Effect of recombinant factor VIIa variant (NN1731) on platelet function, clot structure and force onset time in whole blood from healthy volunteers and haemophilia patients

NN1731 is a novel variant of recombinant factor VIIa (rFVIIa) that binds to activated platelets, but has greater enzymatic activity than rFVIIa in generating FXa and thrombin. The effect of NN1731 on clot structure and platelet function was characterized ex vivo in whole blood from healthy volunteers and haemophilic patients. Blood samples from six healthy volunteers, nine haemophilia A patients with and without inhibitors and one acquired haemophilia A patient, were spiked with increasing concentrations (0.32, 0.64 and 1.28 microg mL(-1)) of rFVIIa and NN1731. Platelet contractile force (PCF) or platelet function, clot elastic modulus (CEM) or clot structure, and force onset time (FOT) or the thrombin generation time (TGT) were determined using the Hemodyne Hemostasis Analysis System (HAS). Baseline PCF, CEM and FOT values in patients were abnormal compared to healthy volunteers' baseline values. Overall, haemophilia blood samples with or without inhibitors spiked with NN1731 had significantly greater PCF, CEM and shorter FOT values relative to samples spiked with corresponding doses of rFVIIa. The variability in response to treatment between patients was greater with rFVIIa compared to NN1731. At 1.28 microg mL(-1) (90 microg kg(-1)), NN1731 normalized PCF, CEM and FOT in nine of 10 patients, while rFVIIa normalized these parameters in four of 10 patients. Increasing in vitro concentrations of NN1731 normalized platelet function, clot structure and thrombin generation consistently in haemophilia blood with or without inhibitors. NN1731 may be a promising haemostatic agent for patients with bleeding disorders. These results should be confirmed in an in vivo study.     

Factor VIIa analogue (V158D/E296V/M298Q-FVIIa) normalises clot formation in whole blood from patients with severe haemophilia A

This study evaluated and compared the haemostatic potential of a recombinant factor VIIa (rFVIIa) analogue (V158D/E296V/M298Q-FVIIa, NN1731, Novo Nordisk, Denmark) with rFVIIa (NovoSeven^®, Novo Nordisk). In vitro studies were performed using freshly drawn whole blood (WB) from 14 patients with severe haemophilia A and two patients with inhibitory antibodies to FVIII, comparing NN1731 and rFVIIa against a buffer control. Fourteen healthy males served as controls. Dynamic WB coagulation profiles were recorded, quantitatively illustrating the initiation [clotting time = CT (s)], propagation [maximum velocity = MaxVel (mm*100/s)] and termination [maximum clot firmness = MCF (mm*100)] as determined by thromboelastography with minute amounts of tissue factor (TF, Innovin^®– final dilution 1:50 000, c. 0·12 pM) serving as activator. WB clot stability was assessed using a separate set-up including TF plus tissue plasminogen activator (final concentration 2 nmol/l), evaluating the MCF as well as the area under the elasticity curve (AUEC) after 60 min (mm*100*s). NN1731 shortened the CT more markedly than rFVIIa. At the dose tested, NN1731 even shortened CT in haemophilia below the value of healthy males. NN1731 accelerated MaxVel giving a value indistinguishable from that in healthy males. Furthermore, NN1731 increased clot stability more markedly than rFVIIa. Altogether, these in vitro studies on WB revealed a favourable haemostatic potential of NN1731 compared with rFVIIa in severe haemophilia A, both in the absence and presence of enhanced fibrinolytic activity.     

Whole blood coagulation in children with thrombocytopenia and the response to platelet replacement, recombinant factor VIIa, and a potent factor VIIa analogue

The present study evaluated dynamic coagulation profiles, platelet aggregation, and thrombin generation in whole blood (WB) from eight children with thrombocytopenia during chemotherapy, and the haemostatic potential of platelets (+60 × 10⁹/l), recombinant factor VIIa (rFVIIa – NovoSeven^®), and a potent rFVIIa analogue (NN1731) both at 1 and 4 μg/ml. Dynamic WB coagulation profiles were recorded by thrombelastometry employing activation with tissue factor (TF – Innovin^®) at low concentrations. The baseline WB coagulation patterns were characterised by a prolonged clotting time (CT) and a pronounced reduction in clot propagation (MaxVel). WB platelet aggregation signal was five times lower in the study group compared with measurements in modelled thrombocytopenic WB from healthy volunteers. In vitro addition of fresh platelets reversed the coagulopathy. Addition of rFVIIa induced no significant changes in the thrombelastographic profile, whereas spiking with NN1731 shortened the CT significantly. The changes in WB thrombin generation reflected the changes in the MaxVel. In modeled thrombocytopenic WB from healthy individuals, both rFVIIa and NN1731 exhibited a pronounced haemostatic effect with NN1731 showing greater potency than rFVIIa. Compromised platelet function in the study group was assumingly responsible for the weakened haemostatic potential of rFVIIa as well as that of NN1731.     

Effects of factor VIII inhibitor bypassing activity (FEIBA), recombinant factor VIIa or both on thrombin generation in normal and haemophilia A plasma

Summary.  Factor VIII inhibitor bypass activity (FEIBA) and recombinant factor VIIa (rFVIIa) are the common bypassing agents for treating haemophilia A or haemophilia B patients who developed an inhibitor to factor VIII or IX, respectively. As these preparations differ in their composition and mode of action, combined therapy, either sequential or simultaneous has recently been used for achieving haemostasis during bleeding episodes in patients who became refractory to FEIBA or rFVIIa when each was given alone. In this in vitro study, we show by a sensitive assay of thrombin generation that phospholipids present in FEIBA and other procoagulants contribute to FEIBA's activity and that exogenous phospholipids are essential for the activity of rFVIIa. We also demonstrate that the combination of FEIBA and rFVIIa has a marked synergistic effect on thrombin generation in plasma of a haemophilia A patient with a high titre of an inhibitor. It is conceivable that simultaneous administration of small doses of FEIBA and rFVIIa may be beneficial in treating haemophilia A patients, with an inhibitor to FVIII, who are resistant to conventional therapy.     

Recombinant factor VIIa for patients with inhibitors to factor VIII or IX or factor VII deficiency

Inhibitors to factor VIII (FVIII) or IX (FIX) in patients with haemophilia A or B create a challenging problem for the treatment of these patients. Recombinant FVIIa (rFVIIa; NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) is a realistic treatment option, owing to its specific mode of action and lack of immunogenicity. This was a multicentre, open-label, compassionate-use trial in patients with severe haemophilia A (FVIII:C < 1%) or B (FIX:C < 1%) with inhibitors, acquired antibodies to FVIII or FIX, or FVII deficiency (FVII:C < 5%), for whom alternative therapies had failed or were contraindicated. Patients received rFVIIa treatment for life- or limb-threatening bleeding episodes or for coverage during essential surgery. The mean rFVIIa dose was approximately 90 microg kg-1 for haemophilia A/B and acquired inhibitor patients, and 25 microg kg-1 for FVII-deficient patients. Efficacy data for 67 treatment episodes (45 bleeding episodes, 22 surgical procedures) are presented; seven patients were treated for a concurrent serious bleeding episode and surgical procedure. At the end of treatment, rFVIIa was effective or partially effective in 85% of serious bleeding episodes. During surgery, bleeding was assessed as none or less than or equivalent to normal in 91% of surgical procedures; postoperatively, 91% of procedures were associated with no or minimal oozing. During 60 separate treatment episodes, 26 adverse events (22 nonserious, four serious) were reported in 15 patients, during 17 bleeding episodes or surgical procedures. Only 10 were considered as having a possible, probable, or unknown relationship with rFVIIa; of these, fever (n=2) and thrombophlebitis (n=3) were the most common. There was no evidence of disseminated intravascular coagulation. In conclusion, rFVIIa is an effective, well-tolerated treatment for serious bleeding episodes and bleeding associated with surgical procedures in patients with severe haemophilia A/B with inhibitors, acquired inhibitors, or FVII deficiency.     

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.     

Concomitant infusion of low doses of rFVIIa and FEIBA in haemophilia patients with inhibitors

Summary.  Patients with severe haemophilia A and an inhibitor may become refractory to FEIBA and/or recombinant factor VIIa (rFVIIa). Sequential therapy with both products has been reported in such patients. In this pilot study, we examined the safety and efficacy of combined rFVIIa and FEIBA therapy in patients with haemophilia A and inhibitors during bleeding episodes. We also tried to evaluate whether thrombin generation (TG), by various mixtures of these agents, can serve as a guide for tailoring therapy. TG was measured in plasma taken from eight haemophilia A patients. Increasing concentrations of rFVIIa, FEIBA or both were added ex vivo to the plasmas, and TG was induced by recalcification. Since low concentrations of rFVIIa and FEIBA had either an additive or a synergistic effect in all patients, the lowest combination, yielding TG comparable or lower than TG achieved with either FEIBA 100 U kg⁻¹ or rFVIIa 160 μg kg⁻¹ alone, was selected for the treatment of bleeding episodes. Five patients with a high titre of an inhibitor (8–1300 BU), including one previously refractory to infusions of rFVIIa at doses up to 400 μg kg⁻¹ X4 daily, were treated with combinations of 30–70 μg kg⁻¹ rFVIIa and 20–30 U kg⁻¹ FEIBA during a total number of 400 bleeding episodes with excellent haemostatic effect. No adverse events and no DIC were observed following these infusions. Concomitant infusion of low-dose rFVIIa and low-dose FEIBA, seems to be safe, efficacious and economical in patients refractory to rFVIIa and probably other haemophilia A patients with an inhibitor.     

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.     

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.     

A novel approach to improving recombinant factor VIIa activity with a preserved platelet preparation

Recombinant activated factor VII (NovoSeven, rFVIIa) is used to abrogate bleeding in haemophiliacs with inhibitors and is hypothesised to work by increasing activated factor X generation on the platelet surface. We hypothesised that rFVIIa activity could be increased by the co-addition of platelet procoagulant surface. This study characterised the ability of a rehydrated, lyophilised (RL) platelet preparation to increase rFVIIa activity in haemophilic conditions. RL platelets supported thrombin generation in the presence of factors VIII and IX but, in the absence of factors VIII and IX, thrombin generation was significantly reduced. RL platelets supported rFVIIa-mediated thrombin generation in a rFVIIa-concentration dependent manner. In a cell-based in vitro model of haemophilia, the presence of RL platelets increased the rFVIIa-dependent thrombin generation rate 2.8-fold compared with rFVIIa alone. Similarly, the addition of RL platelets plus rFVIIa to the in vitro model of haemophilia and to haemophilic platelet-rich plasma shortened the onset of clot formation and increased clot stability in a fibrinolytic environment versus rFVIIa alone. These results suggest that RL platelets can support rFVIIa-mediated thrombin generation, and that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa in some patients.     

Severe and moderate haemophilia A and B in US females

Haemophilia A and B are rare X‐lined hemorrhagic disorders that typically affect men. Women are usually asymptomatic carriers, but may be symptomatic and, rarely, also express severe (factor VIII (FVIII) or factor IX (FIX) <0.01 U mL^?1) or moderately severe (FVIII/FIX 0.01–0.05 U mL^?1) phenotypes. However, data on clinical manifestations, genotype and the psychosocial ramifications of illness in severely affected females remain anecdotal. A national multi‐centre retrospective study was conducted to collect a comprehensive data set on affected US girls and women, and to compare clinical observations to previously published information on haemophilic males of comparable severity and mildly affected haemophilic females . Twenty‐two severe/moderate haemophilia A/B subjects were characterized with respect to clinical manifestations and disease complications; genetic determinants of phenotypic severity; and health‐related quality of life (HR‐QoL). Clinical data were compared as previously indicated. Female patients were older than male patients at diagnosis, but similarly experienced joint haemorrhage, disease‐ and treatment‐related complications and access to treatment. Gynaecological and obstetrical bleeding was unexpectedly infrequent. F8 or F9 mutations, accompanied by extremely skewed X‐chromosome inactivation pattern (XIP), were primary determinants of severity. HR‐QoL was diminished by arthropathy and viral infection. Using systematic case verification of participants in a national surveillance registry, this study elucidated the genetics, clinical phenotype and quality of life issues in female patients with severe/moderate haemophilia. An ongoing international case‐controlled study will further evaluate these observations. Novel mechanistic questions are raised about the relationship between XIP and both age and tissue‐specific FVIII and FIX expression.     

New assays for monitoring haemophilia treatment

Summary.  Precise measurements of factor VIII (FVIII) or factor IX (FIX) activity are believed to be essential for clinical management in haemophilia, although discrepancies between factor levels and clinical severity have been recognized. Clot wave form analysis has demonstrated that different wave form patterns may be evident in severe haemophilia A patients with levels of FVIII activity <1 IU dL⁻¹, and this might explain, in part, the phenotypic heterogeneity seen in these patients. In addition, the relatively new technique of computer-assisted thrombelastography (TEG), in which coagulation is initiated by tissue factor, has revealed a considerable degree of variability in different patients in the presence FVIII levels, which are sufficient to normalize TEG parameters. In contrast, a global thrombin generation test (TGT) has been proposed as a sensitive and reliable method for assessing overall clotting function in haemophilia patients. Several studies have demonstrated a significant correlation between TGT and FVIII/FIX levels, and these measurements also appear to correlate with the clinical phenotype. The TGT may be very useful, therefore, for evaluating overall haemostasis in different clinical situations, although substantial inter-assay and inter-individual variations have been reported. Both the TEG and TGT have been found to be particularly helpful for monitoring haemostatic therapy with bypassing agents or conventional FVIII or FIX concentrates in patients with inhibitors. These global tests enable the selection of appropriate therapeutic agents in individual circumstances and offer the opportunity to tailor the most effective haemostatic treatment even during severe bleeding or major surgery.     

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.     

Factor VIII and IX assays for post-infusion monitoring in hemophilia patients: Guidelines from the French BIMHO group (GFHT)

Replacement therapy with plasma-derived or recombinant FVIII and FIX (pdFVIII/pdFIX or rFVIII/rFIX) concentrates is the standard of treatment in patients with haemophilia A and B, respectively. Measurement of factor VIII (FVIII:C) or factor IX (FIX:C) levels can be done by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). The French study group on the Biology of Hemorrhagic Diseases (a collaborative group of the GFHT and MHEMO network) presents a literature review and proposals for the monitoring of FVIII:C and FIX:C levels in treated haemophilia A and B patients, respectively. The use of CSA is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. Except for rFVIII-Fc, great caution is required when measuring FVIII:C levels by OSA in patients substituted by EHL-rFVIII. The OSA is recommended for the monitoring of patients treated with pdFIX or rFIX. Large discordances in the FIX:C levels measured for extended half-life rFIX (EHL-rFIX), depending on the method and reagents used, must lead to great attention when OSA is used for measuring FIX:C levels in patients substituted by EHL-rFIX. Data of most of recent studies, obtained with spiked plasmas, deserve to be confirmed in plasma samples of treated patients.     

Prophylactic dosing of factor VIII and factor IX from a clinical pharmacokinetic perspective

Summary.  The high cost and limited availability of factor concentrates make dosing of factor VIII (FVIII) or factor IX (FIX) a crucial issue in the prophylactic treatment of haemophilia. It has often been recommended that this treatment should aim to maintain a minimum plasma level of 1% of normal coagulation factor activity (FVIII:C or FIX:C). The dosage needed is commonly given as 25–40 U kg⁻¹ three times weekly for FVIII or twice weekly for FIX. However, these guidelines are valid only with several qualifications. First, the actual trough levels required may vary considerably between patients. The clinical severity of haemophilia may depend on more factors than the endogenous level of FVIII:C or FIX:C. Secondly, interindividual variations in dose requirements are also due to variance in the pharmacokinetics of the coagulation factors. Pharmacokinetic calculations are useful to design optimal dosing schedules to achieve required trough levels of FVIII:C or FIX:C. Moreover, tailoring of the dosing of FVIII or FIX according to their disposition in the individual patient can markedly improve the cost-effectiveness of prophylactic treatment. However, the usefulness of in vivo recovery as a guide for prophylactic dosing seems questionable. It should be clearly understood that maintaining a certain trough level of FVIII:C or FIX:C is not an end in itself. Clinical outcome, not the achieved trough level, determines whether a dosage is adequate. Chiefly for economic reasons, the minimum effective dosage of coagulation factor should be determined and used in every patient. The dose requirement should also be re-evaluated at appropriate times.     

Factor VIII and von Willebrand factor interaction: biological, clinical and therapeutic importance

Summary.  The interaction of factor VIII (FVIII) with von Willebrand Factor (VWF) is of direct clinical significance in the diagnosis and treatment of patients with haemophilia A and von Willebrand disease (VWD). A normal haemostatic response to vascular injury requires both FVIII and VWF. It is well-established that in addition to its role in mediating platelet to platelet and platelet to matrix binding, VWF has a direct role in thrombin and fibrin generation by acting as a carrier molecule for the cofactor FVIII. Recent studies show that the interaction affects not only the biology of both FVIII and VWF, and the pathology of haemophilia and VWD, but also presents opportunities in the treatment of haemophilia. This review details the mechanisms and the molecular determinants of FVIII interaction with VWF, and the role of FVIII–VWF interaction in modulating FVIII interactions with other proteases, cell types and cellular receptors. The effect of defective interaction of FVIII with VWF as a result of mutations in either protein is discussed.     

Age-dependent increase of FVIII:C in mild haemophilia A

Summary.  Variability of FVIII:C levels in healthy individuals and age-dependent increase are a known phenomenon. In haemophilia, increasing FVIII:C levels with age have not been described yet. In our study, we evaluated this issue retrospectively in a cohort older than 45 years of 29 patients with mild haemophilia and 14 patients with moderate or severe haemophilia at last visit at the haemophilia centre Frankfurt. The median duration of observation evaluated in this study was 17 years (range 5–28). Results show a significant correlation of increasing FVIII:C levels with age in mild haemophilia ( P  = 0.000041) and a non-significant tendency to a higher increase in higher age ( P  = 0.085652). The median difference of FVIII:C level between the first and last measurement was 8% of normal plasma concentration (range −3% to +35%). Median FVIII:C level increase of patients younger than 62 years was 7.5% (range −3 to 22), median increase in older patients was 12% (range 0–35). This tendency could not be correlated to decreased number of bleedings, but FVIII substitution dosage should be adapted to changing plasma levels at higher age to prevent overdosing or thrombotic risks. Possible causes and contributing factors for increasing FVIII:C levels are discussed. Statistical significance remains to be confirmed in larger prospective studies also including younger patients.     

Mechanism of action of factor VIIa in the treatment of coagulopathies

Recombinant factor VIIa (rFVIIa) has been developed for treatment of bleeding in patients with hemophilia who have inhibitors against factor VIII (FVIII) or FIX, and has been found to induce hemostasis during major orthopedic surgery. The use of rFVIIa treatment for hemophilia is a new concept and is based on the low-affinity binding of FVIIa to the surface of thrombin-activated platelets. Administration of pharmacologic doses of exogenous rFVIIa enhances thrombin generation on the platelet surface at the site of injury independently of the presence of FVIII or FIX. Pharmacologic doses of rFVIIa induce hemostasis not only in hemophilia patients, but also in patients with thrombocytopenia, functional platelet defects, and with profuse bleeding triggered by extensive surgery or trauma. The general mechanism of action of rFVIIa to induce hemostasis under these conditions may be its capacity to generate a tight fibrin hemostatic plug through increased thrombin generation. A tight fibrin plug will aid in resisting the overwhelming local release of fibrinolytic activity triggered by vast tissue damage occurring in extensive trauma. Local fibrinolytic activity also occurs in the gastrointestinal tract as well as during profuse postpartum bleeding. Pharmacologic doses of rFVIIa induce hemostasis in these cases also.     

Manipulation of prothrombin concentration improves response to high-dose factor VIIa in a cell-based model of haemophilia

Clinical reports suggest that treatment regimens employing both activated prothrombin complex concentrates (aPCCs) and recombinant activated factor VII (rFVIIa) may control the bleeding in patients with haemophilia who fail to respond to either agent alone. We hypothesised that increased concentrations of prothrombin, as may be observed after the infusion of aPCCs, favourably influence parameters of thrombin generation in haemophilia treated with high-dose rFVIIa. We examined the effect of varied prothrombin and rFVIIa concentrations on thrombin generation in a model of haemophilia. At all concentrations of rFVIIa, increased prothrombin concentrations led to increases in the peak and rate of thrombin generation. In assays with the highest concentrations of prothrombin and rFVIIa, peak thrombin actually equalled that measured in the model of normal haemostasis. The significant impact of prothrombin concentration on the effect of rFVIIa in vitro may explain the improved haemostasis reported with concurrent use of aPCCs and rFVIIa. These results imply that persons with plasma prothrombin levels at either end of the 'normal' range could have significantly different responses to similar rFVIIa doses. Furthermore, these results suggest that increasing plasma prothrombin concentration prior to rFVIIa administration may offer advantages over the use of rFVIIa alone in the treatment of haemophilic bleeding.