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Background

  1. Top of page
  2. Background
  3. Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)
  4. Identification of FVIIa as an attractive candidate for use as a haemostatic agent
  5. Purification of human FVIIa for use in patients (1980–1982)
  6. First patients treated with purified human plasma-derived FVIIa (1981–1982)
  7. Development of recombinant FVIIa (rFVIIa) (1985–2000)
  8. Mechanism of action of rFVIIa
  9. Disclosures
  10. References

My story starts in the early 1970s when I was appointed a resident in the Hemophilia Clinic at the Malmö University Hospital, University of Lund, in Sweden, which at that time was headed by Professor Inga Marie Nilsson. This Haemophilia Clinic was very special in combining the clinical investigation and care of haemophilia patients from all over Sweden together with a research programme at the forefront of haemostasis, covering both bleeding and thrombotic disorders. My own research was focused on fibrinolysis, and I presented my dissertation in early 1974. However, in parallel, I was, as the only physician along with Inga Marie in the clinic, deeply involved with the clinical care of haemophilic patients. This involved being on duty most of the time dealing with both inpatient and outpatient care.

In the early 1970s, the most serious problem was to treat haemophilia patients who had developed inhibitors against factor VIII (FVIII) or factor IX (FIX).

Various treatment modalities such as exchange transfusions combined with substitution therapy were tried [1–3]. In 1971, David Green described a combination of simultaneous administration of large amounts of FVIII/FIX and cyclophosphamide. This regimen was used to cover extensive dental surgery in two haemophilia B patients during 1971 [4]. The same treatment modality was successfully used in four haemophilia A patients during 1972 [5], and later in another five patients [6]. In those patients who had an inhibitor titre too high to be suppressed by the administration of large amounts of FVIII/FIX concentrates, the addition of an extracorporal adsorption of the inhibitory gamma globulin as described by Edson et al. [7] was considered.

However, in association with the very high doses of FIX-concentrate (PCC) required in some of the haemophilia B patients, to achieve a neutralization of the inhibitors as well as a haemostatic plasma level of FIX:C, there were signs of thrombin activation with a systemic activation of the coagulation system (high levels of fibrinogen degradation products, decrease of fibrinogen, decreased platelet counts, positive ethanol gelation test, decreased alfa-2-macroglobulin). The addition of antithrombin concentrate did not entirely neutralize these changes [8]. Increasing numbers of reports with similar findings following the use of various PCCs were being published [9,10]. As no pure FIX concentrates were commercially available at the time, this presented a problem for the treatment of haemophilia B patients, especially those with high titre inhibitors, who required large doses of the PCCs for haemostasis.

However, reports appeared in the early 1970s on the use of PCCs in the treatment of mild to moderate bleeding episodes in inhibitor patients. Thus, Fekete et al. [11] reported a haemostatic effect with an ‘activated prothrombin complex concentrate’. This concentrate was claimed to include certain amounts of activated FIX, FX, FVII as well as traces of thrombin. A high in vitro clotting activity was demonstrated, and this was called ‘auto-activated FIX’ [12].

In Malmö, we were not very satisfied with these concentrates when used in the treatment of haemophilia B patients, as we saw changes in the plasma coagulation pattern, indicating activation of systemic coagulation [8], which could cause thrombo-embolic side-effects including disseminated intravascular coagulation (DIC). Furthermore, we did not see any significant clinical effect in patients with inhibitors. At this time, it was suggested that the clot promoting effect in PCCs was caused by the presence of activated coagulation factors, especially FIXa and FXa [10,13,14].

Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)

  1. Top of page
  2. Background
  3. Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)
  4. Identification of FVIIa as an attractive candidate for use as a haemostatic agent
  5. Purification of human FVIIa for use in patients (1980–1982)
  6. First patients treated with purified human plasma-derived FVIIa (1981–1982)
  7. Development of recombinant FVIIa (rFVIIa) (1985–2000)
  8. Mechanism of action of rFVIIa
  9. Disclosures
  10. References

Following the discussions on the risk and benefit of these APCCs and the lack of any solid data regarding factor(s) which might be responsible for any side-effects and/or benefit, I thought it to be relevant to study various PCCs and their ability to induce a systemic activation of the coagulation process in a dog model available at the University Hospital of Malmö, Sweden. In this study, it was demonstrated that various PCCs initiated varying degrees of dose-dependent activation of the coagulation system, but all showed similar changes (decrease in platelet count, fibrinogen level, increase of FDP and signs of thrombin activity in terms of a positive ethanol gelation test) [15]. In a follow-up study, first presented at the International Committee on Thrombosis and Haemostasis before the Vth International Congress on Thrombosis and Haemostasis, June 26–July 2, 1977 in Philadelphia within the Task Force on ‘Factor IX Complex and Factor IXa’, it was shown that the changes in the coagulation pattern was mitigated by addition of a combination of heparin and antithrombin (AT) to the concentrate before infusion, supporting the assumption that the clot promoting activity included the presence of FIXa and FXa, both inactivated by AT and heparin ([16], submitted in 1978, before I joined Earl Davie′s laboratory in Seattle).

Identification of FVIIa as an attractive candidate for use as a haemostatic agent

  1. Top of page
  2. Background
  3. Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)
  4. Identification of FVIIa as an attractive candidate for use as a haemostatic agent
  5. Purification of human FVIIa for use in patients (1980–1982)
  6. First patients treated with purified human plasma-derived FVIIa (1981–1982)
  7. Development of recombinant FVIIa (rFVIIa) (1985–2000)
  8. Mechanism of action of rFVIIa
  9. Disclosures
  10. References

I held a deep frustration about the suboptimal treatment we offered to our haemophilia patients with inhibitors, in spite of the huge amount of contemporary biochemical knowledge. In the second round of the dog experiments, it was obvious to me that the effect leading to signs of a systemic activation of the coagulation system diminished after the addition of heparin and antithrombin to the concentrate administered to the dogs. As a result of these data, I started to look for any activated coagulation protein that was not promptly inhibited by heparin-antithrombin and ended up with FVIIa as a candidate [17]. Furthermore, it had been demonstrated that FVIIa lacked enzymatic activity, unless it was complexed with tissue factor. It had been previously published [18] that the presence of tissue factor highly enhanced the enzymatic activity of FVII/FVIIa in the coagulation system. Thus, it could be hypothesized that injected FVIIa, not active by itself, would be able to find its way to exposed tissue factor at the site of injury, form a complex and initiate local haemostasis.

At this time, the results from the first patients receiving the ‘auto-IX concentrate’ were published by Kuczinski & Penner in 1974 [12]. From Table 1, 2 and Fig. 1 in this publication, it appeared that the concentrate used was especially rich in FVII, and the plasma levels of FVII showed the most striking increases after infusion, which suggested to me that FVIIa might be an attractive candidate for further exploration.

Table 1.   Coagulation factors in activated prothrombin concentrate* [See Kurczynsky & Penner (1974)].
FactorAmount
U mL−1U per bottle
  1. *Average activity obtained from assay of four lots; ranges of values are indicated for thrombin, IXa & Xa.

  2. 1 U defined as the activity present in 1 ml of pooled normal human plasma.

II15.0450.0
V0.061.8
VII200.06,000.0
VIII1.1434.0
IX42.01260.0
X58.01740.0
XI12.012.0
Thrombin0.001–0.0030.03–0.09
IXa3.0–10.090.0–300.0
Xa3.0–8.090.0–240.0
Table 2.   Coagulation data [See Kurczynsky & Penner (1974)].
Case no.Dosage (mL) per 0.5 kgInhibitor level (U mL−1)Partial thromboplastin time (s)Maximum increase after infusion (%)
Before infusionAfter infusion*VIIIXX
  1. *All occurred immediately after infusion or 2 h later.

  2. Peak values (% of normal) reached for each factor level. The peak change occurred between 4 and 12 h after infusion.

  3. Factor IX levels are variable as some inactivation of FVIII occurs almost immediately after mixing with plasmas containing high levels of inhibitor.

10.75117962 10 
0.75115330 15 
23.00>10010656   
31.501051451200420130
0.75104024180160140
40.751012368750180370
1.15610374370120140
0.756926133060170
50.60200 421200(<1)400
1.001259486650(<1)170
1.001309575660(<1)260
61.00328350464140280
70.501009757410190190
0.601005742700180300
0.602196622100  
image

Figure 1.  Partial thromboplastin time and factor V, VII and X after a dose of activated prothrombin complex (case 7) [See Kurczynsky & Penner (1974)].

Download figure to PowerPoint

Although the importance of FVII in initiating haemostasis was stressed much earlier [19], the administration of exogeneous FVII was only considered of importance in patients with liver diseases (Editorial, Lancet II:855, 1975), whereas the presence of FIXa and FXa were thought to be more important for the haemostatic effect observed by APCCs [12,20]. Thus, in the middle of the 1970s, I needed to find out whether FVIIa alone (excluding all the other factors in the PCC/APCCs) would induce haemostasis in vivo.

In my early discussions with Harold Roberts (at the Vth ISTH Congress in Paris 1975; Dr Roberts was at the time Co-chairman of the Task Force on Clinical Use of Factor IX Concentrates meeting on July 20, 1975) and with Earl Davie, when I met him at the Lindeström-Lang Conference, August 25–29, 1975 in Denmark (at this conference a paper by Prydz & Bjørklid on ‘Structure and Function of Thromboplastin’ was presented in which it was stressed that ‘tissue thromboplastin triggered coagulation by forming a complex with factor VII…’) my thoughts of utilizing FVIIa in clinical treatment of haemophilia were met with obvious scepticism. One argument was that haemophilia patients have normal levels of FVII, why should extra FVIIa help them?

This was the situation when I came to Earl′s laboratory in August of 1978, where I came to share office with Walter Kisiel. As he points out in his historical sketch [21], he had been working on the purification of human FVII since 1976, and so, I started to discuss with him the possibilities of purifying FVII to test in animals and later in humans, but he stressed how difficult it was to purify FVII from human plasma. In the meantime, I worked with my project in Earl′s laboratory and followed the ongoing discussion on active components in the ‘auto-IX concentrates’. Much to my delight, I found thoughts expressed in the paper by Seligsohn et al. 1979 [22] reporting on his studies performed in the laboratory of Sam Rapaport, which I thought, supported my idea of the possibility of using FVIIa as a by-pass agent.

Purification of human FVIIa for use in patients (1980–1982)

  1. Top of page
  2. Background
  3. Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)
  4. Identification of FVIIa as an attractive candidate for use as a haemostatic agent
  5. Purification of human FVIIa for use in patients (1980–1982)
  6. First patients treated with purified human plasma-derived FVIIa (1981–1982)
  7. Development of recombinant FVIIa (rFVIIa) (1985–2000)
  8. Mechanism of action of rFVIIa
  9. Disclosures
  10. References

In our shared office in Seattle I discussed haemophilia treatment with Walt (Kisiel), and although I was not able to convince him of the feasibility of using FVIIa, he became interested enough to consider spending some time in the haemophilia clinic in Malmö. We wrote a project plan on the purification of FIX and looking into variants of the FIX molecule also the subject of previous research in Malmö (Wallmark and Hedner; poster at the ISTH in Stockholm 1983). These variants were characterized by different binding capacity to monoclonal antibodies against FIX [23]. I applied to the Swedish Medical Research Council for a fellowship for Walt (Kisiel) to spend 1 year in our clinic in Malmö primarily for these studies. The fellowship was approved, and Walt and his family arrived in Malmö in July of 1980.

An exciting period started with Walt working on the purification of FVII as well as of FIX, and he also learnt more about haemophilia and the daily sufferings of these patients, especially those with inhibitors. This helped him understand my obsession with the idea to find a treatment for these patients as effective as that given to patients without inhibitors. My vision already at this time (late 1970s) was to find treatment in a home treatment setting as well as effective in covering major surgery, contraindicated in inhibitor patients at the time. Thus, I set out to work on making an ex tempore formulation of purified, activated FVII (FVIIa) purified by Walt in our laboratory at the University Hospital of Malmö, according to guidelines and recommendations obtained by personal contact with the Health Authorities in Sweden (personal documents from 1980 to 1981). Furthermore, approval from the Ethical Committee of the University of Lund was obtained.

First patients treated with purified human plasma-derived FVIIa (1981–1982)

  1. Top of page
  2. Background
  3. Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)
  4. Identification of FVIIa as an attractive candidate for use as a haemostatic agent
  5. Purification of human FVIIa for use in patients (1980–1982)
  6. First patients treated with purified human plasma-derived FVIIa (1981–1982)
  7. Development of recombinant FVIIa (rFVIIa) (1985–2000)
  8. Mechanism of action of rFVIIa
  9. Disclosures
  10. References

In March 1981, we had tested our purified FVIIa in the same dog model that I used before to test the APCC, and found no signs of a systemic activation of the coagulation system. During the discussion at a meeting arranged by Immuno AG in Rome, March 31, 1981 [24], I presented our results mentioning that we intended to treat a haemophilia patient with inhibitors as soon as anyone presented with acute bleeding in our Clinic in Malmö. This treatment was performed on April 24, 1981. As mentioned by Walt (Kisiel) in his recollection article [21], the result was very encouraging.

Although the effect on a muscle bleed is difficult to evaluate, it was clear that the patient recovered more quickly this time than after any previous similar bleeding event. Patient number 2 was treated with plasma-derived purified FVIIa prepared in the same way in April 1982 in association with the loss of a primary molar tooth. This patient had previously been treated in-hospital with exchange plasma transfusion and huge amounts of FVIII. Following one dose of the plasma-derived human FVIIa, the patient formed a tight clot in his gum with immediate haemostasis. To me, this was a clear ‘proof of principle’ that the administration of exogenous purified FVIIa would be haemostatically active in severe haemophilia patients with inhibitors [25].

To follow-up on a potential development of FVIIa for use in haemophilia treatment, discussions between KabiVitrum, Stockholm, Sweden, Walter Kisiel and myself were initiated during late 1982. However, nothing materialized, and the project was shelved for some time.

Development of recombinant FVIIa (rFVIIa) (1985–2000)

  1. Top of page
  2. Background
  3. Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)
  4. Identification of FVIIa as an attractive candidate for use as a haemostatic agent
  5. Purification of human FVIIa for use in patients (1980–1982)
  6. First patients treated with purified human plasma-derived FVIIa (1981–1982)
  7. Development of recombinant FVIIa (rFVIIa) (1985–2000)
  8. Mechanism of action of rFVIIa
  9. Disclosures
  10. References

I was recruited by Novo Nordisk A/S, Denmark to establish a haemostasis research group to support the work on antithrombotic therapy in the autumn of 1983. The idea and potential use of FVIIa in the treatment of haemophilia patients with inhibitors was considered. Plasma-derived FVIIa was purified from Finnish plasma bought from the Finnish Red Cross, and tested in four haemophilia patients (three with severe haemophilia A and one with haemophilia B). The results in the patients tested after approval from Health Authorities and Ethical Committees in Denmark and in Sweden were considered encouraging [26]. It became clear that developing FVIIa for clinical use should be based on gene technology to enable large scale production and to avoid transfusion transmitted infection. At this time, the coagulation proteins were cloned in Earl Davie′s laboratory, Department of Biochemistry, University of Washington [27]. Thus, human FVII was expressed in a baby hamster cell-line (BHK) [28].

A project to develop recombinant human FVIIa (rFVIIa) for treatment of haemophilia patients with inhibitors was approved on June 30, 1985, with Novo Nordisk A/S, Copenhagen, Denmark. Our haemostasis research group was the core of this work together with the enzyme research team (responsible for the fermentation of the BHK cells), pharmacology, protein chemistry and many others. Walter Kisiel acted as a scientific consultant to the group. I eventually succeeded in creating a group including pharmaceutical, assay technique, immunology, protein chemistry and large scale production expertise. Although still very small, we were a highly dedicated group prepared to solve all kind of problems. The development of rFVIIa was actually the first time a protein requiring mammalian cells for post-translational modifications was produced in large scale [29].

The first haemophilia patient treated with rFVIIa was subjected to open surgical synovectomy in a knee joint at the Karolinska Hospital, Stockholm, Sweden, on March 9, 1988. He was treated after a patient specific approval had been obtained from the Swedish Health Authority of Sweden by the treating doctor at the Hospital. This approval was granted after a careful examination of the development documents provided by Novo Nordisk. The surgery was a success, and haemostasis was obtained using rFVIIa as the only haemostatic agent together with antifibrinolytic therapy (standard treatment in Sweden in haemophilia surgery) [30]. At this time, rFVIIa also was shown to induce haemostasis in haemophilia dogs at Chapel Hill [31]. Thus, ‘proof of concept’ regarding the potential of rFVIIa as a haemostatic agent had been demonstrated in a human and dogs.

Mechanism of action of rFVIIa

  1. Top of page
  2. Background
  3. Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)
  4. Identification of FVIIa as an attractive candidate for use as a haemostatic agent
  5. Purification of human FVIIa for use in patients (1980–1982)
  6. First patients treated with purified human plasma-derived FVIIa (1981–1982)
  7. Development of recombinant FVIIa (rFVIIa) (1985–2000)
  8. Mechanism of action of rFVIIa
  9. Disclosures
  10. References

It became obvious to us early during the development of rFVIIa that more research regarding the mechanism of action was required to explain, for example, the findings of a normalization of the APTT, after addition of rFVIIa in vitro presented the first time at the ISTH Congress in Brussels 1987 [32]. Based on these initial observations, it was suggested in 1990 that rFVIIa may not only bind to TF but also to phospholipids exposed on thrombin activated platelet surfaces [33]. As rFVIIa at this time was considered a development project, my research group at Novo Nordisk did not get research resources for further studies regarding the mechanism of action of rFVIIa. As a result, I had to establish collaborations with external research groups to be able to follow this up. As part of this strategy, a close collaboration between our haemostasis research group at Novo Nordisk in Copenhagen and the Haemostasis & Thrombosis Center at Chapel Hill, headed by Harold Roberts, was established in the very late 1980s and 1990s. From this collaboration, the cell-based model for studying haemostasis was established [34]. Using this model, it was demonstrated that rFVIIa binds to thrombin-activated platelets suggested previously in 1990 [33], provided pharmacological concentrations were used [35]. In fact, these observations lead to a revised model of haemostasis, stressing its localization to cell surfaces (TF bearing cells and thrombin-activated platelets) [36].

The further development of rFVIIa resulted in the approval of NovoSeven in Europe in 1996, in the US in 1999 and in Japan in 2000.

References

  1. Top of page
  2. Background
  3. Identifications of factors inducing thrombo-embolic side-effects in APCCs (1974–1977)
  4. Identification of FVIIa as an attractive candidate for use as a haemostatic agent
  5. Purification of human FVIIa for use in patients (1980–1982)
  6. First patients treated with purified human plasma-derived FVIIa (1981–1982)
  7. Development of recombinant FVIIa (rFVIIa) (1985–2000)
  8. Mechanism of action of rFVIIa
  9. Disclosures
  10. References
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