Thrombin generation and fibrinolysis in anti‐factor IX treated blood and plasma spiked with factor VIII inhibitor bypassing activity or recombinant factor VIIa

Summary. Activated prothrombin complex concentrates (aPCC) and recombinant activated factor VIIa (rFVIIa) are two important therapies in haemophilia patients with inhibitors and improve clot stability. We hypothesized that potential differences in procoagulant and fibrinolytic actions of aPCC and rFVIIa may lie in the clot stability against fibrinolytic activation. We used thrombin generation, fluorescence detection and thromboelastometry in anti‐factor IXa (FIXa) aptamer‐treated whole blood (WB) and plasma to evaluate: (i) generation of thrombin and activated factor X (FXa) and (ii) viscoelastic properties of blood clots in the presence of tissue plasminogen activator (tPA) after addition of aPCC (0.4 U mL^?1) or rFVIIa (60 nm ). Peak thrombin generation increased from 85 ± 19 nm in aptamer‐treated plasma to 276 ± 83 nm and 119 ± 22 nm after addition of aPCC and rFVIIa respectively (P < 0.001). FXa activity increased within 20 min by 87 ± 6% and by 660 ± 97% after addition of aPCC and rFVIIa respectively (P < 0.001). TPA‐induced lysis time increased from 458 ± 378 s in aptamer‐treated WB to 1597 ± 366 s (P = 0.001) and 1132 ± 214 s (P = 0.075), after addition of aPCC and rFVIIa respectively. In this haemophilia model using the anti‐FIXa aptamer, the larger amount of thrombin was generated with aPCC compared with rFVIIa, while FXa generation was more rapidly increased in the presence of rFVIIa. Furthermore, clot formation in anti‐FIXa aptamer‐treated WB was less susceptible to tPA‐induced fibrinolysis after adding aPCC compared with rFVIIa.     

Tranexamic acid as adjunct therapy to bypassing agents in haemophilia A patients with inhibitors

Haemophilia patients with inhibitors require bypassing agents (BPA) like activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rFVIIa) to control bleeds. Adjunct tranexamic acid (TXA) may improve haemostasis. The objective of this study was to investigate safety and haemostatic effect of TXA given in combination with BPA. Healthy volunteers (N = 5) and haemophilia inhibitor patients (N = 6) were enrolled in a prospective case crossover design. Controls were treated with TXA 20 mg kg^?1 orally (O.R.) Patients were treated with aPCC 75 IU kg^?1 intravenous (I.V.) on day 1 followed by TXA 20 mg kg^?1 O.R. combined with aPCC 75 IU kg^?1 I.V. on day 2. A 14‐day washout occurred before crossover to rFVIIa 90 μg kg^?1 I.V. ±TXA. Safety evaluation and blood sampling processes were performed at baseline, 15, 30, 60, 120, 180 and 240 min post treatment. Primary outcome was maximum clot firmness (MCF) evaluated by whole blood thromboelastometry using a TF + tissue plasminogen activator‐based assay. Healthy controls showed a 20‐fold increase in MCF following TXA. Adjunct TXA to aPCC or rFVIIa induced a significant increase in MCF (P < 0.0001) reaching levels indistinguishable from healthy controls treated with TXA (P > 0.05). Infusion of aPCC or rFVIIa alone induced only 3–10 fold increase in MCF from baseline, with a decline in MCF starting after 60–120 min. TXA did not increase the endogenous thrombin potential. No clinical or laboratory signs of thromboembolic events, disseminated intravascular coagulation, or hypercoagulability were observed. Combination of aPCC or rFVIIa with TXA normalizes clot stability in haemophilia patients with inhibitor as compared to healthy controls. No clinical or laboratory adverse events were observed.     

An in-vitro assessment of tranexamic acid as an adjunct to rFVIII or rFVIIa treatment in haemophilia A

Haemophilia is characterised by defective thrombin generation, reduced clot stability and spontaneous bleeding. Treatment with factor VIII (FVIII) concentrate or bypassing agents (e.g. recombinant factor VIIa (rFVIIa)) is generally effective. Occasionally, haemostasis is not achieved, which may reflect a failure of factor concentrate to normalise clot stability. Tranexamic acid (TXA) is often used to aid haemostasis in surgery (e.g. joint replacements and dental procedures). Used routinely as an adjunct, it may enhance clot stability and allow effective, reliable, and cost-effective treatment at lower doses of factor concentrate. This study hypothesised that clot stabilising adjunct TXA is required in addition to factor substitution to normalise clot stability in whole blood from patients with severe haemophilia A. The in vitro effect of varying concentrations of recombinant FVIII or recombinant FVIIa and adjunct TXA on whole blood clot stability was measured by thromboelastometry. Coagulation was triggered by tissue factor and clots were challenged with tissue plasminogen activator. The area under the elasticity curve was the primary endpoint. High concentrations of FVIII and rFVIIa increased clot stability to levels that were not significantly different from controls (Mean ± SD: control 112,694?±?84,115; FVIII 78,662?±?74,126; rFVIIa 95,918?±?88,492). However, the response was highly variable between individuals and demonstrates why some patients show clinical resistance to treatment. Addition of TXA resulted in normalised clot stability in all individuals, even when combined with the lowest doses of factor concentrate. The results support the concept that a more efficient, reliable and cost effective treatment may be obtained if TXA is combined with factor concentrates to treat individuals with haemophilia.     

Factor (F)VIII/VIIa enhances global haemostatic function in the co‐presence of bypassing agents and FVIII among patients with haemophilia A with inhibitor

Bypassing therapy is essential for the haemostatic management of patients with haemophilia A with inhibitor (PWHA‐inh), but the therapeutic effects are inconsistent. We previously reported that activated prothrombin complex concentrates (aPCC) activated factor (F)VIIIin vitro, and was mediated mainly by the activated FVII (FVIIa) contained in aPCC. We have extended those studies to assess global coagulation in whole blood from 18 PWHA‐inh in the co‐presence of aPCC and FVIII using Ca²⁺‐triggered rotational thromboelastometry. The clot times (CTs) in the presence of both aPCC (0·05 iu/ml) and recombinant (r)FVIII (1 iu/ml) ex vivo were shortened compared to the aPCC alone (P < 0·01). These enhancing effects of rFVIII were observed, irrespective of recognizing inhibitor epitopes; however, the clot formation time and ‘α’‐angle were not significantly different. In samples from 7 PWHA‐inh post‐infusion of aPCC (70‐80 iu/kg), only the CTs were shortened in the presence of rFVIIIex vivo compared to its absence (P < 0·05), indicating that the enhanced activity centred on the initiation phase of coagulation. Furthermore, experiments in the co‐presence of rFVIIa and rFVIII demonstrated that FVIII accelerated only the CTs. We concluded that FVIII/FVIIa‐related coagulation mechanism enhanced global haemostatic function by the co‐presence of bypassing agents and FVIII in PWHA‐inh.     

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.     

Effects of recombinant activated factor VII on thrombin-mediated feedback activation of coagulation

Thrombin is a key hemostatic enzyme, which propagates its own generation by activating factors V, VIII, and XI. Sustained thrombin generation also activates thrombin-activatable fibrinolysis inhibitor (TAFI), which stabilizes fibrin clot against fibrinolysis. Recombinant activated factor VII (rFVIIa) is considered a novel hemostatic intervention for refractory bleeding, but rebleeding episodes related to fibrinolysis still occur. The present study aimed to investigate the antifibrinolytic effects of rFVIIa in relation to thrombin generation. Using thrombelastography, the effects of rFVIIa on thrombin-activated fibrin formation and on fibrinolysis induced by tissue plasminogen activator were evaluated in various factor-deficient plasma samples. A Thrombinoscope was used to quantitate thrombin generation. Thrombin increased antifibrinolytic activity in a concentration-dependent manner as demonstrated by a longer clot lysis time. In plasma deficient in factors V, VIII, IX, X, or XI, clot lysis occurred early (< 20 min), and rFVIIa addition had minimal effect, except for improved antifibrinolytic effect in factor-XI-deficient plasma. A normal clot lysis time was observed in factor-XIII-deficient or dual antithrombin/factor-VIII-deficient plasma. Inhibition of TAFI increased the rate of fibrinolysis. Thrombin generation was delayed or decreased in single factor-deficient plasma except for factor XIII deficiency. After rFVIIa addition, the peak thrombin generation reached over 100 nmol/l in factor-XI-deficient plasma, but not in plasma deficient in factors V, VIII, IX, or X. Thrombin generation and subsequent activation of TAFI were important for clot stability. We conclude that rFVIIa therapy does not compensate for increased susceptibility to fibrinolysis due to lack of factor(s) necessary for the formation of tenase and prothrombinase.     

High dose factor VIIa improves clot structure and stability in a model of haemophilia B

Factor IX (FIX) deficiency results in haemophilia B and high dose recombinant activated factor VII (rFVIIa) can decrease bleeding. Previously, we showed that FIX deficiency results in a reduced rate and peak of thrombin generation. We have now used plasma and an in vitro coagulation model to examine the effect of these changes in thrombin generation on fibrin clot structure and stability. Low FIX delayed the clot formation onset and reduced the fibrin polymerisation rate. Clots formed without FIX were composed of thicker fibrin fibres than normal. rFVIIa shortened the clot formation onset time and improved the fibre structure of haemophilic clots. We also examined clot formation in the presence of a fibrinolytic challenge by including tissue plasminogen activator or plasmin in the reaction milieu. In these assays, normal FIX levels supported clot formation; however, clots did not form in the absence of FIX. rFVIIa partially restored haemophilic clot formation. These results were independent of the effects of the thrombin-activatable fibrinolysis inhibitor. Our data suggest that rFVIIa enhances haemostasis in haemophiliacs by increasing the thrombin generation rate to both promote formation of a structurally normal clot and improve clot formation and stability at sites with high endogenous fibrinolytic activities.     

First 20 years with recombinant FVIIa (NovoSeven)

Summary. This review describes the background for the development of recombinant FVIIa (rFVIIa; NovoSeven) for use in haemophilic patients with inhibitors. The first proof of principle for using pharmacological doses of FVIIa as a haemostatic agent was obtained by producing small amounts of pure plasma‐derived FVIIa, which showed encouraging effect in two patients with haemophilia A and inhibitors. To make pure FVIIa available for use in a larger number of patients, rFVIIa was produced that was approved for use in patients with inhibitors against coagulation factors (congenital haemophilia and acquired haemophilia) in 1996 (EU), 1999 (USA) and 2000 (Japan). The efficacy rate in severe bleedings and in major surgery including major orthopaedic surgery has been found to be around 90% in controlled studies, and no serious safety concerns have been demonstrated. The availability of rFVIIa has facilitated the performance of elective major surgery in haemophilia patients with inhibitors. Further steps along the vision of providing a treatment for inhibitor patients similar to non‐inhibitor patients have been the efficacy of rFVIIa in home‐treatment and recently the encouraging experience in prophylaxis. The concept of using pharmacological doses of rFVIIa as a haemostatic agent is a new one, which has caused difficulties in finding the correct dose. A step forward has been the demonstration that similar efficacy can be achieved after one single dose of 270 μg kg^?1 instead of three injections of a dose of 90 μg kg^?1. The higher clearance rate in children suggests that higher doses may be beneficial in children. The availability of rFVIIa has made advances in the understanding of coagulation processes possible. In a cell‐based in vitro model, it has been shown that rFVIIa binds to preactivated platelets if present in concentrations of 30 nm or higher. By doing so, it activates FX into FXa and enhances the thrombin generation on the activated platelet surface in the absence of FVIII/FIX. Through the increased thrombin generation, a firm, well‐structured fibrin haemostatic plug, which is resistant to premature lysis, is formed. By exploiting this mechanism of action, rFVIIa may also be effective in situations other than haemophilia, characterized by an impaired thrombin generation.     

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.     

Recombinant factor VIIa enhances platelet deposition from flowing haemophilic blood but requires the contact pathway to promote fibrin deposition

In prior microfluidic studies with haemophilic blood perfused over collagen, we found that a severe deficiency (<1% factor level) reduced platelet and fibrin deposition, while a moderate deficiency (1–5%) only reduced fibrin deposition. We investigated: (i) the differential effect of rFVIIa (0.04–20 nm ) on platelet and fibrin deposition, and (ii) the contribution of the contact pathway to rFVIIa‐induced haemophilic blood clotting. Haemophilic or healthy blood with low and high corn trypsin inhibitor (CTI, 4 or 40 μg mL^?1) was perfused over collagen at an initial venous wall shear rate of 100 s^?1. At 100 s^?1 wall shear rate, where FXIIa leads to thrombin production without added tissue factor, FXI‐deficient blood (3%) or severely FVIII‐deficient blood (<1%) produced no fibrin at either CTI level. Whereas rFVIIa potently enhanced platelet deposition, fibrin generation was not rescued. Distinct from the high CTI condition, engagement of the contact pathway (low CTI) in moderately FVIII‐deficient (3%) or moderately FIX‐deficient blood (5%) resulted in enhanced platelet and fibrin deposition following 4 nm rFVIIa supplementation. In mildly FVIII‐deficient blood (15%) at <24 h since haemostatic therapy, rFVIIa enhanced both platelet and fibrin generation in either CTI condition although fibrin was produced more quickly and abundantly in low CTI. For tissue factor‐free conditions of severe haemophilic blood clotting, we conclude that rFVIIa reliably generates low levels of ‘signaling’ thrombin sufficient to enhance platelet deposition on collagen, but is insufficient to drive fibrin polymerization unless potentiated by the contact pathway.     

Measurement of global haemostasis in severe haemophilia A following factor VIII infusion

Patients with haemophilia requires different amounts of FVIII to prevent and treat bleeds. We hypothesise that this is because FVIII has variable effects on individual patients' global haemostasis. Twelve patients with severe haemophilia A were infused with 50 IU/kg FVIII and thrombin generation in platelet rich (PRP) and platelet poor plasma (PPP) and velocity of changing clot elasticity were measured preinfusion and at nine subsequent time points over 72 h. Despite a close correlation between median FVIII and median initial rate of thrombin generation (R(2) 0.94), endogenous thrombin potential (ETP; R(2) 0.94) and peak thrombin (R(2) 0.91) in PPP, there was wide inter-patient variability at each time point. There was, however, a highly predictable intra-patient relationship between FVIII level and thrombin generation. Inter-patient variability was due to both differences in FVIII levels and the variable effect FVIII had on an individuals' thrombin generation. The utility of PRP was limited because, at low-FVIII levels, only rate of thrombin generation was measurable. At low-FVIII concentrations, the rate of thrombin generation in PPP was the most useful test whilst at higher levels ETP and peak thrombin could also be used.     

Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94‐9027) for haemophilia A

Patients with haemophilia (PWH) are usually monitored by the one‐stage activated partial thromboplastin time (aPTT) factor VIII (FVIII) assay. Different aPTT activators may affect clotting time (CT) and FVIII:C levels in patients treated with PEGylated FVIII. To evaluate the characteristics of PEGylated FVIII (BAY 94‐9027) in various aPTT clotting assays, and to identify suitable aPTT reagents for monitoring BAY 94‐9027 during the treatment of PWH, BAY 94‐9027 and World Health Organization (WHO) 8th FVIII standards (WHO‐8) were spiked into pooled and individual severe haemophilia A plasma at 1.0, 0.25 and 0.05 IU mL^?1. Five commercial aPTT reagents widely used in clinical laboratories were compared and evaluated for BAY 94‐9027 activity in plasma from PWH. BAY 94‐9027 and WHO‐8 bestowed similar CT and excellent precision when ellagic acid (SynthAFax, Dade Actin, and Cephascreen) aPTT reagents were used. In contrast, BAY 94‐9027 showed significantly prolonged CT and poor precision compared with WHO‐8 using silica aPTT reagents (APTT‐SP and STA PTT 5). Furthermore, free 60‐kDa polyethylene glycol (PEG), used for the conjugation of FVIII, showed a dose‐dependent prolongation of CT in the APTT‐SP assay. There was no effect on the SynthAFax‐APTT, prothrombin time, or FXIa‐initiated thrombin generation assay, demonstrating that the PEG moiety on FVIII has no general effect on the coagulation cascade. In summary, ellagic aPTT reagents (SynthAFax, Dade Actin, and Cephascreen) are most suitable for evaluating potency of BAY 94‐9027 and should be the preferred aPTT reagents used in clinical laboratories for monitoring FVIII activity after infusion of BAY 94‐9027 to PWH.     

Functional characteristics of N8, a new recombinant FVIII

Summary. The aim of this study was to evaluate the in vitro function of the new recombinant factor VIII (FVIII) compound, N8. The specific activity of N8 as measured in a FVIII:C one‐stage clot assay was 9300 ± 400 IU mg^?1 based on the analysis of seven individual batches. The ratio between the FVIII:C activity measured in clot and chromogenic assays was 1.00 (95% confidence interval 0.97–1.03). N8 bound to von Willebrand factor with K_d values of 0.2 nm when measured by ELISA and by surface plasmon resonance. FVIIIa cofactor activity was determined from the kinetic parameters of factor IXa‐catalysed factor X (FX) activation. The rate of activation of N8 by thrombin as well as K_m and k_cat for FX activation was in the same range as those observed for Advate^®. The rate of activated protein C (APC)‐catalysed inactivation was similar for activated N8 and Advate^®. N8 improved thrombin generation in a dose‐dependent manner and induced similar rates of thrombin generation as Advate^® and the plasma‐derived FVIII product Haemate^®. Using thromboelastography (TEG^®), N8 was shown to improve the clot formation and clot stability in whole blood from haemophilia A patients. Comparable potency and efficacy of N8 and Advate^® was found based on TEG^® parameters. Finally, similar binding profiles to immobilized lipoprotein receptor‐related protein (LRP) of N8 and Advate^® were observed. The study demonstrated that N8 is fully functional in a variety of assays measuring FVIII activity. No functional differences were found between N8 and comparator compounds.     

Results of clot waveform analysis and thrombin generation test for a plasma‐derived factor VIIa and X mixture (MC710) in haemophilia patients with inhibitors—phase I trial: 2nd report

We reported the results of a clinical pharmacological study of MC710 (a mixture of plasma‐derived FVIIa and FX) in haemophilia patients with inhibitors during a non‐haemorrhagic state. This report provides the results of a clot waveform analysis (CWA) and thrombin generation test (TGT) using blood samples obtained in this study. CWA and TGT were conducted using blood samples obtained from a pharmacokinetic and pharmacodynamic study in which MC710 (five dose rates: 20, 40, 80, 100 and 120 μg kg^?1) was compared with NovoSeven (120 μg kg^?1) and FEIBA (two dose rates: 50 and 75 U kg^?1) as control drugs in 11 haemophilia patients with inhibitors without haemorrhagic symptoms. CWA showed that MC710 provided significantly greater improvement than the control drugs in activated partial thromboplastin time (APTT) at 80 μg kg^?1; maximum clot velocity and maximum clot acceleration were more enhanced by MC710 than by control drugs. TGT revealed that MC710 significantly shortened the initiation time of thrombin generation in comparison to FEIBA and induced greater thrombin generation potency than NovoSeven. It was not clear whether or not MC710 caused significant dose‐dependent changes in the two measurements; however, differences between MC710 and the control drugs were clarified. MC710 was confirmed to have superior coagulation activity and thrombin productivity and is expected to have superior bypassing activity.     

Enhancement of fibrinolytic potential in vitro by anticoagulant drugs targeting activated factor X, but not by those inhibiting thrombin or tissue factor.

Tissue factor-induced coagulation leads to the generation of a small amount of thrombin, resulting in the formation of a fibrin clot. After clot formation, thrombin generation continues resulting in the activation of thrombin activatable fibrinolysis inhibitor, leading to downregulation of fibrinolysis. In this study, the effect of anticoagulant drugs targeting different steps in the coagulation cascade on clot formation and subsequent breakdown was investigated using a plasma-based clot lysis assay. All drugs tested significantly delayed clot formation; only those drugs targeting activated factor X (FXa) (tissue factor pathway inhibitor, fondaparinux, and low molecular weight heparin) accelerated fibrinolysis. Anticoagulant drugs targeting tissue factor (active site-inactivated recombinant activated factor VII) or thrombin (hirudin and d-phenylalanyl-l-prolyl-l-arginyl chloromethyl ketone) did not affect clot lysis time. In accordance with these findings, it was shown that total thrombin generation, as quantified by the endogenous thrombin potential, was only affected by anticoagulant drugs targeting FXa when all drugs were used in a concentration resulting in doubling of clotting time. Induction of hyperfibrinolysis by anticoagulant drugs directed against FXa might be beneficial as increased clot breakdown might facilitate thrombolysis or prevent re-occlusion. On the other hand, the induction of hyperfibrinolysis by these compounds might increase the risk of bleeding complications.     

On-demand but not prophylactic treatment with FVIII concentrate increase thrombin activatable fibrinolysis inhibitor activation in severe haemophilia A patients

Introduction: Thrombin activatable fibrinolysis inhibitor (TAFI) down‐regulates fibrinolysis after activation by thrombin/thrombomodulin. We investigated the effect of treatment with FVIII concentrate on plasma levels of pro‐TAFI and activated TAFI in haemophilia A patients. Methods: Samples were collected pre and posttreatment from patients treated prophylactically or on‐demand. Pro‐TAFI, TAFI/TAFIi and FVIII levels were measured in all samples. Results: Treatment had no effect on pro‐TAFI levels. Pro‐TAFI was similar in both patient groups but higher than in controls. Patients from the prophylactic treatment group had measurable FVIII levels pretreatment while in the treatment‐on‐demand group FVIII levels were ≤0.01 IU/mL. In the prophylactic treatment group, the levels of TAFI/TAFIi were significantly lower pre‐ and posttreatment (4.31 ± 3.14 and 3.48 ± 2.65 ng/mL respectively) than in the on‐demand group (13.02 ± 3.47 and 14.87 ± 3.47 ng/mL respectively). This difference may be due to release of tissue factor at the injury site in the on‐demand group. This could induce thrombin and TAFI activation within the clot counterbalancing fibrinolysis in these patients. In the prophylactic group, no injury existed, thus there was insufficient thrombin generation within the clot to activate TAFI. Conclusion: These findings suggest that in patients to whom FVIII is administered on demand the fibrinolysis activity is more down regulated than in patients following a prophylactic treatment regime.     

Evaluation of the overall haemostatic effect of recombinant factor VIIa by measuring thrombin generation and stability of fibrin clots

Summary. It has been reported that thrombin generation test (TGT) may be a useful tool to monitor recombinant factor VIIa (rFVIIa). However, TGT does not reflect the stability of fibrin clot and its resistance to fibrinolysis which are crucial. Using whole‐blood thromboelastography (TEG) and tissue plasminogen activator (tPA), we developed an in‐vitro model to assess fibrin clot stability. Fibrin fibres were thicker in haemophiliacs compared with controls (P < 0.0001). After addition of rFVIIa 90 μg kg^?1, the diameter of fibrin fibres was dramatically decreased (P = 0.006). TEG‐tPA assay showed a dose‐dependent improvement of clot stability in the presence of rFVIIa. These data demonstrate a significant correlation between fibrin clot structure and its stability (P = 0.001). We also showed a correlation between thrombin generating capacity and clot resistance to fibrinolysis. Despite this overall correlation, a relatively large spreading around a general trend was observed, suggesting that the two assays bring complementary information on the haemostatic effect of rFVIIa.     

Platelet-dependent coagulation assays for factor VIII efficacy measurement after substitution therapy in patients with haemophilia A

FVIII therapy for haemophilia A is safe and effective, with the problem of individually sufficient efficacy unsettled. Routine one-stage clotting assays and tests employing chromogenic substrates poorly detect individual haemostatic effects of FVIII due to artificial test conditions. In particular, the use of cell-free and diluted plasma samples neglect the crucial role of platelets for thrombin and fibrin formation. To optimize FVIII substitution therapy, we measured in 40 patients with severe to mild haemophilia A before and after FVIII substitution the FVIII activity in cell-free plasma samples using a one-stage clotting assay as well a chromogenic substrate assay and compared the data with those obtained with cell-based coagulation tests, i.e. thrombin generation in platelet-rich plasma (PRP) and thromboelastography (TEG) in samples of citrated whole blood (WB). To determine the maximum ex vivo haemostatic effect we added 1 unit/ml of FVIII to samples of PRP and WB and measured the maximum thrombin generation in the thrombin generation test (TGT) and the maximum clot firmness (MCF) in TEG. After FVIII substitution we observed a nearly linear relation between the individual FVIII activities administered to the patients and the activities measured in the plasma samples. However, data obtained with TGT and TEG revealed a high inter-individual variation and a very poor correlation to the administered FVIII activity. Actually, it could be shown that FVIII substitution yielding in a FVIII plasma activity of about 30% is sufficient to get an ex vivo haemostatic effect of more that 90% as measured by maximum thrombin generation and MCF. FVIII substitution up to a plasma activity of more than 90% did not further enhance the haemostatic effect. Our data clearly demonstrate that the haemostatic effect of FVIII is not only dependent on the activity that is measured in plasma but also depends on the interplay between coagulation and blood cells, in particular with platelets. The use of cell-based coagulation tests such us TGT or TEG may help to optimize FVIII therapy by determining the individual FVIII dosage that produces a maximum haemostatic effect.     

Cost‐effectiveness of recombinant activated factor VII vs. plasma‐derived activated prothrombin complex concentrate in the treatment of mild‐to‐moderate bleeding episodes in patients with severe haemophilia A and inhibitors in Spain

Several analyses have shown that recombinant activated factor VII (rFVIIa) is a cost‐effective intervention compared with plasma‐derived activated prothrombin complex concentrate (pd‐aPCC) for the on‐demand treatment of mild‐to‐moderate bleeds in haemophilia patients with inhibitors. The aim of the study was to assess the cost‐effectiveness of rFVIIa vs. pd‐aPCC in the treatment of bleeding episodes in severe haemophilia A patients with inhibitors in Spain. A decision analytic model was designed to evaluate the costs and clinical outcomes of using rFVIIa or pd‐aPCC to treat mild‐to‐moderate joint bleeds in children (≤14 years old) and adults with inhibitors. Data were obtained from a published meta‐analysis and a panel of haemophilia experts. The analysis was conducted from the perspective of the Spanish National Healthcare System. One‐way sensitivity analyses were performed to assess the impact of model assumptions on study results. In the Treur meta‐analysis, rFVIIa resulted in cumulative joint bleed resolution of 88% and 95% after 24 and 36 h, respectively, compared with 62% and 76%, respectively, with pd‐aPCC (Treur et al. Haemophilia 2009; 15 : 420–36). Here, the mean cost per bleed was estimated at €8473 and €15 579 in children and adults treated with rFVIIa, vs. €8627 and €15 677 in children and adults treated with pd‐aPCC. rFVIIa treatment was found to be the dominating option (cheaper and more effective). The one‐way sensitivity analysis also confirmed that rFVIIa was less costly than pd‐aPCC. The model suggests that rFVIIa is a cost‐effective option compared with pd‐aPCC for the treatment of mild‐to‐moderate bleeding episodes in a Spanish setting.     

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).