Characterization of F8 defects in haemophilia A in Pakistan: investigation of correlation between mutation type and the in vitro thrombin generation assay

Hereditary haemophilia A is an X‐linked bleeding disorder caused by mutations in the coagulation factor VIII gene (FVIII abbreviates protein, gene symbol F8). The mutation spectrum has been reported in various populations but not in Pakistan. The aims of this study were to (i) characterize F8 mutations in a large haemophilia A cohort from Pakistan and to (ii) investigate whether in vitro thrombin generation (TG) differs according to mutation type (null compared with missense) in severe haemophilia A. One hundred individuals diagnosed with haemophilia A and 100 healthy controls were recruited in Pakistan. Phenotypic measurements were re‐evaulated in Cardiff; the essential regions of F8 were screened for the causative defect. A diagnosis of haemophilia A was confirmed for 92 individuals, 7 were found to have haemophilia B and 1 did not have haemophilia. The F8 defects were characterized for 80 of the 92 haemophilia A individuals and comprised point mutations, inversions (intron 22 and intron 1) and frameshifts. Point mutations (41%) were the most frequent, followed by the intron 22 inversion (20%). Thirty novel variants were identified. Comparison of in vitro TG parameters [velocity index (VI) and peak] was made between severe individuals who had a null mutation (no FVIII) and those with a missense change (dysfunctional FVIII), no significant difference was observed. The spectrum of F8 defects in Pakistan is heterogenous; VI and peak in severe haemophilia A are not influenced by whether the underlying mutation gives rise to dysfunctional FVIII or no coagulation factor at all.     

Thrombin generation in haemophilia A patients with mutations causing factor VIII assay discrepancy

Summary. Up to 40% of patients with mild haemophilia A have a discrepancy whereby factor VIII (FVIII) measurements by a two‐stage chromogenic assay (FVIII:C_CH) are disproportionately reduced compared with the FVIII one‐stage clotting value (FVIII:C). Which assay best reflects the coagulation potential and clinical phenotype in this patient group is of clinical significance, yet remains unclear. We have assessed the global coagulant ability of haemophilia patients with FVIII assay discrepancy using calibrated automated thrombography (CAT). A total of 18 patients with mutations Arg531His/Cys or Arg698Trp causing FVIII discrepancy were investigated, together with 12 haemophilia patients with concordant FVIII values and 15 normal controls. Factor VIII levels in all patients and controls were measured using both one‐stage clotting assay and two‐stage chromogenic assay. Thrombin generation was assessed in platelet‐poor plasma by CAT using a low tissue factor concentration (1 pm ). FVIII:C_CH values were below normal in all patients, and in the discrepant group were between 1.5‐ and 8‐fold lower than FVIII:C values. CAT parameters were affected in all haemophilia patients. The endogenous thrombin potential (ETP) was reduced to 58–67% of the mean normal value (1301 nm min^?1), whereas peak thrombin was further reduced to 27–30% of the mean normal value (178 nm ) in both discrepant and concordant patient groups. Analysis of the discrepant patient group showed the most significant correlation between the one‐stage FVIII:C assay and ETP (r² = 0.44) and peak thrombin parameters (r² = 0.27).     

Thrombin generation and phenotypic correlation in haemophilia A

The clinical phenotype of patients with haemophilia A (HA) often differs between individuals with the same factor VIII (FVIII) gene defect (e.g. within the same family) or the same coagulant activity of FVIII (FVIII:C). We proposed that because the thrombin generation assay in platelet-poor plasma of HA patients provides more information [peak thrombin concentration, endogenous thrombin potential (ETP), rate of thrombin generation and lag-time] than a clot-based FVIII assay it might provide insight into these differences. We therefore investigated the relation between the results of the thrombin generation assay and the clinical severity in nine families with HA (23 patients with different phenotypes). We also examined the contribution of prothrombotic risk factors: (FV Leiden G1691A and prothrombin G20210A), the coagulant activity of FVIII and tissue factor (5'UTR) polymorphisms. Our data detect marked differences between individuals but these did not correlate with the reported clinical phenotype. These differences were also reflected in a marked difference in response to the therapeutic amounts of FVIII. This might account for differences in amounts of treatment consumption. Reduced peak and possibly rate of thrombin generation, rather than FVIII:C or ETP appear to represent the critical defects in FVIII-deficient plasma. We suggest that the analysis of parameters in thrombin generation is a useful tool to detect bleeding tendency in HA but not to predict the modulation of the haemorrhagic tendency in patients within families. However the presence of the other factors such as vessel wall components, protein C and platelets might need to be incorporated into this system.     

Feasibility of using thrombin generation assay (TGA) for monitoring of haemostasis during supplementation therapy in haemophilic patients without inhibitors

Monitoring factor replacement treatment and observing concordance with clinical haemostasis is crucial in vital haemorrhages and major surgeries in haemophilic patients. We aimed to investigate the value of the thrombin generation assay (TGA) and thromboelastography (TEG) for monitoring haemostasis in haemophilic patients during factor replacement treatment. The study group consisted of 29 patients (21 haemophilia A, 8 haemophilia B). All the patients FVIII‐inhibitor were negative. A total of 35 bleeding episodes and/or surgical interventions were evaluated. aPTT, FVIII/FIX activity, TEG and TGA tests were conducted before and after factor therapy during the bleeding episode or surgical prophylaxis of haemophilic patients. Correlations among these tests were evaluated and compared with clinical responses. No correlation was found among aPTT, factor activities and clinical outcome. There were also no correlation found between TEG parameters and clinical outcome. The only significant correlation found between TGA parameters and clinical outcome was the correlation between peak thrombin. In conclusion, we found superiority of TGA‐peak thrombin over other traditional tests for monitoring haemostasis in haemophilic patients in this study.     

Monitoring bypassing agent therapy – a prospective crossover study comparing thromboelastometry and thrombin generation assay

The aim of this study was to evaluate the capability of thromboelastometry (ROTEM) and thrombin generation assay (TGA) to monitor the treatment response of bypassing agent (BPA) therapy and to study whether one method is superior to another. In a prospective crossover study haemophilia A patients with high titre inhibitors were included to receive a dose of 75 U kg^?1 activated prothrombin complex concentrates (aPCC) intravenously. Blood sampling was performed at baseline, 15, 30 min, 1, 2, 3 and 4 h post‐infusion for TGA and ROTEM analysis. After a washout period of 14 days the subjects received recombinant FVIIa (rFVIIa) at a dose of 90 μg kg^?1 and similar blood sampling was performed. Healthy subjects were used as controls. Six haemophilia A patients with inhibitors were included. We found that TGA parameters endogenous thrombin potential (ETP) and peak thrombin increased 2–3 folds from baseline 15–30 min after infusion. ROTEM parameters MaxVel and maximum clot firmness increased to a level comparable to that of healthy controls. An individual difference in response was observed for different parameters among participants. ETP and peak thrombin were almost two‐fold greater following aPCC infusion compared to rFVIIa, whereas ROTEM parameters showed no difference in response between the two products. The study showed that ROTEM and TGA have a great potential to evaluate the effect of BPA in haemophilia patients with inhibitors. TGA seemed to be more sensitive than ROTEM in reflecting the difference in treatment response between aPCC and rFVIIa. Additional prospective clinical studies are needed to clarify which assay and what parameters are clinically predictive.     

Fresh frozen plasma transfusion in patients with cirrhosis and coagulopathy: Effect on conventional coagulation tests and thrombomodulin-modified thrombin generation

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Factor VIII and thrombin generation assays: relevance to pharmacokinetic studies in haemophilia A

Summary.  This article reviews the problems associated with traditional Factor VIII (FVIII) assays in pharmacokinetic studies, and the advantages, disadvantages, and technical aspects of thrombin generation assays as a replacement or additional tool.     

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.     

Combined administration of FVIII and rFVIIa improves haemostasis in haemophilia A patients with high‐responding inhibitors – a thrombin generation‐guided pilot study

Treatment of haemophilia A patients with inhibitors is challenging, and may require individually tailored regimens. Whereas low titre inhibitor patients may respond to high doses of factor VIII (FVIII), high‐responding inhibitor patients render replacement therapy ineffective and often require application of bypassing agents. Thrombin generation (TG) assays may be used to monitor haemostasis and/or predict patients' response to bypass agents. In this study we defined by TG, the potential contribution of FVIII to recombinant activated factor VII (rFVIIa)‐induced haemostasis in inhibitor plasma. Based upon results, prospectively designed individual regimens of coadministration of rFVIIa and FVIII were applied. Plasma samples from 14 haemophilia patients with inhibitors (including high titre inhibitors) were tested. The response to increasing concentrations of FVIII, rFVIIa or both was assayed by TG. Eight patients, chosen following consent and at physician's discretion, comprised the combined FVIII–rFVIIa therapy clinical study cohort. Combined spiking with FVIII/rFVIIa improved TG induced by rFVIIa alone in all inhibitor plasmas. Combined rFVIIa and FVIII therapy was applied during bleeding or immune tolerance to eight patients, for a total of 393 episodes. Following a single combined dose, 90% haemostasis was documented and neither thrombosis nor any complications evolved. During study period decline of inhibitor levels and bleeding frequency were noted. Pre‐analytical studies enabled us to prospectively tailor individual therapy regimens. We confirmed for the first time that the in vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved haemostasis and may safely be applied to inhibitor patients.     

Thrombin generation as objective parameter of treatment response in patients with severe haemophilia A and high‐titre inhibitors

In Mexico, 15% of haemophilia A (HA) patients develop inhibitory alloantibodies in response to replacement therapy with factor VIII (FVIII), requiring bypass therapy such as activated prothrombin complex concentrate (APCC). Because bypass therapy has not been broadly available in Mexico even in recent years, this study aimed to evaluate the thrombin generation assay (TGA) in assessing the response to FVIII or APCC treatment in patients with severe HA positive to inhibitors. We studied 189 patients with severe HA. Clinical severity was verified by one‐stage APTT‐based clotting assay. Inhibitors to FVIII were investigated by the Nijmegen–Bethesda (N–B) method, and type of inhibition was assessed through serial plasma dilutions. Thrombin generation was measured with the calibrated automated thrombogram in inhibitor‐positive plasmas previously spiked and incubated with FVIII or APCC. Data were analysed using anova , Student or Fisher's exact tests. We detected 47 (24.9%) subjects with high‐titre (5–1700 N–B U mL^?1) and 25 (13.2%) subjects with low‐titre inhibitor antibodies (0.6–4.7 N–B U mL^?1). We found an association between kinetic behaviour and clinical response to FVIII (P = 0.0049) or vs. FVIII response evaluated with TGA (P = 0.0007). Global concordance between clinical and in vitro response was 70%. By evaluating the capacity of thrombin formation in a plasma sample, TGA predicts the response to FVIII or APCC therapy and allows individual optimization of resources in patients with severe HA and high‐titre inhibitors. The inhibition pattern of the antibodies to FVIII:C correlated with the TGA parameters and showed an association with the clinical response to FVIII.