Exploring Anti-FVIII Antibodies in Haemophilia A - Role in In Vitro Haemostasis and Clinical Disease

Haemophilia A (HA) is caused by defective synthesis of coagulation factor VIII(FVIII), which has serious effects on haemostasis; joints being the most common site of bleeding. The development of FVIII replacements has improved the situation for patients with haemophilia such that chronic arthropathy can be prevented, and life expectancy and the quality of life have increased. However, approximately 20-30% of patients suffering from severe HA develop neutralizing antibodies (inhibitors) against FVIII. Alternative treatment, using by-passing agents, is available for patients exhibiting inhibitors, although these can only be used for the short-term treatment of acute haemorrhage and as prophylaxis during surgery. Furthermore, the clinical response to by-passing products is unpredictable. Two of the studies included in this thesis evaluated the response to by-passing therapy in plasma from patients with HA. The variation in thrombin production within families was found to be significantly lower than the variation between families, indicating that a familial predisposition may influence thrombin formation in response to by-passing agents (Paper I). Moreover, FVIII clotting factors were found to potentiate the in vitro effect of by-passing agents on thrombin formation in plasma from patients with HA exhibiting inhibitors, indicating that further assessment of this treatment strategy in a clinical context is warranted (Paper II). Not all anti-FVIII antibodies have neutralizing capacity. In the studies presented in Papers III & V, non-neutralizing anti-FVIII antibodies(NNAs) were investigated in two different cohorts, using an enzyme-linked immunosorbent assay (ELISA). NNAs were detected in 18.9% of siblings with HA, and in 12.8% of unrelated HA subjects followed for four years. The antibody response was assayed using three different rFVIII products. The antibody response was found to be heterogeneous, to vary considerably between individuals (Papers III and V), and also over time (Paper V). None of the patients in the cohort with NNAs observed longitudinally developed inhibitors (Paper V). However, in one patient with moderate HA, the detection of Bethesda-negative anti-FVIII antibodies coincided with a change in bleeding phenotype four years prior to FVIII inhibitor development. This finding suggests that immunoassays may be a useful complement in evaluating the immune response to FVIII (Paper IV). The potential clinical impact of NNAs was evaluated in the long term study (Paper V), showing no association between age, F8 mutation, or the influence of immune system challenges on NNA development. Interestingly, patients with NNAs had significantly fewer bleeding episodes than NNA-negative patients (p=0.048), raising questions about the possibility of yet undefined types of anti-FVIII antibodies with protective or potentiating effects on FVIII

At the Cross Section of Thrombotic Microangiopathy and Atypical Hemolytic Uremic Syndrome : A Narrative Review of Differential Diagnostics and a Problematization of Nomenclature

Complement-mediated atypical hemolytic uremic syndrome (aHUS) is a rare disease associated with high mortality and morbidity. Renal biopsies often indicate thrombotic microangiopathy (TMA). The condition is caused by an excessive activation of the alternative pathway leading to depositions of membrane attack complexes (MAC) on host cells. It may depend on mutations in complement components and regulatory proteins, or the formation of complement-specific antibodies. Mainly, an environmental trigger (e.g. infection) is needed for the excessive response to develop. The clinical characteristics are more or less shared with a wide range of diseases manifesting with microangiopathic hemolytic anemia. Because of prior deficits in pathogenic understanding, associated nomenclature has been based on clinical symptoms. New knowledge challenges these symptomatic definitions; however, an outdated terminology is still being applied in clinical practice to various extents. With respect to gained insights, it is more advantageous to rebuild the concepts on etiological and pathogenic grounds. The need for more distinct definitions is even more urgent in the light of the effective treatment regimen with eculizumab for complement-mediated aHUS. This review presents an up-to-date summary of the field of investigation, addresses the need for faster differential diagnostics and proposes a revised nomenclature based on the current pathogenic understanding

Combination of FVIII and by-passing agent potentiates in vitro thrombin production in haemophilia A inhibitor plasma.

The by-passing agents, recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (APCC), are important tools in the treatment of patients with haemophilia A and high-responding inhibitory antibodies. It has been observed clinically that in some patients undergoing immune tolerance induction the bleeding frequency decreases, hypothetically caused by a transient haemostatic effect of infused FVIII not measurable ex vivo. We evaluated how by-passing agents and factor VIII (FVIII) affect thrombin generation (TG) in vitro using plasma from 11 patients with severe haemophilia A and high titre inhibitors. Samples were spiked with combinations of APCC, rFVIIa and five different FVIII products. Combination of APCC and FVIII showed a synergistic effect in eliciting TG (P < 0·005) for four FVIII products. When rFVIIa and FVIII were combined the interaction between the preparations was found to be additive. APCC and rFVIIa were then combined without FVIII, resulting in an additive effect on thrombin production. Each product separately increased TG above baseline. In conclusion, the amount of thrombin formed in vitro by adding a by-passing agent, was higher in the presence of FVIII. Our findings support the use of FVIII in by-passing therapy to optimize the haemostatic effect

Thrombin generation in vitro in the presence of by-passing agents in siblings with severe haemophilia A.

Summary. Previous data have shown an inter-individual difference in the thrombin generating capacity in vitro as well as phenotypic bleeding pattern among patients with severe haemophilia A (FVIII:C activity below 1%). The reason for this is not known. In addition, there are no reports on how thrombin generation may correlate between siblings. In this study, we evaluated and compared thrombin generation in vitro using plasma samples in the presence of by-passing agents (FEIBA((R)) and NovoSeven((R))) in 21 unrelated brother pairs with and without inhibitors enrolled in the Malmö International Brother Study (MIBS). Mean maximum thrombin formation in patients with a current inhibitor titer was 182.0 +/- 52.8 mmol mL(-1) (FEIBA((R))) and 130.7 +/- 54.9 mmol mL(-1) (rFVIIa), respectively, and somewhat higher in those without inhibitors, 222.7 +/-85.5 mmol mL(-1) (FEIBA((R))) and 142.8 +/-53.6mmol mL(-1) (rFVIIa) (P = 0.16 and 0.29). The variance regarding the maximum thrombin production within a family was significantly lower compared with the thrombin production between families (P < 0.001 for both FEIBA((R)) and NovoSeven((R))). Our data indicate that genetically determined factors, other than the FVIII:C activity seems to influence the phenotypic variation in thrombin formation in the presence of by-passing agents. The nature of these determinants remains to be identified

Indications of underdiagnosis of atypical haemolytic uraemic syndrome in a cohort referred to the Coagulation Unit in Malmo, Sweden, for analysis of ADAMTS13 2007–2012

Aim: Complement-mediated atypical haemolytic uraemic syndrome (aHUS) is a rare disease with high mortality and morbidity if left untreated. The diagnostic work-up is complicated and the manifestations overlap with other conditions. Therefore, we hypothesize that complement-mediated aHUS is an under diagnosed disease. Methods: A cohort of 768 referrals referred to the Coagulation Unit in Malmo, Sweden, for analysis of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), 2007–2012, were retrospectively reviewed. Subjects were included on the basis of presence of haemolytic anaemia, thrombocytopaenia, renal failure and ADAMTS13 > 0.05. They were excluded if tested positive for Escherichia coli. Included subjects were categorized as “suspected HUS” with and without potential causes and triggers. Levels of C3 and C4, presence of complement factor H (CFH)-specific antibodies and associated deficiency in complement factor H related protein 1 (CFHR1) were analyzed on frozen samples. Results: In total, 134/316 (42%) unique subjects fulfilled inclusion criteria; 103 were categorized as “suspected HUS associated with potential causes/triggers” and 31 subjects categorized as “suspected HUS” without such association. One case of complement-mediated aHUS had been confirmed during the treatment period. Laboratory analyses performed showed that in total 78 cases had findings consistent with complement-mediated aHUS: 24 cases indicated presence of CFH-specific antibodies whereof five cases had isolated low C3 titres and six cases had deficiency of CFHR1. Additionally 54 cases indicated isolated alternative pathway consumption. Conclusion: The results suggest that the presence of complement-mediated aHUS was under diagnosed in this cohort calling for improvement of diagnostic availability

Bone mineral density in haemophilia – a multicentre study evaluating the impact of different replacement regimens

AimThe aim of this study was to investigate if prophylactic treatment in severe haemophilia impact on bone mineral densisty (BMD) in adults with haemophilia A/B.MethodsSubjects with haemophilia (n = 120) underwent bone-density measurement and clinical data was collected. BMD in subjects with severe haemophilia on high-dose prophylaxis (n = 41) was compared to BMD in subjects with mild haemophilia (n = 33) and to severe haemophilia treated with intermediate-dose prophylaxis (n = 32) or on-demand replacement therapy (n = 14).ResultsSubjects with severe haemophilia on high-dose prophylaxis showed BMD at total hip comparable to subjects with mild haemophilia (median BMD 955.8 and 977.4 mg/cm2 (P = .17), respectively). No difference in BMD was found related to type of prophylactic regimen (median BMD 955.8 and 942.4 mg/cm2, in high-dose and intermediate dose groups, respectively; P = .70). Subjects with severe disease treated on-demand had significantly lower BMD compared to subjects on a high-dose prophylactic regimen (median BMD 771.8 and 955.8 mg/cm2 (P = .001), respectively). BMD decreased significantly with age, regardless of severity of haemophilia disease. In a multivariate analysis, adjusted for disease status and age, type of prophylactic regimen was not significantly associated with osteoporosis development.ConclusionWe show that BMD differs in persons with severe haemophilia on propylaxis as compared to those treated on-demand, but that type of prophylactic regimen does not reflect on BMD. The difference between treatment groups was mainly explained by an age difference between groups. However, patients on prophylaxis displayed a high degree of normal BMD not far from mild haemophilia at comparative age

Antibody formation and specificity in Bethesda-negative brother pairs with haemophilia A.

Antibodies directed towards non-neutralizing epitopes on the factor VIII protein (FVIII) may be detected in patients with haemophilia A. We evaluated the prevalence of non-neutralizing antibodies, in 201 inhibitor-negative brother pairs with severe haemophilia A, enrolled in the Malmö International Brother Study and the Haemophilia Inhibitor Genetics Study. To evaluate binding specificity of the antibodies, ELISA plates were coated with two recombinant full-length (FL) FVIII-products and one recombinant B-domain-deleted (BDD) product. Seventy-nine patients (39.3%) had a history of positive inhibitor titre measured by Bethesda assay, and FVIII antibodies were detected in 20 of them (25.3%). Additional 23 samples from subjects without a history of FVIII inhibitors were ELISA-positive corresponding to a frequency of non-neutralizing antibodies of 18.9%. The antibody response towards the different FVIII products was heterogenous, and was raised not only towards the non-functional B-domain but also towards both FL-rFVIII and BDD-rFVIII. In patients considered successfully treated with immune tolerance induction, 25.4% had remaining FVIII antibodies. The number of families with an antibody response in all siblings was increased when the total antibody response was taken into account, further supporting the concept of a genetic predisposition of the immune response. Further studies and careful monitoring over time are required to appreciate the immune response on the risk of inhibitor development or recurrence in the future