAEMOPHILIA A is generally regarded as a hereditary deficiency of antihaemophilic globulin (factor VIII). Some investigators, however, believe that antihaemophilic globulin may be inhibited rather than deficient. Most recent studies with monospecific antisera against antihaemophilic globulin have shown that patients with haemophilia A possess normal amounts of factor VIII, but which is biologically inactive^2,3. Furthermore, treating plasma from patients with haemophilia A and von Willebrand's disease with succinic acid produced protein fractions with antihaemophilic activity separable by chromatography⁴. These observations indicate that haemophilia A might well be caused by a defective or inhibited factor VIII molecule.     

Plasma exchange and human factor VIII concentrate in managing haemophilia A with factor VIII inhibitors.

Plasma exchanges were combined with human factor VIII concentrate therapy in the treatment of major bleeding episodes in five patients with haemophilia A and factor VIII inhibitors. All patients had a good clinical response to combined treatment. Inhibitor levels showed satisfactory falls before rapid secondary increases of inhibitor levels took place. A sixth patient with von Willebrand''s disease and a factor VIII clotting activity inhibitor was successfully prepared for operation using plasma exchange. Postoperative haemostasis and healing were normal. In two patients the plasma exchanges were relatively more effective than the administered human factor VIII in reducing the levels of factor VIII inhibitor. Combined plasma exchange and human factor VIII treatment may offer a rapidly effective means of reducing factor VIII inhibitor levels in this group of patients, together with significant saving of costs.     

Factor-VIII-related antigen: measurement by enzyme immunoassay.

Factor-VIII-related antigen was measured, both by an enzyme immunoassay using a microplate method and by the Laurell technique, in normal people, patients with von Willebrand''s disease, haemophiliacs, and obligatory haemophilia carriers. The enzyme immunoassay was simpler to perform and gave equally reliable and reproducible results. Many more assays could be carried out at any one time.     

Factor VIII--related protein on vascular intima of patients with chronic renal failure and prolonged bleeding times.

To determine whether the prolonged bleeding time so common to chronic renal failure (CRF) was due to defective factor VIII-related activities, as in von Willebrand''s disease, vascular-factor VIII-related protein was measured in patients with CRF. Factor VIII-related protein was detected by immunofluorescence on the vascular intima of all 13 patients with CRF and greatly prolonged bleeding times. This protein was also present on the vascular intima of a patient with CRF and moderate von Willebrand''s disease. These findings support a previous suggestion that the disturbed haemostasis in patients with CRF is not linked to defective factor VIII-related activities.     

Arthrocentesis of the Knee in Acute Hemophilic Arthropathy

In 27 children and young adults with hemophilia presenting acutely painful distended intra-articular hemorrhages of the knee, aspiration was carried out and the patients were followed for a minimum of 24 months. Seventeen patients with classical hemophilia were found to have less than 1 percent of normal plasmal level of antihemophilic factor (AHF). Of the remainder, five were Factor IX, plasma thromboplastin component (PTG), deficient, whereas two patients had Von Willebrand''s disease. Aspiration was routinely done in an outpatient clinic, followed by immediate discharge with return to regular activity levels within 48 hours. There were no infections nor rehemorrhages attributable to aspiration technique.     

Preparation and platelet aggregating activity of I-labeled ristocetin

The antibiotic ristocetin is a glycoproteoid isolated from the culture media of Nocardia lurida N.R.R.L. 2430 whose structural features have yet to be resolved (1–3). The antibiotic has been studied both for its antibiotic properties (4–6) and, more recently, for its inducement of platelet aggregation in humans, both in vivo and in vitro (7–9). This latter phenomena resulted in the discontinued use of ristocetin in antibiotic therapy (10), but has provided a useful tool for studying the bleeding disorder, von Willebrand's disease. The disease is genetic in origin and is characterized by the lack of a functional plasma protein believed to be required for the adhesion of platelets to vascular epithelia during primary thrombotic events (11). This plasma protein, termed von Willebrand's factor, is also necessary to support ristocetin-induced platelet aggregation of human platelets (12). Thus, ristocetin has found increasing use not only in facilitating the diagnosis of von Willebrand's disease but also in characterizing the plasma protein (13).     

Aspirin-Induced Prolongation of the Ivy Bleeding Time—Its Diagnostic Usefulness

Ivy bleeding time values before and two hours after ingestion of 600 mg of aspirin (aspirin tolerance test) were studied in normal persons, in patients with a disorder of primary hemostasis and in patients with various coagulation factor deficiencies. Aspirin produced a significant prolongation of the bleeding time in patients with von Willebrand''s disease, uremia, and primary platelet disease, and in two patients with Factor XI deficiency. Dextropropoxyphene hydrochloride caused no prolongation of the bleeding time in normal persons.     

Laboratory evaluation of a bleeding patient.

Most causes of abnormal bleeding can be determined from a complete blood count including platelet count and bleeding, prothrombin, activated partial thromboplastin, and thrombin times. Occasionally, further evaluation is necessary, such as tests of factor XIII function, fibrinolysis, and vascular integrity. Possible diagnoses include disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, vitamin K deficiency, von Willebrand''s disease, heparin-induced thrombocytopenia, acquired inhibitors of factor VIII, lupus anticoagulants, and coagulation disorders related to the acquired immunodeficiency syndrome.     

Immunologic dysfunction in patients with classic hemophilia receiving lyophilized factor VIII concentrates and cryoprecipitate.

The occurrence of the acquired immune deficiency syndrome (AIDS) in patients with hemophilia has suggested that an infectious agent transmitted through the frequent use of pooled blood products could be responsible. To determine if the amount or type of factor VIII preparation alters the risk of acquiring immune defects, three groups of asymptomatic heterosexual men were studied: 34 with severe classic hemophilia who were receiving lyophilized factor VIII concentrate, 10 with either mild classic hemophilia or moderately severe von Willebrand''s disease who were receiving cryoprecipitate and 22 normal men who served as controls. Anergy was noted in 68%, 57% and 5% respectively of the three groups. In comparison with the control group, the group treated with lyophilized factor VIII concentrate had a significantly decreased mean ratio of helper to suppressor T lymphocytes, poor responses of the lymphocytes to mitogens, high unstimulated background activity of these cells and significantly elevated serum IgG levels. Although some of the patients with classic hemophilia who were treated with cryoprecipitate were also anergic, they did not manifest these in-vitro abnormalities. The data indicate that a majority of apparently immunocompetent individuals with classic hemophilia show in-vivo and in-vitro evidence of impaired cellular immunity and may be at risk for the development of opportunistic infections and neoplasms.     

Immunological Characterization of Purified Anti-Haemophilic Factor A (Factor VIII) which corrects Abnormal Platelet Retention in Von Willebrand's Disease

ABNORMAL platelet retention in a column of glass beads, which has been described in cases of Von Willebrand's disease (VWD)^1–6, is corrected in vitro by cryoprecipitate prepared from normal plasma and partially purified factor VIII concentrates^6–9 although fibrinogen is ineffective (unpublished results). This suggests that factor VIII is responsible for the correction. We now have further evidence to support this suggestion.     

Shear stress-induced binding of von willebrand factor to platelets

Shear stress-induced platelet aggregation requires von Willebrand factor (vWF), platelet glycoprotein (GP) Ib, GPIIb-IIIa, Ca²⁺, and adenosine diphosphate (ADP). Recent reports using vWF labeled with either ¹²⁵I or fluorescein isothiocyanate (FITC) have demonstrated that in shear-fields, vWF binds to both GPIb and GPHb-IIIa. The sequence of the vWF binding to the two platelet receptors has not been precisely determined in these reports. In this study, a flow cytometry technique using a primary anti-vWF antibody and a secondary FITC IgG antibody was used to measure shear stress-induced vWF binding to platelets. Washed normal platelets suspended at 50,000/μl with purified large VWF multimers were exposed to laminar shear stresses of 15 to 120 dynes/cm² for 30 sec. At this low platelet count, little or no aggregation occurred in the shear fields. A significant increase in post-shear vWF-positive platelets was consistently observed. Experiments with platelets from normal and severe von Willebrand's disease (vWD) (which lack plasma and platelet α-granule vWF) demonstrated that exogenous vWF predominately contributed to the platelet-vWF binding. Blockade of platelet GPIb with the monoclonal anti-GPIb antibody, 6D1, completely inhibited shear stress-induced platelet-vWF attachment. In contrast, blockade of GPIIb-IIIa with monoclonal anti-GPIIb-IIIa antibodies, 10E5 or c7E3, or with the GPIIb-IIIa-blocking tetrapeptide, RGDS, had little or no inhibitory effect on platelet-vWF binding. These data demonstrate that the binding of vWF to GPIb is likely to be the initial shear-induced platelet-ligand binding event. © 1997 Elsevier Science Ltd     

Immunological Studies of Factor VIII (Antihaemophiliac Globulin) in Haemophilia A

PATIENTS with haemophilia A have recently been divided into two groups; one characterized by a functionally defective factor VIII (AHG) molecule in the plasma, which is immunologically similar to normal factor VIII but lacks procoagulant activity^1–3, while the other seems to represent a true deficiency of factor VIII, for both procoagulant and immunological properties of factor VIII are absent. These conclusions are based on the ability of normal and some haemophiliac plasma to neutralize human antibodies from patients with spontaneously occurring inhibitors against factor VIII or from multi-transfuscd haemophiliacs who have developed circulating inhibitors directed against factor VIII. These studies have divided haemophilia A into those with cross reactive material (CRM +) and those without (CRM ?). Other methods of characterizing this genetic polymorphism in haemophilia have not been reported. We have used immunoelectrophoresis and antibody neutralization with a heterologous antibody to show a similar division of haemophilia A into a small group of CRM+ (15%) and a larger group of CRM? (85%). Immunoelectrophoresis and antibody neutralization studies have also been described in factor X polymorphism⁴.     

Proteomic Analysis of Haptoglobin and Amyloid A Protein Levels in Patients with Vivax Malaria

Advancements in the field of proteomics have provided great opportunities for the development of diagnostic and therapeutic tools against human diseases. In this study, we analyzed haptoglobin and amyloid A protein levels of vivax malaria patients with combinations of depletion of the abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, and mass spectrometry in the plasma between normal healthy donors and vivax malaria patients. The results showed that the expression level of haptoglobin had become significantly lower or undetectable in the plasma of vivax malaria patients due to proteolytic cleavage when compared to healthy donors on 2-DE gels. Meanwhile, serum amyloid A protein was significantly increased in vivax malaria patient''s plasma with high statistical values. These 2 proteins are common acute phase reactants and further large scale evaluation with a larger number of patient''s will be necessary to establish the possible clinical meaning of the existential changes of these proteins in vivax malaria patients. However, our proteomic analysis suggests the feasible values of some plasma proteins, such as haptoglobin and serum amyloid A, as associating factor candidates for vivax malaria.     

Plasma Levels of Immunoreactive Corticotrophin in Patients with Cushing's Syndrome

Plasma levels of immunoreactive corticotrophin (A.C.T.H.) have been determined in 56 patients with Cushing''s syndrome by means of a homologous radioimmunoassay. In untreated Cushing''s disease (bilateral adrenal hyperplasia due to excessive A.C.T.H. secretion from the pituitary) plasma values ranged from 40 to 200 μμg./ml., between 8 and 10 a.m., compared with a range in normal subjects of 12 to 60 μμg./ml. Considerably raised levels, often above 2,000 μμg./ml., were found in patients with Cushing''s disease after bilateral adrenalectomy. A.C.T.H. concentrations were usually higher in patients with bilateral adrenal hyperplasia associated with ectopic A.C.T.H. production than in patients with untreated Cushing''s disease; whereas plasma A.C.T.H. was undetectable in the presence of an adrenocortical tumour. All patients with Cushing''s syndrome failed to show the normal circadian rhythm of circulating A.C.T.H. levels.     

Diagnostic value of 9 am plasma adrenocorticotrophic hormone concentrations in Cushing's disease.

Morning plasma adrenocorticotrophic hormone (ACTH) concentrations were measured in 58 normal subjects and seven patients with pituitary-dependent Cushing''s syndrome (Cushing''s disease). Particular note was taken of the time of venepuncture. The range of values for the normal subjects irrespective of timing was 9-77 ng/l. The range between 9 0 am and 9 30 am was 9-24 ng/l. In the patients with Cushing''s disease the ACTH concentrations were in the range 39-109 ng/l. To distinguish patients with Cushing''s disease from normal subjects it is therefore important to define accurately the 9 am normal range, since these results show no overlap.     

A Screening Test for Wilson's Disease and its Application to Psychiatric Patients

Varied modes of onset make the early diagnosis of Wilson''s disease difficult. A deficiency of serum ceruloplasmin, usually characteristic of the disease, was used as the basis for a screening test. Simple test materials and provision for handling about 50 plasma samples simultaneously made this test feasible for large-scale screening.The screening test was applied to 336 persons hospitalized for psychiatric disorders, to detect patients with Wilson''s disease before the classical symptoms appeared. Two patients with ceruloplasmin levels below the normal limits were detected but did not have Wilson''s disease. Further application of the screening test to relatives of patients known to have Wilson''s disease and to individuals with any symptoms of the disease (hepatic disease, extrapyramidal dysfunction, psychiatric disorders, behaviour problems in children) would aid in early diagnosis and more effective treatment.     

Cellular immunity to Hodgkin's splenic tissue measured by leucocyte migration inhibition.

Patients with lymphoreticular malignancy were shown by a leucocyte migration inhibition technique to have cellular immunity to Hodgkin''s splenic tissue. Migration was significantly inhibited in 31 out of 55 patients with Hodgkin''s lymphoma and 19 out of 39 patients with other types of lymphoma. Inhibition was also shown in only three out of 29 patients with other malignancy, one out of 23 normal volunteers, and one out of 25 patients with non-malignant disease. The splenic factor that inhibits leucocyte migration, which has yet to be isolated and identified, may be a helpful diagnostic tool in patients with suspected lymphoma.     

Effect of Hormonal Therapy on Plasma Testosterone Levels in Prostatic Carcinoma

Plasma concentrations of testosterone were estimated in normal men, in patients before treatment for prostatic cancer, and in patients who had had various forms of endocrine treatment for prostatic carcinoma. There was no decline in plasma testosterone levels with age. Patients with non-metastatic disease had levels similar to those of normal controls, but in advanced metastatic disease the levels were low. After orchidectomy the plasma testosterone level fell to that found in normal women. In every patient stilboestrol in doses as small as 1 mg three times a day suppressed plasma testosterone at first to negligible amounts, irrespective of the clinical response. Subsequently a small but significant rise in the concentration was always observed over a period of six months'' oestrogen therapy. Pituitary ablation with yttrium-90 lowered the plasma testosterone concentration again to negligible amounts in patients who had been on stilboestrol. In advanced metastatic disease this was often associated with relief of pain. Preliminary studies with aminoglutethimide indicate that it can produce biochemical and clinical effects similar to those of pituitary ablation.     

Characterisation of a novel high-purity, double virus inactivated von Willebrand Factor and Factor VIII concentrate (Wilate)

This study summarises the biochemical and functional properties of a new generation plasma-derived, double virus inactivated von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate, Wilate, targeted for the treatment of both von Willebrand disease (VWD) and haemophilia A. The manufacturing process comprises two chromatographic steps based on different performance principles, ensuring a high purity of the concentrate (mean specific activity in 15 consecutive production batches: 122 IU FVIII:C/mg total protein) and, thus, minimising the administered protein load to the patient (specification: < or = 15 mg total protein per 900 IU Wilate). The optimised solvent/detergent (S/D) treatment and prolonged terminal dry-heat (PermaHeat) treatment of the lyophilised product at a specified residual moisture (RM) provide two mechanistically independent, effective and robust virus inactivation procedures for enveloped viruses and one step for non-enveloped viruses. These process steps are aggressive enough to inactivate viruses efficiently, but yet gentle enough to maintain the structural integrity and function of the VWF and FVIII molecules, as proven by state-of-the-art assays covering the diverse features of importance. The VWF multimeric pattern is close to the one displayed by normal plasma, with a consistent content of more than 10 multimers, but a relatively lower portion of the very high multimers. The multimeric triplet structure is normal, underlining the gentle and effective manufacturing process, which does not require the addition of protein stabilisers at any step. The balanced activity ratio of VWF to FVIII is close to that of plasma from healthy subjects, rendering Wilate suitable also for the safe and effective treatment of patients with VWD.     

Assay of a placental protein to determine fetal risk.

Plasma concentrations and total amounts of pregnancy associated plasma protein A were determined in 272 patients at 34 weeks'' gestation by immunoelectrophoretic assay. The mean plasma concentration and mean total amount of this protein were closely related (r = 0.9643) and were significantly raised in patients who subsequently developed pre-eclampsia (28 patients), went into premature labour (12), or suffered from antepartum haemorrhage (10). Mean values in all patients delivering growth-retarded babies were also raised, but when the results for such patients who also had other complications were excluded there were no differences between the sets of means. The assays were easily performed, and they may be a useful technique for screening pregnant women to detect those at risk of developing pre-eclamptic toxaemia, although the full potential of these assays cannot be realised until the protein''s function is known.