Effects of recombinant factor VIIa on platelet function and clot structure in blood with deficient prothrombin conversion

While recombinant factor VIIa (rFVIIa) shows promise as a broad-spectrum hemostatic agent, questions remain regarding the most appropriate dose and the best way to monitor its effects. In this study we tested the sensitivity of a thrombin dependent platelet assay, platelet contractile force, to the effects of rFVIIa in normal, factor-deficient, and inhibitor-containing blood samples. Dose dependent effects of rFVIIa on platelet contractile force (PCF) and clot elastic modulus (CEM) were measured in all blood samples. rFVIIa minimally affected PCF and CEM in normal blood clotted with thrombin or batroxobin. While rFVIIa minimally altered PCF and CEM in factor VIII (FVIII) deficient blood clotted with thrombin, rFVIIa increased PCF and CEM and shortened the lag phase in a dose dependent manner in batroxobin-induced clots. The effects of rFVIIa in factor IX (FIX) deficient blood mirrored the effects seen in FVIII deficient samples. Whether clotted with thrombin or batroxobin, baseline PCF and CEM were abnormally low in FVIII deficient samples containing FVIII inhibitors. In such samples, rFVIIa caused dose dependent improvement of PCF, CEM, and lag phases. In one patient with a spontaneous inhibitor, rFVIIa caused dose dependent increases in PCF and CEM in blood clotted with either enzyme. rFVIIa corrects the deficient thrombin generation seen in FVIII and FIX deficiency, and in blood containing FVIII inhibitors. As a consequence, platelet function is improved and clot structure is enhanced. Platelet contractile force and clot elastic modulus measurements are sensitive to the dose dependent effects of rFVIIa.     

Effects of prothrombin complex concentrate and recombinant activated factor VII on vitamin K antagonist induced anticoagulation

INTRODUCTION: Warfarin and its derivatives are widely used for prevention of thrombotic incidents. Prothrombin complex concentrate (PCC) and recombinant activated factor VII (rFVIIa) have been used clinically for the acute reversal of this agent but there is a paucity of data on comparative efficacies of these hemostatic interventions. MATERIALS AND METHODS: Using in vivo rat and in vitro human models of anticoagulation, we compared PCC and rFVIIa on the recovery of endogenous thrombin generation. For in vivo reversal of anticoagulation, saline (control), PCC 50 U ml(-1), or rFVIIa100 mug ml(-1) was given to rats which received phenprocoumon (2.5 mg kg(-1)) orally. For in vitro model, plasma samples from warfarin-treated individuals with INR values of 2.1-6.7 were spiked with PCC (0.2, 0.4, or 0.72 U ml(-1)) or rFVIIa (3.0 mug/ml). The treatments were evaluated using prothrombin time (PT) and thrombin generation (Thrombinoscope). RESULTS: Administration of phenprocoumon to rats prolonged PT (14.7+/-0.5 to 50.43+/-0.7 s) and decreased peak thrombin generation by 89+/-2.3%. Administration of PCC dose dependently reversed the anticoagulation effects both in warfarin-treated human plasma and in phenprocoumon-treated rats by shortening PT and increasing peak thrombin levels. However, rFVIIa only reversed PT, but had minimal effects on peak thrombin levels. CONCLUSION: Both PCC and rFVIIa reverse warfarin anticoagulation based on PT, but only PCC restores overall thrombin generation.     

Factor VIIa and its potential therapeutic use in bleeding-associated pathologies

Recombinant FVIIa (rFVIIa) was developed for treatment of haemophilia patients with inhibitors against FVIII/FIX. The haemostatic efficacy rate of 80-90% including major orthopaedic surgery (dosing of 90-120 microg/kg every other hour [h] for at least the first 24 h) was achieved in these patients. In a home-treatment setting the efficacy rate of haemostasis in mild-moderate bleedings was 92% (average number of 90 microg/kg doses was 2.2). A wide individual variation regarding recovery of rFVIIa (46 +/- 12%; median 43%) as well as of clearance rate (36 +/- 8 ml/kg/h; median 32 ml/kg/h in adults; children 2-3 times higher) has been observed. Thus children may require higher doses than adults. Accordingly the use of a dose of 270 microg/kg in one single injection was approved in the EU. Recent experience indicates that repeated doses of rFVIIa may decrease the number of bleeds in "target joints", and thus may be useful as prophylaxis in severe hemophilia with inhibitors. Pharmacological concentrations of rFVIIa have been shown to enhance the thrombin generation on thrombin activated platelets in a cell-based model. By doing so a tight structured fibrin haemostatic plug resistant against premature lysis is formed. rFVIIa has been shown to induce haemostasis not only in haemophilia but also in other situations characterized by an impaired thrombin generation such as platelet defects, dilution coagulopathy developed as a result of trauma and extensive surgery. A special form of profuse bleeding, that may cause extensive problems is postpartum haemorrhage.     

Reversal of rivaroxaban-induced anticoagulation with prothrombin complex concentrate,activated prothrombin complex concentrate and recombinant activated factor VII in vitro

Introduction

Anticoagulation therapies carry a risk of bleeding; reversal agents may be beneficial in cases of severe bleeding even for anticoagulants with a relatively short half-life, such as the oral factor Xa inhibitor rivaroxaban.

Materials and Methods

We investigated the in vitro reversal effect of prothrombin complex concentrate (PCC; 0.2-1.0 U/mL), activated PCC (aPCC; 0.2–1.0 U/mL) and recombinant activated factor VII (rFVIIa; 5–50 μg/mL) on rivaroxaban-induced (200–1000 ng/mL) changes in prothrombin time (PT) and thrombin generation (TG) in plasma, and in thromboelastometry (clotting time [CT]) in whole blood from healthy subjects.

Results

All three agents were partially effective in reversing rivaroxaban-induced anticoagulation but showed different profiles. rFVIIa and aPCC were more effective than PCC in reversing prolongations of PT, CT and TG lag time; rFVIIa was more effective than aPCC. However, the reversal effect reached a plateau with a maximal effect of approximately 50%. Inhibition of maximum thrombin concentration was slightly reversed by these agents; aPCC was the most effective. In contrast, inhibition of endogenous thrombin potential (ETP) was strongly reversed by aPCC, with significant increases over baseline at low rivaroxaban concentrations. Compared with aPCC, PCC showed a similar but less effective reversal profile. rFVIIa reversed ETP inhibition by approximately 50%.

Conclusions

The extent of reversal by aPCC, PCC and rFVIIa was dependent on the parameter measured in rivaroxaban-anticoagulated plasma or blood. ETP measurements may have predictive power for assessing the reversal potential of PCC or aPCC and may be used to indicate an increased prothrombotic risk.     

Recombinant factor VIIa partially reverses the inhibitory effect of fondaparinux on thrombin generation after tissue factor activation in platelet rich plasma and whole blood

Fondaparinux (Arixtra), a specific AT-dependent FXa inhibitor, is effective and safe in the prevention and treatment of venous thromboembolism, but some major hemorrhagic events may occur. No specific antidote to fondaparinux has been proposed. Recombinant FVIIa (Novoseven) could be used as an haemostatic treatment, but this option has not been well documented. We studied the effect of rFVIIa (1 micro g/ml) on the inhibition of thrombin generation induced by fondaparinux (0.1 micro g/ml to 1 micro g/ml). Coagulation was triggered in platelet rich plasma (PRP) or in whole blood by recalcification in the presence of diluted thromboplastin. In PRP thrombin generation was assessed using the thrombinoscope assay. In whole blood, prothrombin activation was assessed by measuring the kinetics of F(1+2) formation using an ELISA assay. Fondaparinux at concentrations equal or greater than 0.5 micro g/ml prolonged the initiation phase of thrombin generation, and reduced the velocity of prothrombin activation. It also decreased by 60% the endogenous thrombin potential. In the presence of fondaparinux (0.5 micro g/ml to 1 micro g/ml) rFVIIa accelerated the initiation phase of thrombin generation, but it did not significantly increase the endogenous thrombin potential. However, rFVIIa did not completely reverse the inhibitory effect of fondaparinux on the parameters of thrombin generation and prothrombin activation. This study shows that rFVIIa accelerates thrombin generation, but does not completely reverse the inhibitory effect of fondaparinux on thrombin generation. The potential clinical use of rFVIIa as haemostatic treatment of major bleedings related to fondaparinux has to be evaluated.     

Continuous infusion of recombinant factor VIIa for surgery in patients with deficiency of factor VII

The administration of recombinant activated factor VII (rFVIIa) by continuous infusion has provided a safe and convenient alternative to bolus injections in haemophiliacs with inhibitors, but it has only been reported in a single case with congenital factor VII (FVII) deficiency. The results of 12 consecutive surgical procedures in 7 patients with congenital FVII deficiency are reported here. rFVIIa was always given in continuous infusion,aiming at plasma FVII activity of 0.5 IU/mL. Treatment was given for 2 to 7 days with a mean total dose of 7.8 mg rFVIIa. Blood loss was as expected from the different types of procedures and the only thromboembolic complication was a superficial thrombophlebitis at the infusion site. This mode of substitution was therefore safe, effective and well tolerated.     

Roles of platelets and factor XI in the initiation of blood coagulation by thrombin

To account for the variable hemostatic defect in patients with factor XI (FXI) deficiency, with normal hemostasis in contact factor deficiencies, a coagulation paradigm is presented whereby trace quantities of thrombin, generated transiently by exposure of tissue factor at sites of vascular injury, activates FXI bound to the platelet surface in the presence of prothrombin or high Mr kininogen (HK). Tissue factor pathway inhibitor (TFPI) limits the flux of thrombin generated by the tissue factor pathway, and protease nexin II (PNII), released from activated platelets, inhibits solution phase FXIa and localizes FIX activation to the platelet surface where FXIa is protected from inactivation by PNII. Either prothrombin or HK binds to the Apple 1 (A1) domain of FXI, thereby exposing a platelet-binding site in the FXI A3 domain. Dimeric FXI binds to activated platelets directly through the A3 domain of one monomer. After proteolytic activation of platelet-bound FXI by thrombin (or FXIIa), a substrate binding site for FIX is exposed in the opposite monomer that promotes FIX activation on the platelet surface resulting in the local explosive generation of thrombin and the formation of hemostatic thrombi at sites of vascular injury.     

Relationship between factor VII activity and clinical efficacy of recombinant factor VIIa given by continuous infusion to patients with factor VIII inhibitors.

A multicenter prospective study of recombinant activated factor VII (rFVIIa) given by continuous infusion (CI) to treat severe hemorrhages and to handle surgical procedures was carried out. Relations between clinical efficacy, dosages used and levels of FVII coagulant activity (FVII:C) achieved in plasma were also evaluated. Case material included 25 patients with hemophilia (9 children and 16 adults) with high-responding inhibitors and 3 patients with acquired factor VIII inhibitors. Overall, 35 CI courses were given for 10 spontaneous bleeding episodes, 11 major surgical procedures and 14 minor surgical procedures. Bolus doses of 90 to 150 microg/kg (median: 100) were followed by CI given at median rates of 20 microg/kg/h for major surgery and of 17 and 16 microg/kg/h for minor surgery and spontaneous hemorrhages. Satisfactory hemostasis was obtained in 30 of 35 courses (88%). rFVIIa CI was ineffective in 2 hemophiliacs undergoing surgical operations and in another hemophiliac with hemoperitoneum who had to be switched to other treatments (high doses of porcine or human factor VIII concentrates). rFVIIa CI was partially effective in 2 hemophiliacs who had mild local bleeding after minor surgery. The CI rates and the corresponding FVII:C levels in plasma were similar in effective, partially effective and ineffective courses (median rate: 17, 20 and 20 microg/kg/h, respectively; median FVII:C:14, 18 and 18 IU/ml, respectively). A single adverse event was observed, superficial thrombophlebitis. This study confirms that rFVIIa given by CI is effective in a high proportion of patients with factor VIII inhibitors. It also demonstrates that FVII:C levels attained in plasma do not always predict efficacy because similarly high levels were attained during successful treatments and in those that failed.     

Injection of recombinant activated factor VII can induce transient increase in circulating procoagulant microparticles

Recombinant activated factor VII (rFVIIa) is an effective haemostatic treatment in haemophiliacs with inhibitors. In vitro, FVIIa concentrations corresponding to those obtained with therapeutic doses of rFVIIa have been shown to induce normal thrombin generation and platelet activation in the absence of factors VIII or IX. To further study the in vivo haemostatic changes induced by rFVIIa, circulating procoagulant microparticles (MP) were measured in patients treated with discontinuous injections of Novoseven. In 6 out of 15 patients, a transient peak of procoagulant MP was observed after injection, occurring 15 min to 2 h after infusion. It was composed primarily of platelet-derived MP and was of very short duration. This peak was not observed in haemophiliacs without inhibitor, who were treated with conventional replacement therapies. Our results provide further in vivo evidence that rFVIIa specifically activates platelets, either directly or as a consequence of a burst of thrombin generation that could account for its haemostatic efficacy.     

Haemostatic efficacy and safety of bolus and continuous infusion of recombinant factor VIIa are comparable in haemophilia patients with inhibitors undergoing major surgery. Results from an open-label, randomized, multicenter trial

Bolus infusion (BI) recombinant factor VIIa (rFVIIa) administration is safe and effective in the surgical management of haemophilia patients with inhibitors but has not been compared directly with continuous infusion (CI). We conducted an open-label, randomized, multicenter trial comparing the efficacy and safety of rFVIIa administered by BI or CI for the surgical management of haemophilia A or B patients with inhibitors to FVIII or FIX. Safety was compared with that of a control group of non-inhibitor patients receiving FVIII or FIX concentrates for major surgery. All inhibitor subjects received an initial bolus dose of 90 microg/kg rFVIIa and were then randomly assigned to BI (n = 12) or CI (n = 12). The BI group received 90 microg/kg rFVIIa every two hours (h) during surgery through day 5, then every four hours for days 6-10. The CI group received 50 microg/kg/h rFVIIa through day 5, then 25 mg/kg/h for days 6-10. The control group (n = 12) received FVIII or FIX per institutional protocols. Twenty-two major surgeries included orthopedic procedures on the knee (n = 13), hip (n = 3), and abdominal/pelvis procedures (n = 4). One patient with an autoimmune FVIII inhibitor randomized to the BI arm was excluded from efficacy analysis. Haemostatic efficacy of rFVIIa in each group was comparable: effective in 8/11 and 9/12 subjects in the BI and CI arms, respectively, and ineffective in three subjects in each arm. Serious adverse events were related to continued or increased bleeding. In conclusion, haemostatic efficacy and safety of BI and CI of rFVIIa are comparable for the surgical management of haemophilia subjects with inhibitors.     

Pharmacodynamics of recombinant activated factor VII and plasma-derived factor VII in a cohort of severe FVII deficient patients

Recombinant activated factor VII (rFVIIa) and plasma-derived factor VII (pdFVII) are used to prevent bleedings in severe FVII deficient patients, despite their short half-lifes. It is suggested that FVII levels of 15-20 IU/dL are sufficient to maintain hemostasis. We analyzed the pharmacodynamic effects of FVII substitution therapy in the Nijmegen Hemostasis Assay (NHA) that simultaneously measures thrombin and plasmin generation. Ten severe FVII deficient patients were treated with 20 μg/kg rFVIIa or 25 IU/kg pdFVII in a cross-over design.     

Patients with severe factor XI deficiency have a reduced incidence of deep-vein thrombosis

Factor XI (FXI) plays a dual role in haemostasis and thrombosis. It contributes to thrombin generation and promotes inhibition of fibrinolysis. Severe FXI deficiency was shown to confer protection against arterial and venous thrombosis in animal models without compromising haemostasis. We have previously shown that patients with severe FXI deficiency have a low incidence of ischaemic stroke, but display the usual incidence of myocardial infarction. In the present study, we compared the incidence of deep-vein thrombosis (DVT) in 219 unrelated patients with severe FXI deficiency aged 20-94 to the incidence in a large population-based study. No cases of DVT were observed in the FXI-deficient cohort, a result that is significantly lower than the expected number (4.68) computed from the population-based study. The low incidence remains statistically significant when compared to three other population-based studies. These data suggest that severe FXI deficiency provides protection against DVT.     

The effect of combined factor XI deficiency with von Willebrand factor abnormalities on haemorrhagic diathesis

To account for the lack of correlation between the level of factor XI (FXI) in deficient patients and haemorrhagic manifestations, we correlated the prevalence of combined FXI and von Willebrand's factor (vWF) deficiency in 212 FXI-deficient patients. Fifty-four patients had a combined FXI and vWF deficiency: 16 patients had severe and 38 patients had mild FXI deficiency. In a group of 28 patients with comparably mild FXI deficiency, 14 bleeders had significantly lower mean vWF, Ag, ristocetin cofactor and ristocetin induced platelet aggregation than 14 non-bleeders selected on the basis of comparable FXI levels. These findings suggest that the combination of FXI and vWF deficiency is common and may affect the bleeding tendency in mild FXI deficiency.     

The combination of recombinant factor VIIa and fibrinogen correct clotting ex vivo in patient samples obtained following cardiopulmonary bypass surgery

Cardiac surgery involving cardio pulmonary bypass (CPB) may be associated with development of a coagulopathy that increases risk of bleeding. In the present ex vivo study we investigated the influence of fibrinogen and rFVIIa, alone or in combination, on whole blood coagulation thromboelastometry using pre- and postoperative blood samples from 18 consecutive adult patients undergoing CPB surgery. Dynamic thromboelastometric clotting profiles were recorded using citrated whole blood activated with trace amounts of tissue factor (Innovin®, final dilution 1:17000). Blood samples were collected before surgery (control) and postoperative samples were obtained following in vivo neutralization of heparin with protamine sulphate. All blood samples were treated with heparinase to ensure neutralization of possible residual heparin effect. The post-operative blood samples were spiked with buffer, rFVIIa (2 µg/mL), fibrinogen (1 mg/mL), or the combination of rFVIIa and fibrinogen. Despite neutralization of heparin, CPB surgery left a measurable coagulopathy that was thromboelastometrically characterized by prolonged onset of clotting, reduced maximum velocity of clot formation (MaxVel), and decreased maximum clot firmness (MCF). Ex vivo spiking of the postoperative samples with rFVIIa shortened the clotting time. Fibrinogen also shortened the clotting time and, in addition, improved the MaxVel, and MCF. Finally, adding the combination of rFVIIa and fibrinogen to the postoperative samples corrected all thromboelastometric parameters to the preoperative range. In conclusion, the correction of whole blood clotting abnormalities that occurs with rFVIIa and/or fibrinogen suggests that future clinical trials on treatment of bleeding during CPB surgery should study the haemostatic effect of fibrinogen or possibly the combination of rFVIIa and fibrinogen.     

The role of platelets and recombinant factor VIIa on thrombin generation, platelet activation and clot formation

In the present study we assessed the effect of platelet counts and rFVIIa on thrombin generation, platelet activation and clot formation after tissue factor pathway activation in human plasma aiming to investigate the mechanism by which rFVIIa induces haemostasis in patients with severe thrombocytopenia. Plasma samples with platelet counts from 5 x 10(9)/l to 150 x 10(9)/l were spiked with rFVIIa (1 micro g/ml) or buffer. Clotting was initiated in the presence of diluted thromboplastin. Thrombin generation was assessed using the Thrombogram-Thrombinoscope trade mark assay. The kinetics of platelet activation was assessed using flow cytometry to measure the expression the P-selectin on platelet membrane of washed platelets suspended in defibrinated homologous PPP. Thromboelastography was used to evaluate the effect of platelets and rFVIIa on the kinetics of clot formation and clot's firmness. In the presence of low platelet counts the endogenous thrombin potential (ETP) and the maximum concentration of generated thrombin (Cmax) were reduced by 60%-70%. The lag-time of thrombin generation and the time required to reach the Cmax (Tmax) were prolonged, the velocity of platelet activation was decreased and thrombus formation was delayed. Recombinant FVIIa accelerated thrombin generation and platelet activation but it did not significantly modify ETP or Cmax. Recombinant FVIIa enhanced platelet activation in a TF and thrombin dependent manner since its effect on the studied parameters was abolished when TF was omitted or when hirudin was added into the experimental system respectively. Recombinant FVIIa normalized the velocity of clot formation but it did not modify clot firmness, which depended mainly on platelets' count. In conclusion, in experimental conditions simulating severe thrombocytopenia rFVIIa in the presence of low amounts of TF, accelerates thrombin generation, without increasing the maximum amount of generated thrombin, thus leading in enhanced platelet activation and rapid clot formation.     

Factor XI: hemostasis, thrombosis, and antithrombosis

Coagulation factor FXI (FXI), a plasma serine protease zymogen, has important roles in both intrinsic and extrinsic coagulation pathways and bridges the initiation and amplification phases of plasmatic hemostasis. Recent studies have provided new insight into the molecular structure and functional features of FXI and have demonstrated distinct structural and biological differences between activated factor XII (FXIIa)-mediated FXI activation and tissue factor/thrombin-mediated FXI activation. The former is important in thrombosis; the latter is more essential in hemostasis. Activated partial thromboplastin tine (aPTT) artificially reflects FXIIa-initiated intrinsic coagulation pathway in vitro. Conversely, FXIIa-inhibited diluted thromboplastin time assay may reflect tissue factor/thrombin-mediated FXI activation in vivo. Further explication of the genetic mutations of FXI deficiency has improved the understanding of the structure-function relationship of FXI. Besides its procoagulant activity, the antifibrinolytic activity of FXI was well documented in a wealth of literature. Finally, the new emerging concept of inhibiting FXI as a novel antithrombotic approach with an improved benefit-risk ratio has been supported through observations from human FXI deficiency and various animal models. Large- and small-molecule FXI inhibitors have shown promising antithrombotic effects. The present review summarizes the recent advancements in the molecular physiology of FXI and the molecular pathogenesis of FXI deficiency and discusses the evidence and progress of FXI-targeting antithrombotics development.     

A direct thrombin inhibitor studied by dynamic whole blood clot formation. Haemostatic response to ex-vivo addition of recombinant factor VIIa or activated prothrombin complex concentrate

Direct thrombin inhibitors have proven efficacious in prevention of venous thromboembolism. Bleeding complications are rare, but in case of acute serious bleeding, an effective and instant haemostatic intervention may be required. In the present study it was demonstrated that the direct thrombin inhibitor melagatran induces dose-dependent abnormalities in whole blood (WB) clotting profiles as recorded by a recently described modified thrombelastographic model, and that rFVIIa or APCC are capable of improving the haemostatic capacity. Experiments were performed using WB from 30 healthy males. In-vitro titration experiments (n = 10) with addition of melagatran to WB corresponding to plasma concentrations ranging from 0 to 5.0 microM (12 steps) showed a dose-dependent prolongation of the clot initiation and characteristic decrease of the maximum rate of clot propagation. In-vitro intervention studies (n = 20) were completed with four different concentrations of melagatran as well as addition of four different levels of rFVIIa or APCC. At all tested concentrations of melagatran, rFVIIa significantly shortened the melagatran-induced prolonged clot initiation but induced only minor improvements of the reduced clot propagation. In contrast, APCC significantly and dose-dependently shortened the clot initiation and accelerated the clot propagation. In conclusion, our thrombelastographic model appears useful for evaluating the effect of direct thrombin inhibitors on dynamicWB clot formation and rFVIIa, but especially APCC significantly improved theWB clot formation. The pronounced stabilizing effect of APCC may be caused by its content of prothrombin and activated coagulation factors.     

The role of factor VII in haemostasis: infusion studies of factor VIIa in a canine model of factor VIII deficiency

The role of factor VIIa in haemostasis has been studied using a canine model of factor VIII deficiency. Highly purified human factor VIIa was administered to dogs at a dosage of 0.5 microgram/kg. At selected times pre- and post-infusion, haemostasis was evaluated by the cuticle bleeding time. Plasma was collected for the assay of various parameters, including fibrinopeptide A (FPA) as a marker for thrombin generation in vivo. Factor VIIa infusion resulted in a 6-fold increase of factor VII clotting activity with a t1/2 of 2 h. FPA levels, which were 1.4 ng/ml before infusion, did not increase significantly in haemophilic dogs. In normal dogs, however, FPA levels rose to a mean value of 190 ng/ml 30 min post-infusion. It appeared that thrombin generation by factor VIIa infusion had occurred mainly via the intrinsic, factor VIII-dependent pathway. In factor VIII-deficient dogs, factor VIIa infusion did not correct cuticle bleeding, but an inconsistent haemostatic effect was observed 15-30 min post-infusion. Similar results were obtained in haemophilic dogs with circulating antibodies against factor VIII. The haemostatic effectivity could not be improved by increasing the factor VIIa dosage up to 40-fold. Although these data suggest that the extrinsic, factor VII-dependent factor X activation provides only a minor pathway of thrombin generation in vivo, it is possible that the suboptimal haemostatic effect noted may be promoted in bleeding situations where tissue factor availability is less limited. As such, factor VIIa may prove useful in the treatment of haemophilia A patients with acquired inhibitors to factor VIII.     

Does recombinant factor VIIa, apart from overall hemostasis, regulate TAFI dependent fibrinolysis? In vitro analysis using overall hemostasis potential (OHP) assay

Using the recently developed overall hemostatic potential (OHP) assay, we investigated the effects of different concentrations of recombinant factor VIIa on overall hemostasis and on thrombin activatable fibrinolysis inhibitor (TAFI) dependent fibrinolysis in different factor deficient plasmas. rFVIIa increased OHP in FV, FVIII and FIX deficient plasmas but not up to the levels in normal pooled plasma (NPP). Maximal levels were found at 2.4 microg/mL of rFVIIa, while higher doses did not further increase OHP. In FXII deficient plasma, OHP is higher than in NPP and addition of rVIIa further increased it. Even very high concentrations of rFVIIa (9.6 microg/mL) did not induce a significant increase of OHP in NPP. Higher concentrations of rVIIa down-regulated fibrinolysis in FVIII, FIX and FXI deficient plasmas to values obtained in NPP. Using potato tuber carboxy-peptidase inhibitor (PTCI), a specific inhibitor of TAFI, it was found that TAFI's influence on fibrinolysis down-regulation in FVIII and FIX deficient plasmas increased after addition of higher concentrations of rFVIIa. From this in vitro study it seems that rFVIIa in a concentration of 2.4 microg/mL improves overall hemostasis in FV, FVIII and FIX deficient plasmas. rFVIIa, even at very high (supra-therapeutic) concentrations, does not induce hypercoagulability either in NPP or in deficient plasmas. Higher concentrations of rVIIa induce, at least partly, TAFI dependent down-regulation of fibrinolysis in FVIII and FIX deficient plasmas. These results, together with some previous ex vivo studies, point to OHP assay as a possible diagnostic tool for evaluating the hemostatic effects of rFVIIa.     

Immunogenicity of novel recombinant human activated factor VII analogues on factor VII neonatally-tolerized rats

Recombinant activated factor VII (rFVIIa; NovoSeven) has been widely used to treat bleeding in patients with haemophilia with inhibitors. To increase the intrinsic activity, analogues of rFVIIa (rFVIIa Q, rFVIIa DVQ, and rFVIIa DVQA) with altered amino acid sequence at or near the active centre have been developed. The immunogenicity of these analogues was tested in a rat immune tolerance model. Neonatal rats received rFVIIa intraperitoneally on post-natal Day 1 and were subsequently challenged with rFVIIa in Freunds Incomplete Adjuvant subcutaneously on Days 10 and 24. Rats were tested for tolerance on Day 32; the tolerant cohort and a parallel cohort of untreated control rats were challenged with rFVIIa, rFVIIa Q, rFVIIa DVQ, or rFVIIa DVQA on Days 46 and 76. Immune responses determined by enzymelinked immunosorbent assay (ELISA) on Day 84 showed no statistically significant difference between the responses in the four control cohorts. Immune responses were higher in the control than in the tolerant cohort. Compared with rFVIIa (4/16), there was no difference in the proportion of rats that broke tolerance following challenge with rFVIIa DVQ (3/16) and rFVIIa DVQA (7/16), whereas a statistically significant greater proportion broke tolerance after challenge with rFVIIa Q (11/16). Therefore, in this model rFVIIa DVQ or rFVIIa DVQA were not more immunogenic than rFVIIa.