In this chapter, it is described how I learned to know hemophilia by working as a laboratory technician at the very small coagulation laboratory situated at the Malmö General Hospital during the summer after my first year at medical school. A lifelong interest was born, and from 1972 until 1983 I worked as a physician in the same place that had grown with time. I got involved with the clinical care of patients with hemophilia and started to study how to develop improved treatment, especially for those patients who had developed inhibitors against the coagulation factor they were missing. Based on different experiences, I draw the conclusion that activated coagulation factor VII might be an attractive candidate as a new hemostatic agent that should be independent on both factors VIII and IX and thus be beneficial in hemophilia patients with inhibitors.

The further development of rFVIIa during the 2000s showed no safety problems by using a dose of 270 µg/kg rFVIIa administered as a bolus instead of the 90 µg/kg in three doses with a 2- to 3-hour interval previously recommended. Based on the clinical experiences late in the 1990s, it was obvious that some patients required higher doses to stop an upcoming bleeding with one single injection. Furthermore, an increased clearance rate of rFVIIa was found in children. Thus, the higher dose was recommended in children and in patients with heavy bleedings. Furthermore, the large distribution volume of rFVIIa observed already early in the 1990s was followed-up by studies of the extravascular distribution of rFVIIa in mice. Intravenously injected rFVIIa disappeared quickly from the blood and could be traced to various tissues, in some up to 7 days after injection. This would indicate a mechanism for a long-term hemostatic effect in stopping microhemorrhages in the microvasculature. A trial with daily doses of rFVIIa also showed a decrease of joint bleeds in a group of heavily bleeding hemophilia patients with inhibitors.

This chapter describes the launch of rFVIIa after approval in EU in 1996, first in France. The interest from the clinicians was high and the sales of rFVIIa increased by 40% per year in the first years. After approval in the United States in 1999, it increased by 63%. Soon after the approval, rFVIIa was successfully used in patients with Glanzmann's thrombasthenia and was registered for this indication in 2004 in EU. Furthermore, a hemostatic effect in patients with liver disease was seen, but the indication was never pursued. It was, however, used in heavily bleeding patients with a basically normal hemostatic mechanism and later in heavily traumatized patients. One placebo-controlled, randomized, double-blind study showed no side effects and indicated a lower transfusion requirement in the rFVIIa-treated group. A larger study was not pursued. A number of case stories of survival in heavily traumatized patients (terrorist attacks) were published. Finally, rFVIIa (20 µg/kg) was shown to decrease the expansion of intracerebral bleedings without any increase in thromboembolic side effects. In patients receiving a dose of 80 µg/kg, an increased number of arterial thromboembolic side effects (9% vs 4% in the placebo group) were observed.

This chapter describes how I ended up with Novo Nordisk in 1983, when I was recruited to establish a research laboratory in the area of hemostasis. A project to develop a modified heparin, low molecular weight heparin (LMWH), was already started and the further development became my first project at Novo Nordisk. The LMWH of Novo Nordisk (Tinzaparin and later Logiparin) was prepared by enzymatic degradation of heparin using the enzyme heparinase. We demonstrated that Tinzaparin as the first LMWH was effective in the treatment of existing blood clots. In addition, lower cancer mortality was seen in the patients who had received Tinzaparin. Despite the good results, Tinzaparin was sold off to Leo Pharmaceuticals. Furthermore, the confirmatory small study of plasma-derived purified FVIIa and the use in a few patients with decreased number of platelets are described. The encouraging results are presented.

In this chapter a boy considered to be resistant to rFVIIa treatment became a high responder when the rFVIIa dose was increased. A small pharmacokinetic study, initiated in the Coagulation Clinic of Malmö in early 1996, including children younger than 15 years demonstrated a clearance rate for rFVIIa up to three times as rapid as what had been found in adults. At this time, it was clear that the recommended dose was suboptimal in some patients. The boy described in this chapter had the highest clearance rate in our study. Thus, the dose necessary for an adequate response in an acute bleeding episode seemed to be dependent on the patient’s clearance rate of rFVIIa. By using this higher dose rFVIIa, an orthopedic traction of his knee joint contractions was successfully performed. Furthermore, administration of a daily dose of rFVIIa, a prolonged intensive physiotherapy following the traction was successful. Thus rFVIIa prevented any bleeding episodes, indicating that rFVIIa might also prevent bleedings in general.

Activated coagulation proteins in the circulation have always been associated with fear of a systemic activation of the coagulation system (disseminated intravascular coagulation, DIC). There were no signs of a systemic activation of the coagulation system by rFVIIa in animal experiments including rabbits preinjected with endotoxin. Also there were no signs of DIC observed in patients treated with rFVIIa except for one patient subjected to extensive surgery to remove a large hip abscess. The trigger seemed to be a release of proteolytic enzymes from the damaged tissue rather than rFVIIa. Safety follow-ups have shown low incidence of thrombotic events (less than 1%). In the follow-up of 2008 it was 3.75 per 100,000 infusions. The localization of rFVIIa to the site of injury through its binding to tissue factor and preactivated platelets is suggested to be the reason for the low thromboembolic incidence. rFVIIa makes major orthopedic surgery in hemophilia patients with inhibitors possible. An economic evaluation from 2008 draws the conclusion that major knee surgery with rFVIIa in such patients might be cost saving in the long term. The importance of comparing total cost of treatment and not only the cost per vial of drug used is emphasized.

This chapter describes how rFVIIa was accepted as an "orphan drug" according to the Orphan Drug Act (Public Law 97.414) in January 1988. Furthermore, the long drawn out and demanding procedure on the patent dispute with Baxter Travenol who had filed a patent on a Therapeutic method for treating blood clotting defects with factor VIIa, on June 25, 1981, is described. It seemed unbelievable to us that Baxter would be able to get a patent on something I had presented on a meeting in March the same year at an international meeting in Rome. However, despite numerous submissions to both the American and European patent authorities, Novo Nordisk finally lost in the European Patents Court. They did appeal the outcome, but finally at the end of the 1990s, it was withdrawn after an agreement eventually was reached between Novo Nordisk and Baxter in which Novo Nordisk was to pay a certain license fee to Baxter.

This chapter describes the prompt use of rFVIIa following the first successful use in a hemophilia A patient in 1988. To make rFVIIa available to inhibitor patients suffering from life-threatening bleedings, a "Compassionate Use" study was started in early 1989. In 1993, 112 patients had been treated within this study including patients with congenital hemophilia and inhibitors, some with acquired hemophilia, one with factor XI deficiency and one with FVII deficiency. Following the approval of an "Investigation of a New Drug, IND," January 1989, a study of effect and safety of rFVIIa in patients with or without ongoing bleeding was initiated. Following the merger between Novo and Nordisk Gentofte, an extensive reorganization was undertaken leading to a dramatic decrease of research support for rFVIIa. To compensate a collaboration with the research group at Chapel Hill was initiated and resulted in a changed concept of the hemostatic mechanism. The development during the early 1990s was complicated due to the disruption of functioning teams and disagreements between different parts of the organization. Finally the rFVIIa file was submitted in 1995 in the United States and rFVIIa approved in 1999. In Europe it was approved in 1996 and in Japan in 2000.

The development of techniques to transfer capacity to produce coagulation proteins into mammalian cells started with a hybrid genome construction (one part of the FVII gene and one part of the FIX gene). This hybrid genome was introduced in a baby hamster kidney (BHK) cell. The complete DNA sequence of human FVII was constructed at Novo, and the final BHK cell-line containing this genome produced a normally functioning FVII molecule. In June 1985 a project to develop rFVIIa for the treatment of hemophilia patients with inhibitors was approved at Novo in Copenhagen. A working group, including people from different functional areas, was formed, and within 3 years enough rFVIIa to treat the first patient was produced. The FVII molecule was spontaneously activated into FVIIa during the purification procedure, and the final product consisted solely of pure rFVIIa. The rFVIIa was hemostatically active in hemophilia dogs, and the first patient successfully underwent orthopedic surgery in March 1988.

In this chapter the mechanism of action of FVIIa in hemostasis is presented and discussed. The role of FVII in the clotting process had not been seriously considered previously. The availability of rFVIIa helped this process in providing the molecule for further research. In the 1980s several research groups had suggested that FVII did not only depend on TF for its activity. The establishment of a cell-based model was crucial to approach this issue. With the help of the cell-based model worked out by the Chapel Hill group, a low-affinity binding site for FVII on the preactivated platelets was identified. Pharmacologically doses of rFVIIa did enhance the thrombin generation on these platelets thereby at least partially compensating for the lack of FVIII/FIX mimicking hemophilia. Similar doses were hemostatically effective in hemophilia patients supporting the importance of rFVIIa binding to preactivated platelet. Thus, hemostasis was localized to cell surfaces, initially the TF-expressing cells and then the thrombin-activated platelet surface. Other views of the potential mechanism of action are mentioned and discussed. The problems in finding the correct dosing of rFVIIa are dealt with based on experiments in hemophilia dogs as well as in hemophilia patient studies.

Hemophilia, the classical bleeding disease, was first described in 1803, but was known much earlier. The characteristics are spontaneous bleedings, especially joint bleeds. Hemophilia is a congenital, inherited disease. The most well-known family carrying the hemophilia gene is the family of the British Queen Victoria. The last tsarevich of Russia, Alexis, had hemophilia. This chapter describes the family stories of Queen Victoria and that of her granddaughter, Alexandra, Empress of Russia, typical for the disease before any effective treatment to stop the repeated bleedings was available. The development of such treatment started in the 1940s. The first concentrate for use in hemophilia A was developed in Sweden in the 1950s. The availability of this concentrate made surgery in hemophilia patients possible. Thus, orthopedic correction surgery in hemophilia patients with severe joint defects is described. Also, prophylactic treatment of hemophilia to decrease the number of joint bleeds, thereby minimizing the development of joint damages is described.

In this chapter three versions of the development of rFVIIa are presented. The first one is written by Novo Nordisk in the early 2000s, The NovoSeven® Story. This story gives a chronological overview of the project's history from 1983 when it was decided by Novo Nordisk to expand in the area of hemostasis. The technology is described, and a short review of the clinical development and finalizing with the license application is presented. The second version is an examination paper in information science using the translation model by Bruno Latour. It is based on interviews of a number of Novo Nordisk employees involved in the rFVIIa project from early 1980s until licensing in the 1990s. This gives a special view on the development of the project. Finally, my own description is given. This is based on various sources such as documents, memos, publications, and my own recollections partly written down, which gives it a special view from the inside of the daily project work.

Recombinant activated factor VII (rFVIIa) was initially developed to treat bleeding episodes in patients with congenital haemophilia and inhibitors. The story of its development began in the 1970s, when FVIIa was identified as one of the activated coagulation factors that has minimal potential for inducing thromboembolic side-effects. Extensive research over the last 30 years has greatly increased our knowledge of the characteristics of FVII, its activation, and the mechanisms by which rFVIIa restores haemostasis. In haemophilia, the haemostatic effect of rFVIIa is mediated via binding to thrombin-activated platelets at the site of injury, thereby enhancing thrombin generation also in the absence of factor (F) VIII or FIX. The mechanism of action of rFVIIa has also allowed its successful use in other clinical scenarios characterised by impaired thrombin generation, and its licensed uses have now been extended to acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia.

Although effective therapies for haemophilia have been available for decades, the prevention and treatment of joint disease remain major clinical concerns for all haemophilia patients. Early identification of joint disease is vital to initiate or modify treatment, and prevent arthropathy. However, there remains a need for more sensitive and accurate methods, which may also detect improvement in patient outcome with new therapies or different prophylaxis regimens. These topics were explored at the Ninth Zürich Haemophilia Forum. A summary of our shared views on the limitations of current assessment methods, and the potential advantages of more recently developed tools, is provided. Ultrasonography enables more frequent routine monitoring and the early detection of joint disease. In addition, serological markers may provide suitable biomarkers of early arthropathy. To prevent arthropathy, in our opinion, prophylaxis is key to prevent joint bleeds and subsequent initiation of the ’vicious circle of joint disease’. However, issues remain, including when prophylaxis should be started, stopped, and if it is efficacious for inhibitor patients. Once joint bleeding has occurred, enhanced on-demand treatment should be considered. For more advanced stages of joint disease, the issues regarding the treatment options available are explored. Radiosynovectomy should be performed to treat chronic synovitis, and may prevent the need for elective orthopaedic surgery (EOS). Ultimately, however, EOS can be considered once all other treatment options have been explored. While, bypassing agents have facilitated the use of EOS in inhibitor patients, a multidisciplinary approach and careful surveillance is required for good patient outcome.

Previous work has shown that normalized haemostasis only at the time of an injury is not sufficient to promote optimal wound healing in haemophilia B (HB) mice. However, the duration of treatment required for optimal healing has not been established. The goal of these studies was to determine the effect of different durations of replacement or bypassing therapy [factor IX(FIX) or factor VIIa (FVIIa)] on wound healing parameters in a mouse model of HB. A dermal wound was placed on the back of HB mice. Animals were either untreated or pretreated and then subsequently treated for 3 days, 5 days, or 7 days with FIX or FVIIa. Wound area, time to wound healing, haematoma formation and iron deposition were measured. All treated animals showed shortened time to healing relative to untreated animals. Haematoma formation was prevented by treatment and bleeding into the wounds, measured by iron scores, was reduced by treatment. In addition, there was a progressive improvement in healing with 7 days of treatment more effective than 5 days which was more effective than 3 days. Replacement therapy with FIX had slightly shorter healing times than bypassing therapy with FVIIa. HB mice treated with FIX had slightly smaller wound area than untreated animals; by contrast, FVIIa-treated animals had much smaller wound areas that were close to the wound areas seen in wild-type animals. The data suggest that sustained therapy is required for normal wound healing.

SummaryRecent in vitro studies have shown that the zymogen and activated form of factor (F)VII bind to endothelial cell protein C receptor (EPCR). At present, there is no evidence that FVIIa binds to EPCR on vascular endothelium in vivo in the presence of circulating protein C, a primary ligand for EPCR. The present study was carried out to investigate the interaction of murine and human ligands with murine EPCR both in vivo and in vitro. Measurement of endogenous plasma levels of FVII in wild-type, EPCR-deficient and EPCR-over expressing mice showed slightly lower levels of FVII in EPCR-over expressing mice. However, infusion of high concentrations of competing ligands, either human APCi or FVIIai, to EPCR-over expressing mice failed to increase plasma levels of mouse FVII whereas they increased the plasma levels of protein C by two- to three-fold. Examining the association of exogenously administered mouse FVIIa or human FVIIa by immunohistochemistry revealed that human, but not murine FVIIa, binds to the murine endothelium in an EPCR-dependent manner. In vitro binding studies performed using surface plasmon resonance and endothelial cells revealed that murine FVIIa binds murine EPCR negligibly. Human FVIIa binding to EPCR, particularly to mouse EPCR, is markedly enhanced by availability of Mg2+ ions. In summary, our data show that murine FVIIa binds poorly to murine EPCR, whereas human FVIIa binds efficiently to both murine and human EPCR. Our data suggest that one should consider the use of human FVIIa in mouse models to investigate the significance of FVIIa and EPCR interaction.

BACKGROUND:Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown. OBJECTIVE:The aim of the present study was to investigate the role of the FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues. METHODS:Wild-type, EPCR-deficient or ECPR-over-expressing mice were injected with human recombinant (r)FVIIa (120 μg kg(-1) body weight) via the tail vein. At varying time intervals after rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR. RESULTS:The data reveal that, after intravenous (i.v.) injection, rFVIIa rapidly disappears from the blood and associates with the endothelium in an EPCR-dependent manner. Immunohistochemical analyses revealed that the association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. The FVIIa association with the endothelium was undetectable at time points exceeding 24 h post-FVIIa administration. The levels of rFVIIa accumulated in tissue correlate with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days. CONCLUSIONS:The observation that an EPCR-dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides.

Both plasma factor (F)VII and FVIIa are cleared relatively fast from circulation with a half‐life of 3–6 h in humans [1Loeliger E.A. van der Esch B. Ter Haar Romney‐Wachter C.C. Booij H.L. Factor VII its turnover rate and its possible role in thrombogenesis.Thromb Diath Haemorrh. 1960; 4: 196-200PubMed Google Scholar, 2Erhardtsen E. Pharmacokinetics of recombinant activated factor VII (rFVIIa).Semin Thromb Hemost. 2000; 26: 385-91Crossref PubMed Google Scholar]. The mechanism(s) responsible for the clearance of FVII/FVIIa from circulation are unknown. Pharmacokinetic studies in mice have shown that FVII, FVIIa, active‐site blocked FVIIa and pre‐formed FVIIa–antithrombin complexes are cleared with similar rates, indicating that plasma elimination kinetics for FVII were independent of its activation and subsequent inactivation by plasma inhibitors [3Petersen L.C. Elm T. Ezban M. Krogh T.N. Karpf D.M. Steino A. Olsen E.H. Sorensen B.B. Plasma elimination kinetics for factor VII are independent of its activation to factor VIIa and complex formation with plasma inhibitors.Thromb Haemost. 2009; 101: 818-26Crossref PubMed Scopus (27) Google Scholar]. Nonetheless, recent studies have implicated that a substantial fraction of pharmacologically administered FVIIa activity is inactivated by antithrombin (AT) in humans and dogs, which could explain differences observed in FVIIa activity and antigen clearance curves in these previous studies [4Agerso H. Brophy D.F. Pelzer H. Martin E.J. Carr M. Hedner U. Ezban M. Recombinant human factor VIIa (rFVIIa) cleared principally by antithrombin following intravenous administration in hemophilia patients.J Thromb Haemost. 2011; 9: 330-8Abstract Full Text Full Text PDF Scopus (38) Google Scholar, 5Agerso H. Kristensen N.R. Ostergaard H. Karpf D.M. Hermit M.B. Pelzer H. Petersen L.C. Ezban M. Clearance of rFVIIa and NN1731 after intravenous administration of Beagle dogs.Eur J Pharm Sci. 2011; 42: 578-83Crossref PubMed Scopus (0) Google Scholar]. Recent studies from our laboratory and others showed that endothelial cell protein C receptor (EPCR) acts as a true cellular receptor for FVII or FVIIa [6Preston R.J. Ajzner E. Razzari C. Karageorgi S. Dua S. Dahlback B. Lane D.A. Multifunctional specificity of the protein C/activated protein C Gla domain.J Biol Chem. 2006; 281: 28850-7Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 7Ghosh S. Pendurthi U.R. Steinoe A. Esmon C.T. Rao L.V. Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium.J Biol Chem. 2007; 282: 11849-57Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8Lopez‐Sagaseta J. Montes R. Puy C. Diez N. Fukudome K. Hermida J. Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity.J Thromb Haemost. 2007; 5: 1817-24Crossref PubMed Scopus (0) Google Scholar] and promotes FVIIa endocytosis in cell model systems [7Ghosh S. Pendurthi U.R. Steinoe A. Esmon C.T. Rao L.V. Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium.J Biol Chem. 2007; 282: 11849-57Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 9Nayak R.C. Sen P. Ghosh S. Gopalakrishnan R. Esmon C.T. Pendurthi U.R. Rao L.V.M. Endothelial cell protein C receptor cellular localization and trafficking.Blood. 2009; 114: 1974-86Crossref PubMed Scopus (0) Google Scholar]. Administration of murine EPCR blocking antibodies was shown to reduce FVIIa clearance from circulation, particularly in the initial phase (α‐phase) of clearance, indicating that EPCR may play a role in FVII clearance in vivo [9Nayak R.C. Sen P. Ghosh S. Gopalakrishnan R. Esmon C.T. Pendurthi U.R. Rao L.V.M. Endothelial cell protein C receptor cellular localization and trafficking.Blood. 2009; 114: 1974-86Crossref PubMed Scopus (0) Google Scholar]. However, EPCR blocking antibodies that could fully block FVIIa binding to murine EPCR prolonged the circulatory half‐time of FVIIa only modestly and did not block the clearance of FVIIa from circulation [9Nayak R.C. Sen P. Ghosh S. Gopalakrishnan R. Esmon C.T. Pendurthi U.R. Rao L.V.M. Endothelial cell protein C receptor cellular localization and trafficking.Blood. 2009; 114: 1974-86Crossref PubMed Scopus (0) Google Scholar]. Together these data suggest EPCR may play a role in the initial, rapid phase of FVIIa clearance but other mechanism(s) may be responsible for its clearance in the terminal phase. To further investigate the potential role of EPCR in FVIIa clearance in a more stringent model system, in the present study we evaluated plasma elimination kinetics of FVIIa in wild‐type, EPCR‐deficient and EPCR‐over expressing mice. Given that human FVIIa interacts well with murine EPCR whereas murine FVIIa binds only negligibly [10Puy C. Hermida J. Montes R. Factor X and factor VII binding to endothelial protein C receptor differs between species.J Thromb Haemost. 2011; 9: 1255-7Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 11Sen P. Clark C.A. Gopalakrishnan R. Hedner U. Esmon C.T. Pendurthi U.R. Rao L.V.M. Factor VIIa binding to endothelial cell protien C receptor: differences between mouse and human systems.Thromb Haemost. 2012; Crossref PubMed Scopus (23) Google Scholar], we have used human FVIIa in the present study. Wild‐type littermate controls, EPCR‐deficient mice [12Li W. Zheng X. Gu J.M. Ferrell G.L. Brady M. Esmon N.L. Esmon C.T. Extraembryonic expression of EPCR is essential for embryonic viability.Blood. 2005; 106: 2716-22Crossref PubMed Scopus (83) Google Scholar] or EPCR‐over expressing mice [13Li W. Zheng X. Gu J. Hunter J. Ferrell G.L. Lupu F. Esmon N.L. Esmon C.T. Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin.J Thromb Haemost. 2005; 3: 1351-9Crossref PubMed Scopus (113) Google Scholar] were injected with 125I‐labeled human FVIIa (5 μg kg−1) as a single intravenous bolus via the tail vein. The low concentration of FVIIa dosing was chosen to reflect elimination kinetics of FVII at its plasma concentration. All mice were bled retro‐orbitally at 3 min after FVIIa administration and thereafter at one or two pre‐set time points. Mice were anesthetized by isoflurane gas for tail vein injection and blood sampling, and experiments were conducted in accordance with the animal welfare guidelines set forth in the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use and Care Committee. Except in a rare instance, three or more mice were used per time point. Blood (45 μL) was collected into citrate anticoagulant (5 μL of 0.13 m tri‐sodium citrate) and plasma was obtained by centrifugation at 4000 × g for 5 min using a table‐top Eppendorf centrifuge. FVIIa concentration in plasma was quantified by measuring radioactivity. Pharmacokinetics were evaluated by a standard non‐compartmental method or fitting the data to a two‐compartmental model using the NONMEM modeling program (GloboMax/ICON, Ellicott City, MD, USA). As shown in Fig. 1A, relatively large differences were observed among the genotypes at the first sampling time point (3 min). At this point, approximately 20% more FVIIa was recovered in the circulation of EPCR‐deficient mice compared with the wild‐type littermates. In contrast, FVIIa recovery in plasma of EPCR‐over expressing mice was reduced by 30% compared with wild‐type mice. These data indicate that a fraction of FVIIa administered to wild‐type and more so to EPCR‐over expressing mice was removed from circulation almost instantaneously after dosing. This indicates that FVIIa administered to mice readily associates with EPCR on the vascular endothelium. These data also suggest that a substantial fraction of EPCR on the vascular endothelium is left unoccupied by protein C/activated protein C (APC) at their plasma concentrations and exogenously administered FVIIa is capable of binding to unoccupied EPCR in vivo. Interestingly, when FVIIa elimination kinetics in plasma were normalized to the mean maximum concentration of FVIIa in plasma measured at 3 min, the pharmacokinetic curves were almost identical among all three genotypes (Fig. 1B). Analysis of the data using the standard non‐compartmental method showed a very similar half‐life of FVIIa, between 2.2 and 2.4 h, in all three genotypes but relatively large differences in the Cmax (wild type, 60 ng mL−1; EPCR deficient, 78 ng mL−1; EPCR overexpressors, 45 ng mL−1). When the data were fitted to a two‐compartmental model using Nonmem modeling, it confirmed the variation in bioavailability of FVIIa among the wild‐type, EPCR‐deficient and EPCR‐over expressing mice. The bioavailability of FVIIa in plasma was increased by about 30% in EPCR‐deficient mice, compared with wild‐type mice, suggesting that less FVIIa was sequestered in these mice. In EPCR‐over expressing mice, the bioavailability of FVIIa in plasma was decreased by 30%, indicating that a larger fraction of FVIIa was bound to EPCR immediately after its administration. Only minor differences were found in the CL (clearance) values among the wild‐type (0.018 mL min−1), EPCR‐deficient (0.017 mL min−1) and EPCR‐over expressing mice (0.014 mL min−1), indicating that FVIIa is cleared in a similar profile in these mice. After the initial disparate sequestration trends in EPCR transgenic mice, FVIIa was eliminated from circulation thereafter in all three genotypes essentially with a similar half‐life, t1/2α, 0.12 h and t1/2ß, 2.2–2.8 h. Other pharmacokinetics values, such as Q, V1 and V2, were identical among the three genotypes. Taken as a whole these data indicate that EPCR may play a role in modulation of FVII(a) levels in the circulation by sequestering it on the vascular endothelium but it is unlikely to influence the rate of FVII(a) clearance. Overall, FVIIa pharmacokinetics observed in the present study were similar to that reported earlier [3Petersen L.C. Elm T. Ezban M. Krogh T.N. Karpf D.M. Steino A. Olsen E.H. Sorensen B.B. Plasma elimination kinetics for factor VII are independent of its activation to factor VIIa and complex formation with plasma inhibitors.Thromb Haemost. 2009; 101: 818-26Crossref PubMed Scopus (27) Google Scholar]. Any minor variation in CL, V1 and V2 values reported in the present study and the previous study could reflect variation in the methods for measuring FVIIa concentration in plasma (radioactivity vs. clotting activity) and a difference in the dosage levels (5 vs. 10 mg kg−1). In an earlier study, we found that blockade of EPCR with EPCR‐blocking antibody prolonged the t1/2α of FVIIa clearance from 19 to 31 min [9Nayak R.C. Sen P. Ghosh S. Gopalakrishnan R. Esmon C.T. Pendurthi U.R. Rao L.V.M. Endothelial cell protein C receptor cellular localization and trafficking.Blood. 2009; 114: 1974-86Crossref PubMed Scopus (0) Google Scholar]. These data were interpreted as EPCR serving a role in FVIIa clearance. However, based on the present study that showed no significant differences in FVIIa clearance rates among wild‐type, EPCR‐deficient and EPCR‐over expressing mice, it is unlikely that EPCR‐mediated FVIIa internalization plays a significant role in FVII(a) clearance in vivo, at least at concentrations close to the endogenous plasma concentration of FVII. In our earlier study [9Nayak R.C. Sen P. Ghosh S. Gopalakrishnan R. Esmon C.T. Pendurthi U.R. Rao L.V.M. Endothelial cell protein C receptor cellular localization and trafficking.Blood. 2009; 114: 1974-86Crossref PubMed Scopus (0) Google Scholar], the reversible nature of antibody binding to the receptor which could allow the exchange between antibody and ligand binding to EPCR coupled with potential differences in the bioavailability of FVIIa in control and EPCR blocking antibody‐treated mice, might have given the impression that blockade of EPCR prolonged the initial phase of FVIIa clearance modestly but statistically significantly. Although the present data suggest that EPCR does not appear to play a significant role in the rate of FVIIa clearance from plasma, we can not completely rule out the possibility of EPCR influencing FVII clearance in humans as human EPCR may behave differently than murine EPCR. Irrespective of its role (or lack there of) in FVII clearance from plasma, EPCR still could play an important role in the continual, prolonged transport of a small but physiologically meaningful amount of FVIIa from circulation into extravascular compartments. Studies investigating this possibility are in progress within the authors’ laboratories. This work is partly supported by a grant from Novo Nordisk. One of the authors (H. Agersø) is an employee of Novo Nordisk, Denmark. U. Hedner is a consultant to Novo Nordisk A/S, Zurich, Switzerland. This work has been partly supported by a grant from Novo Nordisk and NHLBI grants (HL 58869 and 107483).