Mechanism of action, development and clinical experience of recombinant FVIIa

Journal of Biotechnology 124 (2006) 747–757

Mechanism of action, development and clinical experience of recombinant FVIIa Ulla Hedner a,b,∗ b

a University of Lund, Sweden Research & Development, Novo Nordisk A/S, Bagsværd, Denmark

Received 25 October 2005; received in revised form 23 January 2006; accepted 29 March 2006

Abstract Recombinant FVIIa has been developed for treatment of bleedings in hemophilia patients with inhibitors, and has been found to induce hemostasis even during major surgery such as major orthopedic surgery. Recombinant FVIIa is being produced in BHK cell cultures and has been shown to be very similar to plasma-derived FVIIa. The use of rFVIIa in hemophilia treatment is a new concept of treatment and is based on the low affinity binding of FVIIa to the surface of thrombin activated platelets demonstrated in a cell-based in vitro model. By the administration of pharmacological doses of exogenous rFVIIa the thrombin generation on the platelet surface at the site of injury is enhanced independently of the presence of FVIII/FIX. As a result of the increased and rapid thrombin formation, a tight fibrin hemostatic plug is being formed. A tight fibrin structure has been found to be more resistant to fibrinolytic degradation thereby helping to maintain hemostasis. The general mechanism of action of pharmacological doses of rFVIIa shown to induce hemostasis not only in hemophilia, but also in patients with platelet defects, and with profuse bleedings triggered by extensive surgery or trauma, may very well be the capacity of generating a tight fibrin hemostatic plug through the increased thrombin generation. Such a fibrin plug will help to resist the overwhelming mostly local release of fibrinolytic activity triggered by the vast tissue damage occurring in extensive trauma. A release of fibrinlytic activity locally has also been demonstrated to occur in the gastrointestinal tract as well as during profuse postpartum bleedings. Pharmacological doses of rFVIIa have in fact, also been shown to induce hemostasis in such cases. © 2006 Elsevier B.V. All rights reserved. Keywords: Recombinant FVIIa; Bleeding disorders; Hemophilia treatment; Glanzmann’s thrombasthenia

1. Introduction

∗ Correspondence to: Caritasgatan 19, SE-218 20 Malm¨ o, Sweden. Tel.: +46 40 162869; fax: +46 40 162819. E-mail address: [email protected].

0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.03.042

Patients with severe hemophilia need regular treatment with a hemostatically effective product in order to avoid the development of a chronic athropathy developing as a result of repeated joint bleedings. They

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also require immediate effective treatment of bleedings associated with trauma or essential surgery. Around 20% of patients with severe hemophilia A develop inhibitory antibodies against the coagulation factor they are missing. The administration of factor VIII (FVIII) or factor IX (FIX) concentrates is not effective in these patients, since the respective coagulation factor is neutralized by the antibodies. Much effort has, therefore, been focused on finding a treatment which is hemostatically effective independent of the presence of FVIII/FIX. Activated prothrombin complex concentrates (APCC) containing both activated coagulation proteins and zymogens are still widely used to achieve such a FVIII by-passing effect. A hemostatic effect of these concentrates averaging between 50 and 65% was reported (Lusher et al., 1983; Sjamsoedin et al., 1981). Also, side-effects in terms of thromboembolic events have been reported (Lusher, 1991). A dog model was used to identify some of the factors involved in the development of these side-effects (Hedner et al., 1979). Based on these studies, activated coagulation FVII (FVIIa) was identified as an attractive candidate for a hemostatic agent independent of FVIII/FIX, for use in hemophilia patients. Purified FVIIa was later shown to be free of such effects in a similar dog model (Hedner and Kisiel, 1983). Furthermore, purified plasma-derived FVIIa was shown to induce hemostasis in a few severe hemophilia patients (Hedner and Kisiel, 1983; Hedner et al., 1989). It was suggested at the time that pharmacological doses of FVIIa bind to tissue factor (TF) exposed at the site of injury, activating FX and provide thrombin locally at the site of injury (Hedner and Kisiel, 1983).

beta-hydroxy-aspartic acid in position 63 was found, neither in the plasma-derived FVIIa nor in the rFVIIa. Furthermore, 9 out of the 10 possible Gla residues were fully gamma-carboxylated and one partially (approximately 50%) in the rFVIIa (Thim et al., 1988). Both of the two O-glycosylated sites of plasma-derived FVIIa were similarly O-glycosylated in rFVIIa, but the relative amounts of the three O-linked structures differed slightly between plasma-derived and rFVIIa (Bjoern et al., 1991). Both potential N-glycosylation sites were occupied in plasma-derived as well as in rFVIIa. Minor quantitative differences were seen in the carbohydrate composition of the two FVIIa molecules, the most pronounced difference being a higher fucose content and a lower sialic acid content of rFVIIa compared with those for plasma-derived FVIIa (Thim et al., 1988). A full characterization of the N-linked carbohydrate structures of rFVIIa has later been performed (Klausen and Kornfelt, 1995; Weber et al., 1995; Klausen et al., 1996). The rFVIIa is produced in a mammalian expression system using baby hamster kidney cells (Hagen et al., 1986). A master cell bank (MCB) was established and found to provide BHK cells capable of a stable expression of FVII. Further production using a working cell bank (WCB) for cultivation of the cells as described previously (Jurlander et al., 2001) is being used for production of rFVIIa. The single-chain rFVII molecule is spontaneously activated into rFVIIa during the purification process (Bjoern and Thim, 1986). The purification process is essentially performed as being described (Jurlander et al., 2001).

3. Preclinical development 2. Development of recombinant FVIIa Based on the observed hemostatic effect of plasmaderived purified FVIIa in severe hemophilia patients, recombinant FVIIa (rFVIIa) was developed for use in the treatment of severe hemophilia complicated with inhibitors against FVIII/FIX (Thim et al., 1988; Hagen et al., 1986). The amino acid sequence and posttranslational modifications of rFVIIa from the culture medium of a transfected baby hamster kidney cell line was compared to human plasma FVIIa. The two molecules were identical as for amino acid sequence and identical to that predicted from the cDNA sequence. No

The hemostatic effect of rFVIIa was demonstrated in hemophilia dogs (Brinkhous et al., 1989) as well as in warfarin treated rats (Diness et al., 1990). No systemic activation of the coagulation was found following the injection of rFVIIa into rabbits using a stasis model originally developed as a thrombosis model. For a comparison it was shown in the same model that the injection of activated prothrombin complex concentrate (FEIBA) did induce lowering of the platelet counts as well as of the fibrinogen plasma concentration considered as signs of a systemic activation of the coagulation system (Diness et al., 1992). Administration of

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rFVIIa in doses up to 300 ␮g/kg to rabbits previously exposed to endotoxin did not result in any significant hematologic changes, compared with rabbits treated with endotoxin alone (Diness et al., 1992).

4. Clinical development The first patient treated with rFVIIa underwent open surgical synovectomy in March 1988 without any complications and no per- or postoperative abnormal bleeding (Hedner et al., 1988). An efficacy rate of 90–100% was later confirmed in several series of severe hemophilia patients subjected to surgery including major orthopedic surgery (Ingerslev et al., 1996; Shapiro et al., 1998). A review of patients with severe hemophilia with antibodies including close to 500 subjects treated at around 1900 bleeding episodes showed an efficacy rate in limb- and life threatening bleeds between 76 and 84% (Lusher et al., 1998; Abshire and Kenet, 2004). The level of inhibitor to FVIII/FIX does not influence the efficacy of rFVIIa, nor does it induce an anamnestic response in FVIII/FIX-deficient patients (Johannessen et al., 2000). Furthermore, a follow up of 267 patients including 222 hemophilia A and 16 hemophilia B patients, 16 non-hemophilia patients with inhibitors, and 13 FVII-deficient patients, for 6 years regarding a potential development of inhibitors against FVII was performed. The study revealed that pre-treatment samples from 5% of the hemophilia A patients had values above the normal range as measured in a direct enzyme-linked immunosorbent assay in which the cut-off was defined as two standard deviations above the mean value, obtained from analysis of pre-treatment samples from hemophilia patients and healthy donors (Nicolaisen et al., 1996), but none of the reactions were specific. Increased post-treatment values were observed in two FVII-deficient patients; both of whom had been previously treated with plasmaderived FVII. The FVII-specific antibodies in these two patients reacted equally well with plasma FVII and rFVIIa. The overall result from antibody determination shows no indication of antibody formation against rFVIIa in hemophilia A or B patients or in nonhemophilia patients with acquired inhibitors. However, FVII-deficient patients represent a risk group for development of antibodies against FVII (Nicolaisen, 1998).

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The incidence of thrombotic events with the use of rFVIIa has been found to be extremely low. Since the licensing of rFVIIa in 1996, more than 700,000 standard doses (90 ␮g/kg for a 40 kg patient) of rFVIIa have been administered to patients with congenital hemophilia with inhibitors, or acquired hemophilia. Among these patients, 16 thrombotic events have been reported. In addition, six events were reported in association with clinical trials. All these events have been reviewed in detail. In no case, it could be clearly determined that rFVIIa was definitely causally related to the thrombotic event, and known factors predisposing to thrombosis were present in 20 of the 25 (80%) hemophilia patients reported spontaneously or who developed a thrombosis during a clinical trial (Abshire and Kenet, 2004). The final product (rFVIIa; NovoSeven) was approved for use in hemophilia with inhibitors and in patients with acquired hemophilia in 1996 in Europe, 1999 in USA and 2000 in Japan.

5. Mechanism of action 5.1. Normal hemostasis According to current concept, hemostasis occurs on principally two types of surfaces, the tissue factor (TF) expressing cell and the thrombin activated platelet (Monroe et al., 2002). TF is expressed by a number of various cells localized in the deeper layers of the vessel wall and normally not exposed to the circulating blood. TF is a true receptor protein and FVII/FVIIa is its ligand. As a result of tissue injury, TF is exposed to the circulation and forms a complex with FVII or FVIIa on the surface of the TF-bearing cell (Rapaport and Rao, 1992). TF may also originate from circulating blood in the form of encrypted TF carried by cell elements such as WBC or microparticles through a P-selectin/P-selectin glycoprotein ligand 1 mediated process (Giesen et al., 1999; Himber et al., 2003; Polgar et al., 2005). Recently, a thiol-oxidizing agent was shown to promote activation of TF, implying that a disulphide-bond is reduced in the cryptic form of TF and that formation of the disulphide results in activation and facilitating the binding of FIX and FX (Chen et al., 2005). Approximately, 1% of the FVII protein mass is normally present in an activated form

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in the circulation (Wildgoose et al., 1992; Morrissey et al., 1993). Furthermore, FVII and other vitamin K-dependent coagulation proteins are present in the interstitial fluid (Le et al., 1998), and the, in plasma normally occurring limited amounts of FIX, FX, and prothrombin activation peptides (Bauer et al., 1990), may therefore originate from an interstitial activation of the hemostatic system. The limited amount of thrombin generated by the priming phase of hemostasis initiated by the TF–FVII/FVIIa complexes, activates platelets resulting in a surface that supports the binding of coagulation factors thereby facilitating the full thrombin burst necessary for effective hemostasis (Monroe et al., 2002). The binding of coagulation proteins such as FV, FVIII, FIX, and FX was found to be facilitated by the combined stimulation of the platelet collagen receptor (GPVI) and thrombin receptors due to the development of a subpopulation of platelets with an increased binding capacity (Kempton et al., 2005). Full activation of the thrombin-activable fibrinolytic inhibitor (TAFI) requires a thrombin concentration around 500 nM (Bajzar et al., 1995). TAFI is crosslinked to fibrinogen through the activity of FXIII (Valnickova and Enghild, 1998), and it cleaves lysine and arginine residues on fibrin leading to a reduced binding of plasminogen and plasminogen activator to fibrin (Wang et al., 1998), thereby protecting the hemostatic plug against premature lysis. To achieve necessary thrombin generation to allow full TAFI activation, FVIII, FIX as well as FXI are required explaining the susceptibility to fibrinolysis observed in hemophilia and in patients with FXI-deficiency (Broze and Higuchi, 1996; van dem Borne et al., 1997). A full thrombin burst is also important for the fibrin structure of the hemostatic plug. Fibrin porosity and structure have been demonstrated to vary as a function of added thrombin (Blomb¨ack et al., 1989; Carr and Alving, 1995). Furthermore, increased prothrombin concentrations result in increased thrombin generated as well as in increased initial rate of thrombin generation (Allen et al., 1999), which resulted in a clot structure with reduced fibrin mass-to-length ratios (Wohlberg et al., 2003). However, not only the fiber thickness, but also the fibrin network configuration and the number of fibers per volume of clot seem to have a substantial impact on the rate of fibrinolysis (Carr and Alving, 1995; Collet et al., 2003). Also, the fibrinogen concen-

tration has been found to play a key role. Thus, the rate of fibrinopeptide A cleavage increases with increasing fibrinogen concentration (Okada and Blomb¨ack, 1983), which is associated with a shorter lag phase and a more dense and tight fibrin network (Blomb¨ack, 1994). High fibrinogen levels also result in the formation of thicker fibers (Carr and Hermans, 1978; Blomb¨ack et al., 1989), and interfere with the binding of plasminogen to its receptor, reducing fibrinolysis (Koenig, 2003). In summary, a full thrombin burst is essential for the formation of a stable fibrin hemostatic plug, that is resistant to premature fibrinolysis, thus providing a reliable and maintained hemostasis. In the process of thrombin generation the coagulation proteins are of great importance, increasing prothrombin as well as fibrinogen concentrations resulting in enhanced thrombin generation and a tighter fibrin plug. High fibrinogen concentration also helps to reduce fibrinolysis. 5.2. Role of pharmacological doses of rFVIIa Hemophilia patients have normal initiation or priming phase of hemostasis with a normal basis formation of FX and prothrombin activation peptides. The intact TF pathway including the formation of initial TF–FVIIa complexes results in a normal initial thrombin formation leading to a normal initial platelet activation. Hemophilia patients lack FVIII (hemophilia A) or FIX (hemophilia B), and therefore are unable to form the FIX–FVIII complexes on the surface of the activated platelets. Such complexes are necessary for the full FX activation and thrombin generation occurring on the surface of thrombin activated platelets. The impaired thrombin generation in hemophilia patients leads to the formation of a defective fibrin plug as well as suboptimal activation of TAFI and FXIII. The impaired hemostasis characteristic of hemophilia, thus, depends on the formation of defective hemostatic plugs that are sensitive to lysis and therefore fail to sustain hemostasis. The administration of pharmacological doses of rFVIIa reaching plasma concentrations of 25 nM or higher, induces hemostasis in the absence of FVIII or FIX, most probably by enhancing the thrombin generation on the thrombin activate platelet surface in the end leading to the formation of a tight, stable fibrin hemostatic plug. In a cell-based in vitro model, it was shown that rFVIIa binds to thrombin activated

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platelet surface with a low affinity, requiring higher concentrations of rFVIIa than those found normally in circulating blood. The bound rFVIIa activates FX on the activated platelet surface independent of the presence of FVIII or FIX (Monroe et al., 1997). In the same cell-based in vitro model, the thrombin generation in the absence of FIX was substantially improved, following addition of rFVIIa in concentrations of 50 nM and up to 100 nM, although never totally normalized as compared to, if the system included also FXI in physiological concentrations (Allen et al., 2000). However, rFVIIa recently was found to prolong the clot lysis time in vitro in hemophilia A plasma being dependent on activation of TAFI (Lisman et al., 2002a), supporting the capability of pharmacological doses of rFVIIa to substantially enhance the thrombin generation in spite of the fact that these experiments were performed in the presence of artificial phospholipids instead of platelets. A more efficient thrombin generation in terms of both shortening of lag time, the rate, and the total yield in the presence of platelets than if artificial phospholipids were used, was previously shown (Walsh, 2003). An enhanced effect on the prolonged clot lysis time was demonstrated by a modified rFVIIa molecule having an increased TF-independent FX-activating activity (Lisman et al., 2003) supporting the importance of FX-activation on the thrombin activated platelet surface and thereby thrombin generation. These experiments were performed in the presence of artificial phospholipids as well as in platelet rich plasma. Furthermore, a normalization of the fibrin permeability also was achieved by addition of rFVIIa to hemophilia plasma containing platelets, an effect that was reflected in a tighter fibrin structure (He et al., 2003). The hemostatic effect of exogenously added rFVIIa in pharmacological doses, thus, seems to be mediated by enhancing the rate of thrombin generation on thrombin activated platelet surfaces resulting in an enhanced further activation of platelets (increased exposure of phospholipids) at the site of injury, increased platelet adhesion suggested to involve an enhanced platelet–platelet interaction initiated by thrombin binding to GPIb as well as other mechanisms (Lisman et al., 2005). The enhanced thrombin generation also ensures the formation of a tight fibrin structure of the hemostatic plug as well as full activation of TAFI and FXIII necessary for maintaining hemostasis.

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In a different model system using artificial phospholipids in a test tube system it was demonstrated that rFVIIa was able to overcome the inhibitory effect of zymogen FVII on the TF-dependent thrombin generation. Based on these results it was suggested that the rationale for a therapeutical effect of rFVIIa in hemophilia patients may be at least partly overcoming the inhibitory effect of physiologic concentrations of zymogen FVII on TF-dependent hemostasis (Van’t Veer et al., 2000; Butenas et al., 2002). However, the distinct difference between artificial phospholipids and platelets has been stressed (Walsh, 2003; Monroe et al., 2002). The hemostatic effect of rFVIIa is obviously dependent on TF initially for providing the initial limited amount of thrombin necessary for activation of the platelet to occur. The requirement for thrombin activated platelets in the enhancing effect on thrombin generation by rFVIIa is underlined by several studies (Butenas et al., 2002; He et al., 2005). Furthermore, the doses required clinically for hemostasis in hemophilia seem to be in the ranges of amount of rFVIIa that gave a substantial effect on thrombin generation in the cellbased model in the presence of platelets (Allen et al., 2000).

6. Clinical experience with rFVIIa in hemophilia patients with Inhibitors Hemophilia patients lacking FVIII (hemophilia A) or FIX (hemophilia B) develop severe, spontaneous bleedings deep in the body, characterized by being essentially non-responding to local pressure and likely to recur several hours later. Thrombin formation is markedly impaired in hemophilia, underscoring the importance of full thrombin generation for optimal hemostasis. The administration of pharmacological doses of rFVIIa increasing the plasma level of FVII:C from the normal value of around 1 U/ml of plasma to levels varying between 30 and 80 U/ml have been demonstrated to induce hemostasis in serious bleedings with an efficacy rate of between 62 and 88% (Lusher et al., 1998), while the overall effective response after 8 and 24 h in the first 55 consecutive bleeds was 91 and 90%, respectively (Glazer et al., 1995). Major surgery in hemophiliacs require full substitution therapy with FVIII or FIX in order to avoid uncontrollable bleeding. Accordingly, elective surgery

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has been contraindicated in hemophilia patients with inhibitors against FVIII or FIX, since FVIII/FIX concentrates are ineffective in such patients. The administration of rFVIIa has been demonstrated to induce hemostasis with an efficacy rate of around 90% provided doses of 90–120 ␮g/kg are given as bolus every second hour for the first 48 h, and thereafter, the same dose every 2–6 h for an additional 3–5 days depending on type of surgery (Ingerslev et al., 1996; Shapiro et al., 1998). These results strongly support the capacity of rFVIIa in pharmacological doses to generate enough thrombin to compensate for the lack of FVIII/FIX.

7. Clinical experience with rFVIIa in other situations with an impaired thrombin generation than hemophilia The availability of platelet procoagulant phospholipids has been demonstrated to be rate limiting for thrombin generation and accordingly, an impaired thrombin generation was described in patients with thrombocytopenia in the 1950s by Biggs and MacFarlane (1962). In accordance with this, the dependence of thrombin generation on platelet counts in a cell-based in vitro model was shown (Kjalke et al., 1999, 2001). In the same cell-based model the addition of rFVIIa in concentrations of 50–500 nM was demonstrated to cause a dose-dependent increase in the initial rate of thrombin generation, and a shortening of the lag-phase of platelet activation in the presence of platelet counts down to at least 10,000 ␮l−1 (Kjalke et al., 2001). The addition of rFVIIa did not, however, normalize the total thrombin generation. It was postulated that the initial rate of thrombin may be of special importance due to its effect on ensuring a tight fibrin structure. It also was demonstrated in a flowchamber model using whole blood made thrombocytopenic (<6000 platelets/␮l), that the addition of rFVIIa increased the fibrin deposition on the thrombogenic surface used in the model (Gal´an et al., 2003). Fibrin added to platelet-rich plasma has, in fact, been demonstrated to increase the thrombin generation by binding thrombin, resulting in the activation of coagulation factors VIII, V, XI as well as platelets (Kumar et al., 1994). In this process also the vWillebrand factor may play an essential role (B´eguin and Kumar, 1997). The increased

fibrin deposition in the flow-chamber model initiated by rFVIIa, may well contribute to the hemostatic effect of rFVIIa observed in patients with thrombocytopenia as well as with various forms of thrombasthenia. Furthermore, it was later demonstrated in a similar model using reconstituted blood and collagen or fibrinogen covered surface, that the addition of rFVIIa increased platelet adhesion and aggregation as well as the exposure of procoagulant phospholipids on the platelets available (Lisman et al., 2003, 2005). The final step in hemostasis is the formation of a stable fibrin hemostatic plug and the addition of rFVIIa to plasma containing low platelet counts normalized the fibrin structure. A normalization of the fibrin structure following the addition of rFVIIa was also found to occur in plasma from a patient with Glanzmann’s thrombasthenia (He et al., 2005). Limited clinical experience of the successful use of rFVIIa in patients with thrombocytopenia has been published (Kristensen et al., 1996; Vidarsson and Onundarson, 2000; Gerotziafas et al., 2002), supported by a study in thrombocytopenic rabbits showing reduced bleeding after the administration of rFVIIa (Tranholm et al., 2003). This later study confirmed the preliminary results reported previously (Hedner et al., 1985). The dosing of rFVIIa in these patients have mostly been similar to the recommended dose in hemophilia, 70–120 ␮g/kg every 2 h in serious bleeds. Recently, a placebo-controlled study of rFVIIa in patients with bleeding complications following hematopoietic stem cell transplantation was published (Pihusch et al., 2005). The dosing of rFVIIa in this study was 40, 80 or 160 ␮g/kg given every 6 h for 36 h (total seven doses). No overall effect of rFVIIa treatment was observed, although a post hoc analysis showed an improvement in the control of bleeding for a dose of 80 ␮g/kg rFVIIa versus standard hemostatic treatment. It cannot be excluded that the long intervals between the doses especially initially may have prevented the formation of a firm, stable fibrin hemostatic plug strong enough to resist the heavy exposure of fibrinolytic activity especially in cases with hemorrhagic cystitis and profuse gastrointestinal bleedings. Successful use of rFVIIa in patients with Glanzmann’s thrombasthenia has been reported and gathered in a Registry (Poon et al., 2004). The dosing used was similar to hemophilia dosing and the conclusion was that most patients who got an adequate dosing showed effect of rFVIIa. Based on the data provided, rFVIIa

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was approved for use in patients with Glanzmann’s thrombasthenia in EU since spring 2005. As pointed out by Lisman et al. (2005), the potency of rFVIIa to enhance platelet adhesion and aggregation also in the presence of lowered number of platelets may be at least part of the therapeutic effect of rFVIIa, not only in hemophilia, but also in a wide scope of situations characterized by increased bleeding as previously described (Hedner, 2000). The enhancing effect of rFVIIa on the thrombin generation on the thrombin activated platelets, thus mediates the effect on platelet adhesion involving GPIb as well as on platelet aggregation. A feed-back effect leading to more platelet activation in terms of increased availability of the phospholipids of the platelet membrane, will further enhance the thrombin generation on the platelet surface. The enhanced local thrombin generation mediates the formation of a tightly knit fibrin clot structure, as well as activation of both FXIII and TAFI, all contributing to make the hemostatic fibrin plug more resistant against premature lysis. In summary, the enhancing effect of pharmacological doses of rFVIIa on the local thrombin generation on thrombin activated platelets resulting in the formation of a tight fibrin structured hemostatic plug as well as full activation of FXIII and TAFI, most likely explains the hemostatic effect of rFVIIa in situations characterized by an impaired thrombin generation. Such situations include congenital disorders such as hemophilia, platelet disorders as well as acquired disorders with lowered concentrations of coagulation proteins and therefore a compromised procoagulant potential. One such disorder would be patients with liver disease having an impaired synthesis of vitamin Kdependent coagulation proteins as well as a defective platelet function (Paramo and Rocha, 1993). Conflicting reports on a potential hemostatic effect of rFVIIa in patients with severe liver disease were published (Hendriks et al., 2001; Lodge et al., 2005; Planinsic et al., 2005). In a flow-chamber ex vivo model Tonda et al. (2003) demonstrated a defective adhesion of platelets from whole blood drawn from patients with liver cirrhosis. In the same study, they were able to show an increase of the fibrin deposition following the addition of rFVIIa to the cirrhotic blood. The potential hemostatic effect in patients with liver disease has been claimed to be partly dependent on a down-regulation of the fibrinolytic capacity due to an improved activation

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of TAFI as a result of the enhanced thrombin generation (Lisman et al., 2002a). Such an effect was not confirmed in a study using an in vitro diluted prothrombin time, although, an increased initial thrombin generation was observed in plasma from cirrhotic patients after addition of rFVIIa (Lisman et al., 2002b). The enhanced thrombin generation, thus, may enhance fibrin formation and generate a tight fibrin structure of the hemostatic plugs formed in the presence of rFVIIa. Another situation with a potential for impaired thrombin generation due to a complex hemostatic pattern would be patients subjected to multiple transfusions including plasma substitutes and other types of fluid. This would refer to patients having been exposed to major trauma or extensive surgery with vast tissue and cell damage. As a result of the primary cause of bleeding and the therapeutical measures, such patients most often end up having a dilution coagulopathy with lowered plasma levels of several coagulation proteins, as well as of normally functioning platelets. In addition, the vast cell disrupture results in release of a host of proteolytic enzymes degrading coagulation proteins. By being localized at the sites of tissue damage, an excessive binding of such enzymes, especially those involved with the fibrinolytic system, tPA, plasminogen and leucocyte enzymes, to any fibrin formed at the sites, will occur. As a result of the dilution coagulopathy thrombin generation may be impaired leading to the formation of loose, porous fibrin deposits easily dissolved by the bound fibrinolytic enzymes. The result may very well be a profuse, diffuse bleeding from extensive surfaces of damaged tissue. This process may still be mainly localized without signs of a generally increased fibrinolytic activity in the circulation. Low pH may aggravate the situation by slowing down the release of fibrinopeptide A (FPA) and thereby the formation of fibrin from fibrinogen adding to the generation of defective fibrin plugs. The formation of fibrin gels with large pores easily dissolved is also favoured by low pH (Okada and Blomb¨ack, 1983). Since 1999, a number of reports on the successful use of rFVIIa in patients with bleedings from extensive trauma (Kenet et al., 1999; Martinowitz et al., 2002; Martinowitz and Michaelson, 2005), as well as from an array of other bleeding situations such as postpartum hemorrhage, general surgery in adult and pediatric patients, cardiac surgery etc (Segal et al., 2003; Grounds, 2003; Eikelboom et al., 2003; Ahonen

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and Jokela, 2005). Recently, a randomized, placebocontrolled, double-blind clinical trial including 301 bleeding trauma patients was published. A significant reduction in RBC transfusion in severe blunt trauma in the group treated with rFVIIa in doses of 200 ␮g/kg initially, followed by 100 ␮g/kg, and another dose of 100 ␮g/kg 1 and 3 h later was found (Boffard et al., 2005). rFVIIa has also been used successfully in patients without a preformed coagulation disorder in order to prevent bleeding or to prevent/mitigate a further expansion of an already established hemorrhage. Thus, a double-blind placebo-controlled randomized trial including 36 patients undergoing retropubic prostatectomy was found to reduce the requirement of transfusion. None of the patients receiving one single dose of 40 ␮g/kg of rFVIIa just before the enucleation of the prostate gland was initiated required transfusion (Friederich et al., 2003). Prostatectomy is often associated with major blood loss being the result of high content of fibrinolytic activators being released from the prostate gland (Astrup, 1991). Furthermore, an extensive contact between the prostate cavity and the urine containing high concentrations of urokinase postoperatively enhance the fibrinolytic stress locally (Andersson, 1964, 1974). It may be speculated that the administration of rFVIIa to these patients just before the release of the fibrinolytic activators would help to generate extra thrombin, resulting in the formation of tight fibrin plugs in the numerous blood vessels in the prostatic bed opened up during this procedure, resistant against the fulminant fibrinolysis occurring locally. By doing so, the bleeding during the postoperative period may be prevented or mitigated. The administration of one single dose of rFVIIa was reported to limit the growth of an intracerebral hematoma, reduce mortality, and improve functional outcomes at 90 days in patients with intracerebral hemorrhage diagnosed by CT within 3 h after onset in a double-blind, placebo-controlled trial including 399 patients (Mayer et al., 2005). The volume of the hematoma is a critical determinant of mortality and functional outcome after intracerebral hemorrhage (Broderick et al., 1993). Early hematoma growth has been demonstrated in 38% of patients on repeated CT, such a hematoma growth occurs in the absence of coagulopathy and has been assigned to continued bleeding or rebleeding at multiple site within the first

few hours after onset (Mayer, 2003). Cerebral tissues have been shown to possess fibrinolytic activity, and in intracerebral bleeding, fibrinolytic activity was found in the cerebrospinal fluid (Takashima et al., 1969). The favourable effect of one dose of rFVIIa administered within 3 h after onset of the hemorrhage may contribute to the formation of stable fibrin plugs, that are resistant against the surrounding firbrinolytic activity, thereby preventing rebleeding or ongoing bleeding from multiple sites. In conclusion, the use of rFVIIa may be an effective method to induce hemostasis in patients with a defective thrombin generation resulting in profuse and severe bleeding following various stimuli. Pharmacological doses of rFVIIa enhances the thrombin generation on thrombin activated platelets. By doing so, it enhances platelet adhesion and aggregation as well as increases the availability of phospholipids. As a result of the enhanced thrombin generation a tight fibrin network plug is formed, which will be resistant against premature lysis. The enhanced thrombin generation also will ensure a full activation of TAFI as well as of FXIII. References Abshire, T., Kenet, G., 2004. Recombinant factor VIIa: review of efficacy, dosing regimens and safety in patients with congenital and acquired factor VIII or IX inhibitors. J. Thromb. Haemost. 2, 899–909. Ahonen, J., Jokela, R., 2005. Recombinant factor VIIa for lifethreatening post-partum haemorrhage. Br. J. Anaesth. 94, 592–595. Allen, G.A., Monroe III, D.M., Roberts, H.R., Hoffman, M., 2000. The effect of factor X level on thrombin generation and the procoagulant effect of activated factor VII in a cell-based model of coagulation. Blood Coagul. Fibrinolysis 11 (Suppl. 1), S3–S7. Allen, G.A., Wolberg, A.S., Oliver, J.A., Hoffman, M., Roberts, H.R., Monroe, D.M., 1999. Effect of varied procoagulant concentration on thrombin generation in a model system. Thromb. Haemost. 319 (Suppl.). Andersson, L., 1964. Antifibrinolytic treatment with epsilonaminocaproic acid in connection with prostatectomy. Acta Chir. Scand. 127, 552. Andersson, L., 1974. In: Nilsson, I.M. (Ed.), Haemorrhagic and Thrombotic Diseases. John Wiley & Sons, London, pp. 155– 182. Astrup, T., 1991. Fibrinolysis: past and present, a reflection of fifty years. Semin. Thromb. Hemost. 17, 161–174. Bajzar, L., Manuel, R., Nesheim, M.E., 1995. Purification and characterization of TAFI, a thrombin activable fibrinolysis inhibitor. J. Biol. Chem. 270, 14477–14484.

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