Some laboratory and clinical aspects of haemophilia A.

Abstract

The hereditary bleeding disorder haemophilia A is caused by a deficiency or functional defect of coagulation factor VIII (FVIII). FVIII replacement therapy enables effective treatment of bleeding episodes. However, the care for these patients remains complex, particularly from challenges arising from the development of inhibitors (allo-antibodies) to infused FVIII. To further advance current practice in the management of Haemophilia A, a better understanding of the molecular pathogenesis of the disease is required, including the study of functional epitopes and immunogenicity of the FVIII molecule. In addition, the evaluation of coagulation deficiency by the effect of FVIII deficiency on the thrombin generation profile in a thrombin generation assay may provide novel insights into the usefulness of the thrombin generation assay (TGA) in the characterisation of, in particular, the milder form of Haemophilia A. In contrast to severe haemophilia where haemophilic osteoarthropathy is common in multiple joints (particularly knees, ankles, shoulders and elbows), we have observed that in our patients with mild haemophilia arthropathy occurs only in the ankle joints, and is often disabling. We considered that the incidence of ankle arthropathy in the milder form of haemophilia A should be properly studied. The inhibitors are often directed against several dominant epitopes in the A2 and C2 domains of the FVIII molecule. We undertook the expression of recombinant FVIII peptides with the amino acid sequence of the C1 and C2 domains in mammalian cell expression systems. We were successful in expressing the C1 and C2 peptides in COS-7 cells. However the peptides were mostly confined to the cell pellet. The small amounts excreted into the supernatant were insufficient for further work that was planned to examine the ability of the peptides to neutralise FVIII inhibitors. We also obtained a murine monoclonal anti-FVIII antibody by immunising mice with recombinant human FVIII and performing fusion experiments with harvested spleen cells and myeloma cells. The monoclonal antibody did not inhibit factor VIII function, indicating that it may be against a non functional epitope of factor VIII. The recombinant C1 or C2 peptides bound to the murine polyclonal anti-FVIII antibodies (post-immunisation mouse serum) in Western blot, but not to the purified monoclonal antibody. To evaluate the thrombin generation assay as a possible method for the laboratory assessment of FVIII deficiency we established an in-house TGA using a fluorogenic substrate. Changes in fluorescence in plasma were measured in a microtitre plate using an automated reader. The development of fluorescence over a time course was evaluated by studying the rate of increase of fluorescence, which was represented by four derived parameters including the lag time, peak time, peak thrombin generation and area under the curve (AUC). We compared the two different reagents to trigger thrombin production in plasma: the activated partial thromboplastin time (APTT) reagent and the tissue factor (TF), and applied both methods in 18 normal subjects and 42 patients with mild/moderate haemophilia A. Using APTT reagent, the lag time (R = -0.72) and peak time (R = -0.72) demonstrated a significant negative correlation with the one-stage FVIII level. In contrast, using TF activation there was a significant positive correlation of the peak thrombin concentration (R = 0.73) and AUC (R = 0.71) to the FVIII level. For the 42 patients with mild/moderate haemophilia A, there were significant correlations between their FVIII levels (either by one- or two-stage assay) and the TGA results by both triggers. A clinical study of patients with mild haemophilia A to document the frequency and severity of arthropathy has not been previously published. We conducted a clinical study on ankle arthropathy in 34 patients with mild/moderate haemophilia A. Clinical and radiological evaluation systems for the assessment of haemophilic arthropathy recommended by the World Federation of Haemophilia (WFH) were used, in addition to the visual analogue scale (VAS) pain score. The prevalence of ankle arthropathy in the study group was 52% by the radiology scoring system alone, and 48% by both the physical and radiological systems. In many of the patients pain and disability were major problems. Of 34 patients, nearly half had constant ankle pain and the severity of pain was moderate to severe in nine patients. The most significant physical finding in the 26 patients with a positive physical ankle score was the loss of free range of motion (ROM) of the ankle joint, characterised predominantly by loss of dorsiflexion. The symptom of ankle pain, present in half of the patients, had a high sensitivity (88%) and specificity (94%) for prediction of ankle arthropathy by the radiology system. The presence and severity of ankle arthropathy was most common in patients with a FVIII level of less than 11 IU/dl by the one-stage assay (6 IU/dl by the two-stage assay). There was a significant relationship between the presence of ankle arthropathy and a history of bleeds into the ankle joint as a child. We conclude that arthropathy of the ankle in these patients is common, is often severe and disabling, and is due to the episodes of bleeding into the ankle joint during childhood. The current study highlights some important areas of research about haemophilia A, both on the molecular biological level of the basic sciences, and the clinical aspects of diagnostic approach and disease severity. For patients with mild/moderate haemophilia, it is now important to recommend the education to ensure the awareness of the risks of ankle arthropathy, and the consideration by clinicians for early and aggressive treatment of ankle bleeding.