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Semin Thromb Hemost 2010; 36(5): 471-476
DOI: 10.1055/s-0030-1255440
PREFACE

© Thieme Medical Publishers

Recombinants in Thrombosis and Hemostasis: From Basic Research to Clinical Therapy

Giuseppe Lippi1 , Emmanuel J. Favaloro2
  • 1U.O. Diagnostica Ematochimica, Dipartimento di Patologia e Medicina di Laboratorio, Azienda Ospedaliero-Universitaria di Parma, Parma, Italy
  • 2Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Westmead, Australia
Further Information

Publication History

Publication Date:
14 July 2010 (online)

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Welcome to another issue of Seminars in Thrombosis and Hemostasis, this one devoted to the development and use of recombinant proteins in thrombosis and hemostasis.

Genetic recombination is a process by which molecules of nucleic acid (usually DNA but can also be RNA) are separated and then joined to different nucleic acids. Recombination can occur between similar (i.e., homologous recombination) or dissimilar (nonhomologous recombination) molecules of DNA (or RNA). Although this phenomenon occurs naturally, artificial (deliberate) recombination of different strands of DNA, even from different organisms, can culminate in the creation of “recombinant DNA” and is defined by the term genetic engineering.

The use of recombinant proteins has increased greatly in recent years, as has the number of techniques and products used for their amplification and purification. The development and application of recombinant proteins is advantageous in biomedical research, in that it permits the study of specific genes or altered structures and their biological effects, as well as developing new proteins of biological interest (i.e., protein engineering). The more that it is known about the biochemical features of a protein, the more easily it can be produced, isolated, and purified.

A variety of host systems can be used for recombinant technology, including phage, bacteria, yeast, plants, filamentous fungi, insect or mammalian cells grown in culture, as well as various transgenic animals. The final choice of which is utilized depends on specific aspects that characterize the product, including specific requirements and applications, desired degree of purity, biological integrity and functionality, posttranslational modifications, and potential toxicity. The selection of the vector family is also critical, mostly governed by the host and influenced by the specific requirements of the application as well as the behavior of the target protein.

What has driven the wide diffusion of recombinant proteins in science is that many of them, now currently used for medical treatment or research, can be usually purified only at very low concentrations from other living sources (i.e., insufficient for need by several orders of magnitude). Recombinant DNA technology therefore permits production of much larger quantity of proteins with potential for good consistency both in function as well as supply, and therefore enables a greater degree of safety and predictability in the final products. In the main, this can be attributed to their production and purification from cultured cells rather than from a human or animal source.

The number of recombinant products being used for research, clinical and even inappropriate applications (e.g., doping) grows incessantly, as does the availability of recombinant proteins in the field of hemostasis and thrombosis. More than 25 recombinant proteins have already been licensed for clinical use in this field, and these products have been largely shown to be safe and effective in the treatment of a variety of disorders. Expectedly, recombinant technology is now also used for producing proteins that target the entire hemostatic system (coagulation, anticoagulation, fibrinolysis) and can be used for research, diagnostics, and therapy. It is not a surprise, therefore, that the global market of recombinant coagulation factors (i.e., factor VIIa, VIII, and IX) posted strong 2006 sales of more than US $4.6 billion, and is likely to soon exceed that of human plasma-derived products.

The achievement of a stable hemostatic response is paramount to all surgical procedures. Although intraoperative hemostasis is traditionally achieved through suture ligation or electrocautery, certain cases are nonamenable, however, especially when there is diffuse raw surface bleeding, so that alternative methods have to be considered, including the use of topical thrombin. Bowman et al[1] therefore introduce the concepts related to use of recombinants in the field of thrombosis and hemostasis in the first chapter of this issue by highlighting that topical thrombin preparations have been used for controlling surgical bleeding for decades, and that a recombinant form (rhThrombin) was finally approved by the Food and Drug Administration (FDA) in 2008. The efficacy of this recombinant product has already been demonstrated in randomized controlled trials, whereby effective hemostasis could be achieved within 10 minutes of administration for mild to moderate diffuse raw surface bleeding, especially in vascular, hepatic, and spinal procedures. Bowman et al also emphasize that although rhThrombin appears to be as effective as bovine thrombin, it carries a significantly lower risk of immunogenicity, as well as a lowered theoretical risk of disease transmission, so that economic parameters, efficacy, and safety profiles would support its use over other bovine thrombin, as well as plasma-derived human thrombin. Thus, although previous application of plasma-derived bovine thrombin carried substantial risk for development of various coagulation inhibitors (including those to factor V),[2] [3] as well as theoretical potential for transmission of disease,[3] [4] recombinant thrombins are considered to have similar clinical efficacy but a much safer profile.

In the second article of this issue, Franchini and Lippi[5] describe the current understanding of the mechanisms of action and a brief overview of clinical experience of recombinant activated factor VII (rFVIIa), which was initially developed to overcome the limitations of existing treatments for patients with congenital hemophilia and inhibitors.[6] However, the ongoing great achievements in this area, joined with an increased understanding of its mechanisms for controlling bleeding, supported the experimental use of rFVIIa in other coagulopathies (e.g., life-threatening obstetric postpartum hemorrhage[7] and Glanzmann's thrombasthenia[8]) potentially characterized by impaired thrombin generation, as well as to its eventual FDA approval for use in acquired hemophilia, factor VII deficiency, and Glanzmann's thrombasthenia. The agent's safety profile is also described, along with recent advances in rFVIIa dosing and storage that may help improve both clinical outcomes and patient quality of life.

In the following article, the same authors review current knowledge on the commercially available recombinant factor VIII (rFVIII) concentrates, which were the first recombinant products to be developed and used in hemostasis, nearly 20 years ago, and representing at that time a major breakthrough in the treatment of patients with hemophilia A.[9] Currently, first-, second- and third-generation rFVIII products are available in the market. These basically differ in formulation, with second-generation products containing sucrose instead of albumin as a stabilizer, whereas third-generation formulations are produced without human or animal plasma proteins. Franchini and Lippi also emphasize that the use of rFVIII concentrates has enormously improved the safety profile of replacement therapy in hemophilia A, virtually abolishing the risk of blood-borne pathogen transmission, so that the most challenging aspect of hemophilia A management has now become the development and management of FVIII inhibitors,[6] [10] [11] [12] [13] [14] and perhaps also issues of dosing and monitoring.[15] Current and future research is now driven to the development of rFVIII concentrates with a longer duration of biological activity or a more convenient route of administration. Gene therapy is another area of great interest in hemophilia treatment, although several hurdles are yet to be fully overcome[16] before this proves to be as effective as current treatments, including demonstration of long-term efficacy and safety (e.g., the risk of overexpression, insertional mutagenesis, immune response, and development of inhibitors).

Soon after the introduction of rFVIII, the next significant advance in the therapy of hemophilia was the introduction of recombinant factor IX (rFIX) for hemophilia B, as reported in the next article by Monahan and Di Paola.[17] As such, the possibility to generate recombinant DNA sequences for the expression of rFIX has greatly facilitated the study of the protein's structure, function, and protein-to-protein interactions, enabling contextually an advanced level of safety from potential infectious contaminants of plasma-derived clotting factors. This recombinant DNA technology has been further applied to engineer an expanding spectrum of novel FIX therapies that are now increasingly translating into clinical trials. In their article, Monahan and Di Paola review the experience with the existing rFIX product, which has demonstrated very good to excellent clinical efficacy for stopping and preventing bleeding, although some drawbacks still remain, including evading immune responses to the delivery of foreign DNA sequences for constitutive expression in host cells. The current research toward developing products characterized by the potential to improve the quality of life for individuals with hemophilia B is also mentioned, including gene therapy for correcting deficient FIX activity.

The next two articles are devoted to recombinant von Willebrand factor (rVWF), which has had a somewhat long journey on its path toward potential clinical utility. Indeed, rVWF was the subject of two previous publications in Seminars in Thrombosis and Hemostasis nearly a decade ago.[18] [19] It is unclear what is largely delaying the clinical studies that will ultimately permit its clinical use, although there may be several potential explanations. Certainly, VWF is a complex protein, and development of an rVWF would likely represent a greater challenge to development of smaller less complex proteins, and potentially represents the greatest recombinant challenge within hemostasis. There have also been some changes in the development and formulation of rVWF along this journey. Of particular interest to us is the fact that rVWF by nature of its production has never been exposed to cleavage by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin repeats, number 13). This means that rVWF contains some ultra-large VWF molecules, and this has perhaps led the manufacturer to be overly cautious because of potential concerns that this may impart a “pro-thrombotic” element to rVWF, perhaps akin to the situation seen in TTP (thrombotic thrombocytopenic purpura) and other microangiopathies.[20] [21] [22] Although caution is certainly appropriate as the manufacturer heads toward “the first time in human” studies, it should be remembered that platelet and endothelial stored VWF has similarly never been exposed to plasma ADAMTS13, and that this stored VWF represents the major form of VWF released into plasma in response to stress or injury, or as a result of desmopressin (DDAVP) challenge.[23] Accordingly, rather than representing a prothrombotic risk, it is conversely possible that rVWF may instead simply represent a superior presentation for therapy in von Willebrand disease (VWD).

In the first article on rVWF in this issue, Turecek et al[24] review the technological challenge for the production of rVWF (i.e., the complex structure, large size, and multiple posttranslational modifications). They present results of a form of rVWF expressly developed for eventual treatment of VWD, whereupon the rVWF protein is coexpressed with rFVIII in Chinese hamster ovary (CHO) cells as normally used to produce rFVIII for the treatment of hemophilia A. The authors comprehensively describe the characterization of the structure and function of this rVWF product, focusing on its in vitro platelet aggregation and matrix protein binding functions. The preliminary results are certainly encouraging because rVWF reflects similar activities to those of native plasma-derived protein, highlighting that the high potency of this product is due to the presence of intact, highly functional multimers that do not appear, however, to promote exaggerated platelet aggregation.

In the second article dealing with rVWF, Muchitsch et al[25] describe the preclinical testing of human rVWF, focusing on the ADAMTS13 cleavage capacity of animals as a criterion for species suitability. As already mentioned briefly, ADAMTS13 regulates the hemostatic activity of VWF by reducing its multimeric size in the human system. Although both in vitro and ex vivo studies have shown that human rVWF is virtually resistant to the proteolytic activity of murine ADAMTS13, rabbit and cynomolgus ADAMTS13 are instead able to cleave human rVWF. The authors have performed a series of investigations using three mouse strains (ADAMTS13 deficient, C57BL/6J [wild type], VWF deficient), as well as rats, rabbits, and cynomolgus monkeys. An exaggerated pharmacological effect of rVWF was recorded in mice, with the ADAMTS-13 knockout mouse the most sensitive strain. Similar findings—with decreased incidence and severity—were also observed in normal C57BL/6J mice and also in VWF-deficient mice. An exaggerated pharmacological effect was also observed in rats after 14 doses, but not in rabbits and cynomolgus monkeys. On the whole, the marked differences recorded among the different species provide valuable biological information, suggesting that the efficiency of ADAMTS13 to cleave rVWF might influence the severity of clinical, laboratory, and pathohistological findings. As such, these data are essential to streamline species' suitability for the generation of meaningful preclinical data with rVWF because only animals with a sufficient rVWF cleavage capacity by endogenous ADAMTS13 would be considered appropriate for preclinical evaluation of the rVWF products.

Thrombolytic therapy plays a pivotal role in several vascular pathologies, including ST-elevated myocardial infarction (STEMI), venous thromboembolism (VTE), ischemic stroke, peripheral arterial occlusive disease (PAOD), and clearance of occluded vascular access devices.[26] Recombinant peptides used for this purpose are mainly represented by recombinant forms (alteplase [t-PA], reteplase [r-PA], and tenecteplase [TNK]) of tissue plasminogen activator (t-PA), a proteolytic enzyme that catalyzes the conversion of plasminogen into active plasmin, which then functions to dissolve clots. Whereas alteplase shares the structure of native t-PA, the other two peptides have been modified to enhance half-life and fibrin specificity. In the article by Campbell and Hilleman,[27] the leading indications for thrombolytic therapy are reviewed, along with the dosage, metabolism, clinical outcomes, and the potential side effects. It is thereby concluded that thrombolysis is able to provide critical reperfusion in STEMI patients without access to immediate percutaneous coronary intervention (PCI). Likewise, catheter-based systems to deliver the active thrombolytic drug may permit an increased use of recombinant peptides in the treatment of VTE and PAOD due to a lower risk of bleeding and improved outcomes in selected patients. Thrombolytic peptides might also be useful in high-risk pulmonary embolism (PE) patients and in the treatment of ischemic stroke when administered within 4.5 hours of the onset of symptoms, especially using catheter-based technology, which might increase the therapeutic window. Last but not least, thrombolytic recombinant peptides might also be useful for the treatment of occluded central venous access devices.

The last three articles of this issue are devoted to speculative and/or unlicensed use of recombinant peptides in hemostasis and thrombosis. The development of a variety of erythropoiesis-stimulating agents (ESAs), including recombinant human erythropoietin (rHuEPO) and the newest continuous erythropoietin receptor activator (CERA), represents a valuable therapeutic option for the treatment of patients with various forms of anemia, including that of chronic renal disease, malignancy, hematologic disorders, prematurity, and acquired immune deficiency syndrome (AIDS). These agents were proven effective in sustaining the hematologic response and thereby reduce the number of red blood cells transfusion. Nevertheless, in their article Lippi et al[28] highlight that the incidence of side effects is uncertain, and several recent studies have emphasized problems related to an increased trend of tumor progression, mortality, and thrombotic complications, especially VTE. As such, the use of ESAs is an important topic in hemostasis in thrombosis inasmuch as the biological basis of their potential prothrombotic effects is multifaceted and involves polycythemia/hyperviscosity syndrome, hypertension, thrombocytosis, platelet hyperactivity, and activation of blood coagulation, although it also seems to require the presence of additional largely unknown but major prothrombotic factors. This clinical and biological evidence might lead to the conclusion that therapy with ESAs might not be as safe as is commonly perceived, so that these drugs should not be routinely used as an alternative to blood transfusion unless future studies confirm their safety against the development of thromboembolic complications as well as definitive clinical benefits on the major end point (i.e., mortality).

The multifaceted interplay between inflammation and coagulation has an important role in the pathogenesis of sepsis, where inflammation leads to activation of coagulation, but coagulation also considerably affects inflammatory activity. As such, Levi et al[29] discuss the intricate relationship between inflammation and coagulation, highlighting that it might produce important consequences for the pathogenesis of microvascular failure and subsequent multiple organ failure, describing most of the molecular pathways that contribute to inflammation-induced activation of coagulation (especially endothelial-bound anticoagulant mechanisms such as disruption of the antithrombin, [activated] protein C/thrombomodulin system and tissue factor pathway systems). The reestablishment of these anticoagulant pathways by administration of recombinant peptides (e.g., recombinant antithrombin, recombinant protein C, recombinant soluble thrombomodulin, recombinant tissue factor pathway inhibitor) is currently being evaluated in several clinical studies that are summarized in this article. Basically, it can be concluded that the simultaneous modulation of both coagulation and inflammation, rather than specific therapies aimed at one single component, might provide more concrete and favorable results.

The final article of this issue by Lippi and Favaloro[30] is somewhat provocative and reviews the biochemistry, metabolism, and function or recombinant platelet factor 4 (rPF4), concluding that this protein and its derivative peptides might be potentially used in the future as therapeutic and anti-neoplastic agents. Cancer and hemostasis is closely intertwined, as previous explored n several past articles from Seminars in Thrombosis and Hemostasis.[31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] Angiogenesis plays a pivotal role in tumor growth and metastasis as well as in other serious disorders such as atherosclerosis, diabetes, arthritis, psoriasis, nephropathy, and retinopathy. Although rPF4 was originally developed and evaluated as a therapeutic alternative to protamine for neutralization of heparin therapy, some recent scientific evidences support a role in inhibiting tumor growth and spread because PF4 interferes with several steps of endothelial cell proliferation, migration, and angiogenesis, regulates apoptotic death through activation of distinct signal transduction pathways, inhibits growth factor receptor binding, amplifies the inflammatory response of natural killer cells through regulation of cytokines production, and induces and maintains a nonspecific immune response to cancer cells. This biological evidence led to the production and commercialization of PF4-based angiostatic agents (e.g., carboxyl-terminal fragments of recombinant human PF4, modified and chimeric peptides), raising the possibility of an alternative approach for preventing and treating growth and metastasis of solid tumors. Innovative means of delivering this antiangiogenic agent are also being attempted to obtain higher concentrations of the therapeutic agent in a localized area and for a longer period of time (i.e., PF4-bearing polymeric microspheres, vector-mediated PF4 transduction, transgene transfection into oncolytic viruses, molecular targeting therapy against PF4 and rHuPF4 conjugates).

We think it is important to highlight here that the collection of articles in the current issue of Seminars in Thrombosis and Hemostasis by no means defines the limit of this technology for the investigation or treatment of thrombosis or haemostatic failures. Indeed, contributions from several other researchers were sought for the current issue on other proteins of interest in this field including recombinant hirudin, recombinant factor XIII, and recombinant ADAMTS13. Despite interest, potential contributors were not able to provide contributions within a time frame permissible to ensure timely publication of this issue. Nevertheless, the importance of this field mandates a reinvestigation of the state of play in the near future, and we will facilitate the publication of relevant articles in future issues.

In conclusion, we hope that you enjoy the current collection of articles, devoted to the use of recombinant proteins in hemostasis, and we would like to sincerely thank all the authors for their most interesting and timely contributions.

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