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Oligodeoxyribonucleotides derived from salmon sperm DNA: An alternative to defibrotide
Biologicals 41 (2013) 190e196
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Biologicals journal homepage: www.elsevier.com/locate/biologicals
Oligodeoxyribonucleotides derived from salmon sperm DNA: An alternative to defibrotide Chang-Ye Hui a, *, Yan Guo b, Xi Zhang c, Jian-Hua Shao d, Xue-Qin Yang a, Wen Zhang a a
Shenzhen Prevention and Treatment Center for Occupational Disease, Shenzhen, Guangdong 518001, China Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China c Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China d College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 July 2012 Received in revised form 29 November 2012 Accepted 19 December 2012
Defibrotide is a single-stranded nucleic acid polymer originally derived from porcine mucosa. Cheap salmon sperm DNA is commercially available and widely used in drug production. In this study, oligodeoxyribonucleotides were successfully obtained from the controlled depolymerization of salmon sperm DNA. The obtained product shared similar chemical and biological properties with defibrotide produced by Gentium SpA, Italy. It was also found that oligodeoxyribonucleotides derived from non-mammalian origins could also directly stimulate tissue plasminogen activator (t-PA) release from cultured human endothelial cells, and enhance fibrinolytic activity in the rabbit. Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Defibrotide Oligonucleotide Salmon sperm DNA Depolymerization
1. Introduction
formula of random sequence: P1-5,(dAp)12-24,(dGp)10-20,(dTp)13purines/pirymidines mol > 0.85; hyperchromicity (the rearrangement of defibrotide monitored by thermal denaturation) ¼ 12e18%. Due to a bright prospect for medical application, it is crucial to expand the source of defibrotide and to reduce its production costs. DNA extracted from salmon testes, organs typically as industrial waste, is used as raw material for some drugs and health foods. In this paper, a simple approach was established to generate oligodeoxyribonucleotides from salmon sperm DNA. Its chemical and biological properties were identical to defibrotide from mammalian organs.
26,(dCp)10-20;
Defibrotide is an investigational drug candidate and has been investigated as a treatment for different vascular diseases in various stages of clinical development. It has extensive pharmacological activities due to its anti-ischemic, anti-inflammatory, pro-fibrinolytic and anti-thrombotic properties both in vitro and in vivo. Although its precise role in hemostasis and cancer diseases has not yet been clarified, it is believed to be associated with its polyanionic nature [1]. Defibrotide is usually tolerated when administered orally or intravenously. Several clinical trials have indicated that defibrotide could provide benefits for the treatment and prophylaxis of severe hepatic veno-occlusive disease and multiorgan failure, resulting from stem cell transplantation and chemotherapy [2]. The efficacy of defibrotide as an antitumor agent is still under investigation. Defibrotide is formed by a polydisperse mixture of singlestranded oligodeoxyribonucleotides (length, 9-80 mer; average molecular mass, 16.5 kDa), generally derived from the controlled depolymerization of porcine intestinal mucosal genomic DNA. The depolymerization is done at controlled temperature and pH, followed by denaturation of the double stranded filaments. Defibrotide has well defined chemical and physical properties, such as the
* Corresponding author. Tel.: þ86 15815539746. E-mail address: [email protected] (C.-Y. Hui).
2. Materials and methods 2.1. Pretreatment of salmon sperm DNA Commercially available salmon sperm DNA sodium salt was purchased from Wako Pure Chemical Industries (Osaka, Japan) with further purification. A 5% salmon sperm DNA solution was prepared by dissolution with water, and brought to pH 4.0 adding HCl. Residual protamine impurities were removed after passing through a cation exchanger (Amberlite IR120). Next, NaAc was added to the DNA solution in order to achieve a 1.5 mol/L concentration of salt. High molecular weight DNA was precipitated by the addition of an equal volume of ethanol. After dehydration with anhydrous ethanol, purified DNA was subjected to depolymerization.
1045-1056/$36.00 Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biologicals.2012.12.003
C.-Y. Hui et al. / Biologicals 41 (2013) 190e196
2.2. Preparation of porcine intestinal genomic DNA Total genomic DNA was extracted via enzymatic hydrolysis and ultrafiltration as previously described, with a minor adjustment [3]. Fresh porcine intestinal mucosa was ground and subjected to complete proteolysis by papain. The filtered solution of proteolysate was passed through a Mr 100,000 cut-off membrane (Pellicon 0.5 m2, Millipore), and CaCl2 was added to the retentate at a concentration of 0.3 mol/L, pH 3.0. The precipitation was collected and dissolved in 1.5 mol/L NaAc, pH 8.0, and ethanol was added to the resulting solution to precipitate the DNA sodium salt. 2.3. Controlled thermal depolymerization of DNA Porcine intestinal mucosal DNA and salmon sperm DNA were dissolved in pH 4.2 HAc-NaAc buffer (3 mol/L) at a concentration of 3% and 5%, respectively. Depolymerization was done at 751 C and monitored by HPLC. For HPLC sample preparation, at 15e30 min intervals, the reaction solution was diluted in 0.05 mol/L NaOH to disrupt hydrogen bonds stabilizing double-stranded DNA. The depolymerization process was stopped until the degradation product had similar molecular weight distribution to defibrotide (Gentium SpA, Italy). After thermal denaturation, insoluble particulates were removed by a filtration membrane (0.22 mm, Millipore). The products were finally precipitated with alcohol and dried under vacuum. 2.4. Size exclusion HPLC Polymeric substances can be separated according to their molecular weights via size exclusion chromatography. HPLC analyses were performed with a Shimadzu Corporation (Tokyo, Japan) liquid chromatograph system, equipped with a TSK G4000SW column (used for high molecular weight DNA analysis) and a TSK G3000SW column (for oligonucleotide analysis), both obtained from Tosoh Corporation (Tokyo, Japan). The mobile phase was isocratic and consisted of the following: 50 mM sodium phosphate buffer and 100 mM NaCl pH 7.0, at a flow rate of 0.7 ml/min. Nucleic acid in the eluate was determined by spectrophotometry at 260 nm. 2.5. Anion exchange HPLC Oligodeoxyribonucleotide is a polyanion and has a particular net negative charge based on the number of phosphodiester groups in the molecule. Anion-exchange chromatography could be another choice for analysis of such biochemical materials. Depolymerization products were performed on a Gen-PakÔ FAX column, 4.6 100 mm (Waters; WAT015490). The eluents were as follows: buffer A, 10 mM TriseHCl, 1 mM EDTA, pH 8.0; buffer B, 1 M NaCl in buffer A. The gradient pattern for separation was a 30e85% linear gradient of eluent B for 35 min, and the flow rate was held constant at 0.5 ml/min. Elution was monitored by absorbance at 260 nm. 2.6. Chromogenic microtitre plate assay The biological activities of oligonucleotides derived from different animal origins were compared by using a chromogenic microtitre plate assay [4]. The kinetics of human plasmin (Sigmae Aldrich Corp., St. Louis, USA) activity was measured in the presence of oligonucleotides (1e8 mg/ml). A 96-well plate containing oligonucleotides and 0.6 mmol/L of the chromogenic substrate HD-Val-Leu-Lys-pNA (S-2251, Chromogenix, Sweden) was incubated at 37 C. Plasmin was added to each well to a final concentration 8 mU/ml. The plate was then incubated at 37 C for 100 min, and the rate of change of A405 was monitored with a spectrophotometer.
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The plasmin activity levels were expressed as the slopes of absorbance versus time curves. 2.7. Human umbilical vein endothelial cell fibrinolytic parameters To evaluate the effect of oligodeoxyribonucleotides on fibrinolytic properties of endothelial cells, human umbilical vein endothelial cells (HUVECs) were cultured and treated as previously described [5]. Confluent monolayers of HUVECs grown in a 24-well plate were incubated with various oligonucleotides (200 mg/ml) or the vehicle (control). After 48 h of incubation at 37 C, conditioned media (CM) were harvested and filtered through a 0.22 mm filter. Then the levels of tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen and t-PA activity in CM were measured. A human t-PA total antigen assay kit (Boster, Cat No. EK0897, China) and t-PA activity assay kit (Gentaur, Cat No. CT1001, Belgium) were used to detect the total t-PA antigen and active t-PA level in CM, respectively. PAI-1 was also measured in CM by ELISA (Boster, Cat No. EK0859, China). All assays were performed strictly as recommended by the manufactures. Results for the antigens are expressed as ng/105 cells, and IU/105 cells for activity. 2.8. Euglobulin clot lysis time assay The study was performed on male New Zealand white rabbits, weighing 1.5e2.0 kg, receiving a standard diet and water. Sterility and absence of pyrogens contamination was verified for all agents. Various oligonucleotides were dissolved in saline and injected intravenously into rabbits at 60 mg/kg. The control group is given linear polyanion polystyrene sulfonate (PSS, 6e15 kDa, Shanghai Institute of Materia Medica, China) at 60 mg/kg. Blood was collected from the marginal ear vein before and 2 h after administration. The euglobulin clot lysis time (ELT) was assessed, based on the method described previously [6,7]. Briefly, 400 ml of plasma was diluted with distilled water up to 4 ml. pH was adjusted to 5.6 with 0.25% acetic acid and incubated in an ice bath for 20 min. After 1 min centrifugation at 14,900 g, the euglobulin precipitate was dissolved in 400 ml buffer (13.4 mM KH2PO4, 53.6 mM Na2HPO4). The clot formation started when 0.2 IU thrombin was added. The fraction was put at 37 C and the time for complete lysis of the clot (ELT) was recorded. Results were finally expressed as a percentage of ELT obtained before treatment [100 (ELT at 2 h after administration/ELT before treatment)]. 3. Results 3.1. Ethanol fractionation of salmon sperm DNA Salmon testes, as DNA-rich waste, are abundant in the food processing industry. Salmon sperm DNA is commercially available as powder containing some oligonucleotides with a molecular weight lower than defibrotide (Fig. 1a). After utilization of the ethanol fractionation process, oligonucleotides were completely removed (Fig. 1b). Otherwise, residual oligonucleotides will change the molecular weight distribution of the depolymerization products, the chromatogram of which is a non-normal distribution curve (data not shown). 3.2. High molecular weight genomic DNA was extracted from porcine intestinal mucosa Genomic DNA, using the modified method, was successfully isolated from porcine intestinal mucosa. After tissue homogenization, the porcine intestine was completely digested. The resultant proteolysate was mainly composed of genomic DNA (Fig. 2a, peak 1),
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Fig. 1. Chromatogram of salmon sperm DNA and of defibrotide, produced by Gentium SpA. Molecular size analysis of salmon sperm DNA was carried out by gel filtration (TSK G4000SW, with the detection of UV absorbance at 260 nm). Commercial available salmon sperm DNA (a) usually contained deoxyribonucleic acid of both higher and lower molecular weight than defibrotide (c). High molecular weight DNA (b), isolated by fractional precipitation from an aqueous solution of salmon sperm DNA in the presence of salt by addition of ethanol, was then subjected to depolymerization. Combining (a), (b) and (c) to obtain (d).
oligopeptides and amino acids (Fig. 2a, peak 2). Protein hydrolysis products were effectively removed by ultrafiltration (Fig. 2b). Due to its polydispersity and polyanionic nature, intestinal polysaccharides are very difficult to separate from DNA. Heparin, the main fraction of intestinal oligosaccharides, can increase the risk of bleeding. Therefore its residue should be strictly controlled in the final product. Watersoluble heparin calcium can be effectively separated from DNA calcium salt, as the latter is poorly soluble inwater. It is also demonstrated that different extraction methods greatly affect the molecular weight of DNA (Fig. 2c). In order to obtain high molecular weight DNA, mechanical shear should be avoided as much as possible. 3.3. Fragments derived by chemical depolymerization of DNA Depolymerization of DNA sodium salts in acidic buffer was performed at 75 C. The salt concentration and pH conditions of the aqueous buffer affect the molecular weight distribution of the product, and the rate of depolymerization, respectively. Therefore, these key parameters have been optimized firstly. Depolymerization was controlled by monitoring the polymer molecular weight at fixed intervals, and carried out until the molecular weight fell within the limit of defibrotide (Fig. 3a). Finally, Oligodeoxyribonucleotides were formed by removing the hydrogen bonds in the double-stranded DNA fragments. The time required for depolymerization mainly depends on the molecular weight of the starting material. Reaction time required for intestinal mucosal DNA depolymerization is about 4 h (Fig. 3c), in contrast to 1 h for salmon sperm DNA (Fig. 3b).
Fig. 2. Preparation of genomic DNA from porcine intestinal mucosa. Fractions taken from each step were analyzed by TSK G4000SW. The filtered solution of proteolysate contained two molecular species (a). The identity of peak 1 (genomic DNA) and peak 2 (oligopeptides and amino acid) were confirmed by amino acid composition analysis. Genomic DNA was fully retained by the ultrafiltration membrane, with a nominal molecular weight cut-off of 100,000 Da (b). (c) 2% agar electrophoresis analysis of DNA samples. Markers were run in lane 1, and their molecular mass values are indicated. Lane 2: calf thymus DNA (SigmaeAldrich); lane 3: porcine intestinal mucosal genomic DNA extracted in this paper; lane 4: salmon sperm DNA.
3.4. Quality control of oligonucleotides from different biological origins Despite the polydisperse nature of the mixture, defibrotide still has well defined chemico-physical properties. In this study, we compared the chemical properties of oligonucleotides derived from salmon sperm DNA (ONT-S) and porcine intestinal mucosal genomic DNA (ONT-P). The molecular weight distribution of them was nearly identical to defibrotide from Gentium SpA (Fig. 3d). In addition, they all share similar chemical properties, as shown in Table 1. Besides chain length, the polyanionic nature is another important property of defibrotide, as it is associated with biological activities. Oligonucleotides prepared in this study were also
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Fig. 3. Preparation of chemically defined and reproducible single-stranded oligodeoxyribonucleotides. Polymerized DNA degradation was analyzed with a TSK G3000SW gel permeation column, and the eluate was monitored at 260 nm. Controlling the depolymerization through the measurement of the variation of the polymer molecular weight until it achieves the value of defibrotide, which as control (a). Depolymerization of highly polymerized DNA derived from salmon sperm (b) and intestinal mucosa (c) was compared. Combining the chromatograms of defibrotide from Gentium SpA (a), depolymerization products obtained from salmon sperm DNA (b) and intestinal mucosal DNA (c) to obtain (d).
characterized by anion-exchange chromatography (Fig. 4). Both of them showed similar elution curve.
(control versus treatment group, P < 0.05; Fig. 6c). The difference between treatment groups was not statistically significant.
3.5. Effect of oligonucleotides on plasmin activity
3.7. Effect of oligonucleotides on euglobulin lysis time
Although ONT-S was chemically identical to oligonucleotide extracted from mammalian organs, a biological activity assay was necessary due to its intrinsic biopolymeric nature. Defibrotide was demonstrated to have a stimulatory effect on plasmin activity, which led to the development of in vitro activity assay of defibrotide [4]. As we expected, oligonucleotides derived from different origins all produced a concentration-dependent enhancement of plasmin activity (Fig. 5).
Determination of clot lysis time on euglobulin fraction was used to assess the overall activity of the fibrinolytic system. After a single intravenous dose of defibrotide, shortening of ELT was observed in both healthy volunteers and peripheral arterial disease patients [9]. Defibrotide administered intravenously was also reported to enhance the fibrinolytic activity of the euglobulin fraction in many animal species [6,9]. Although the fibrinolytic activity assay is related only to one among the activities of defibrotide, it is a biological calibration usually done by manufacturer (Gentium SpA, Italy) [4,9]. Intravenous administration of ONT-P or ONT-S (60 mg/ kg) to rabbits all caused a significant decrease in ELT measured ex vivo. Similar to the defibrotide group, the decrease of ELT was about 14e18% (Fig. 7). Polyanionic molecules such as sulfated polyvinylacrylates have been suggested to be associated with plasminogen activation and plasmin activity [10]. However, polyanion PSS did not have significant effect on ELT, as shown in Fig. 7.
3.6. Effect of oligonucleotides on the fibrinolytic properties of HUVECs Endothelium is believed to be the major target for defibrotide activity, and in vitro studies have demonstrated that defibrotide significantly influenced the fibrinolytic properties of both macrovascular and microvascular endothelial cells [5,8]. HUVECs were incubated for 48 h with 200 mg/ml defibrotide, ONT-P and ONT-S. All oligonucleotides did not significantly affect PAI-1 antigen secretion compared with control cells (Fig. 6a), while a remarkable increase of t-PA antigen was found in the CM of HUVECs stimulated with all agents (control:0.520 0.036, defibrotide: 0.807 0.038, ONT-P: 0.830 0.031, ONT-S: 0.733 0.043 ng/105 cells; P < 0.05 versus control; Fig. 6b). A progressive increase in the level of t-PA activity was also observed after oligonucleotides stimulation
4. Discussion In the present study, Oligonucleotides derived from salmon sperm DNA were successfully prepared. Its chemical and physical properties, especially the molecular weight and the negative charge property, were similar to those of defibrotide. More importantly, oligonucleotides derived from non-mammalian origins have shown
Table 1 Chemical characteristics of the oligonucleotide preparations. Item
Oligodeoxyribonucleotides
Appearance DNA content Phosphorus Nitrogen (A þ G)/(C þ T) mol P/(A þ G þ C þ T) mol Hyperchromicity
Light yellow, hygroscopic, amorphous powder 91.2% 90.6% 8.65% 8.54% 13.91% 13.20% 0.96 0.94 1.07 1.04 13.9% 14.1%
(Gentium SpA)
Method (Porcine intestine)
(Salmon sperm) 91.7% 8.66% 13.73% 0.95 1.06 13.6%
Calculation from a phosphorus content Fiske-Subbarow method Kjeldahl method HPLC Alkaline denaturation
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Fig. 4. Elution profiles of various oligonucleotides on a DEAE-resin column. Gentium’s defibrotide (a), ONT-S (b) and ONT-P (c) were injected onto an anion-exchange column equilibrated with 25 mM TriseHCl (pH 8.0) containing 1 mM EDTA, and eluted at a speed of 0.5 ml/min with a 17.5-ml linear NaCl gradient (from 0.3 to 0.85 M) in the same buffer. The eluate was monitored at 260 nm. Combining (a), (b) and (c) to obtain (d).
promising activity as an alternative to defibrotide for further development. Salmon sperm is a liquid with a very high density of DNA that is considered a waste product of the fishing industry. Thus, salmon sperm DNA is an affordable and abundant alternative to mammalian genomic DNA. In addition, the GC content of salmon sperm genome is 44.4% [11], similar to that of swine genome (38.2%) [12]. Oligonucleotides sequence features and base compositions are at least partially related to GC content. Oligonucleotides derived from other materials, such as microbial genomic DNA with similar GC
Fig. 5. Effect of various oligonucleotides on plasmin activity. Increasing concentrations of oligonucleotides were added to the standard assay of plasmin activity with substrate S-2251. Defibrotides from Gentium SpA, ONT-P and ONT-S could increase the activity of plasmin to degrade the S-2251 substrate, which contains the preferred cleavage site for plasmin. Upon cleavage, the release of p-nitroaniline was followed spectrophotometrically at 405 nm. Levels of activity of plasmin were calculated as the slopes of the linear portion of the absorbance versus time curves in 5 min steps for a period of 20e 60 min. The test was performed three times in at least triplicate, and the figure shows data from one representative experiment.
content, are also under investigation. Defibrotide is actually a mixture of oligodeoxyribonucleotides with different molecular chain lengths. The reversible hyperchromicity is a peculiar optical property of oligonucleotides [3]. This property is very obvious in the double helix DNA, whereas in the case of a 15-mer oligonucleotide, this value is almost zero. The hyperchromicity is a measure of the capacity of oligonucleotides rearrangement (DNA base pairing) in the solution, which makes oligonucleotides different from other polyanionic biologicals, such as heparin and pentosan polysulfate. The hyperchromicity is mainly determined by oligonucleotide length and base composition. There is evidence that oligonucleotide chain length (the optimum value of hyperchromicity) is important for the biological activity. The high molecular weight fractions of defibrotide were responsible for the endotheliumdependent fibrinolytic effect [7]. However, a hyperchromicity higher than 20% involved risks of undesirable side effects [3]. Therefore, the molecular weight distribution is crucial for quality control of defibrotide and its oligonucleotide analog. The mammalian plasminogen (Plg)-plasmin system has a central role in fibrinolysis. The proenzyme Plg circulates in plasma at high concentration and can be converted to plasmin, a potent and nonspecific serine protease [13]. Previous studies have shown that defibrotide exerts a length-dependent stimulatory effect on plasmin activity in vitro, that is reflected in an increase in the kcat of plasmin [4]. The stimulation of plasmin activity by oligonucleotides is closely associated with their mean molecular weights. The stimulatory effect increases as the oligonucleotides chain length increases. It is believed that oligonucleotides interact with a conserved lysine binding region of the kringle domains, producing a structural change that enhances its catalytic ability [4]. This is different from systemic anticoagulants and thrombolitycs, including t-PA and heparin, which are always associated with excessive bleeding complications [14,15]. Defibrotide exerts no obvious
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Fig. 7. Oligonucleotides increased overall plasma fibrinolytic activity in rabbits. New Zealand white male rabbits (4 or 5 rabbits per treatment group) were given various oligonucleotides or polyanion PSS intravenously in a dose of 60 mg/kg. Blood samples were collected before and 2 h after the injection, and ELT was then determined. Results are presented as percentage of ELT after treatment relative to ELT before treatment. The error bars represent the standard deviation of the mean, *P < 0.05 by unpaired t test, significantly different from PSS group. There was no statistically significant difference between oligonucleotides groups (P > 0.05).
Fig. 6. Effect of oligonucleotides on fibrinolytic protein expression by HUVECs. HUVECs were treated with 200 mg/ml defibrotide, ONT-P and ONT-S for 48 h, and subsequently CM was collected and tested for PAI-1 and t-PA antigens and for t-PA activity. Various oligonucleotides didn’t significantly affect the expression of PAI-1, but they all significantly increased t-PA antigen and activity. Each value represents mean of three independent assays done in triplicate SD. The asterisks indicate significant differences compared to the control (*P < 0.05). P values are determined by paired t-test.
systemic anticoagulant effects, and it has emerged as a safe treatment option for deep-venous thrombosis and hepatic venoocclusive disease [2,16]. In the preclinical research and development, defibrotide was reported to enhance the fibrinolytic activity of the euglobulin fraction in some animal species [6,17]. Following intravenous administration of defibrotide to Wistar rats, a dose-dependent shorten of the plasma ELT was observed [18]. In an early clinical trial, defibrotide administration was associated with shortening of the ELT both in healthy volunteers and peripheral obliterative arterial disease patients, in whom an increase in t-PA level was also verificated [19]. Numerous studies in human and in animal model show that defibrotide affects the expression of plasma proteins with antithrombotic activities, such as t-PA and its inhibitor, tissue factor pathway inhibitor, prostacyclin, P-selectin and thrombomodulin, and reduces platelet and neutrophil functions [16,20].
Defibrotide appears to have a more substantial effect on t-PA activity. Defibrotide dose-dependently counteracted the proinflammatory stimulus induced decrease in t-PA activity expression, and significantly increased t-PA antigen expression in resting endothelial cells [5]. ONT-S and ONT-P treatment also significantly increased t-PA expression in HUVEC, but didn’t significantly affect PAI-1 release. The activation of the fibrinolytic system is another important antithrombotic mechanism induced by oligonucleotides, which is mainly attributed to the increase of t-PA in the arterial wall [1]. Multiple myeloma alters the profibrinolytic potential of endothelial cells decreasing t-PA and increasing PAI levels, which is potentiated by thalidomide, Defibrotide was able to counteract these effects [8]. These studies suggested that endothelium may be the major target of oligonucleotides. Adenosine receptors A1 and A2 were demonstrated to be their receptors in vascular endothelium. They belong to a nucleotide receptors family that is involved in endothelial cell regulation and response to injury [21]. Urokinase (u-PA) is the main PA in eukaryotic cell migration processes, but tissue-type plasminogen activator (t-PA) appears to be the primary PA in fibrinolysis [22]. Heparin is known to bind various components of the fibrinolytic system, especially t-PA, u-PA and Plg. The activation of Plg by t-PA and u-PA can be increased by heparin [23]. Similarly, in an in vitro study conducted by Echart et al., t-PA or u-PA was added to a mixture of Plg and S-2251, and then incubated with and without defibrotide. Defibrotide was demonstrated to stimulate t-PA and u-PA mediated plasminogen activation [4]. Oligonucleotides may contain various aptamers responsible for their diverse biological activities. The three aptamers identified from defibrotide happened to be potent inhibitors of thrombin [24]. Oligonucleotide aptamers inhibit Toll-like receptor liganddependent dendritic cells activation and is blocked by adenosine receptor antagonist [25]. For the time being, only limited aptamers have been identified in defibrotide. Oligonucleotides derived from animal genome seem to be an aptamer database. Various potent oligomers and second-generation derivative oligomers may offer a more effective alternative to defibrotide and its analog in the future.
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Oligodeoxyribonucleotides derived from salmon sperm DNA: An alternative to defibrotide Chang-Ye Hui a, *, Yan Guo b, Xi Zhang c, Jian-Hua Shao d, Xue-Qin Yang a, Wen Zhang a a
Shenzhen Prevention and Treatment Center for Occupational Disease, Shenzhen, Guangdong 518001, China Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China c Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China d College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 July 2012 Received in revised form 29 November 2012 Accepted 19 December 2012
Defibrotide is a single-stranded nucleic acid polymer originally derived from porcine mucosa. Cheap salmon sperm DNA is commercially available and widely used in drug production. In this study, oligodeoxyribonucleotides were successfully obtained from the controlled depolymerization of salmon sperm DNA. The obtained product shared similar chemical and biological properties with defibrotide produced by Gentium SpA, Italy. It was also found that oligodeoxyribonucleotides derived from non-mammalian origins could also directly stimulate tissue plasminogen activator (t-PA) release from cultured human endothelial cells, and enhance fibrinolytic activity in the rabbit. Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Defibrotide Oligonucleotide Salmon sperm DNA Depolymerization
1. Introduction
formula of random sequence: P1-5,(dAp)12-24,(dGp)10-20,(dTp)13purines/pirymidines mol > 0.85; hyperchromicity (the rearrangement of defibrotide monitored by thermal denaturation) ¼ 12e18%. Due to a bright prospect for medical application, it is crucial to expand the source of defibrotide and to reduce its production costs. DNA extracted from salmon testes, organs typically as industrial waste, is used as raw material for some drugs and health foods. In this paper, a simple approach was established to generate oligodeoxyribonucleotides from salmon sperm DNA. Its chemical and biological properties were identical to defibrotide from mammalian organs.
26,(dCp)10-20;
Defibrotide is an investigational drug candidate and has been investigated as a treatment for different vascular diseases in various stages of clinical development. It has extensive pharmacological activities due to its anti-ischemic, anti-inflammatory, pro-fibrinolytic and anti-thrombotic properties both in vitro and in vivo. Although its precise role in hemostasis and cancer diseases has not yet been clarified, it is believed to be associated with its polyanionic nature [1]. Defibrotide is usually tolerated when administered orally or intravenously. Several clinical trials have indicated that defibrotide could provide benefits for the treatment and prophylaxis of severe hepatic veno-occlusive disease and multiorgan failure, resulting from stem cell transplantation and chemotherapy [2]. The efficacy of defibrotide as an antitumor agent is still under investigation. Defibrotide is formed by a polydisperse mixture of singlestranded oligodeoxyribonucleotides (length, 9-80 mer; average molecular mass, 16.5 kDa), generally derived from the controlled depolymerization of porcine intestinal mucosal genomic DNA. The depolymerization is done at controlled temperature and pH, followed by denaturation of the double stranded filaments. Defibrotide has well defined chemical and physical properties, such as the
* Corresponding author. Tel.: þ86 15815539746. E-mail address: [email protected] (C.-Y. Hui).
2. Materials and methods 2.1. Pretreatment of salmon sperm DNA Commercially available salmon sperm DNA sodium salt was purchased from Wako Pure Chemical Industries (Osaka, Japan) with further purification. A 5% salmon sperm DNA solution was prepared by dissolution with water, and brought to pH 4.0 adding HCl. Residual protamine impurities were removed after passing through a cation exchanger (Amberlite IR120). Next, NaAc was added to the DNA solution in order to achieve a 1.5 mol/L concentration of salt. High molecular weight DNA was precipitated by the addition of an equal volume of ethanol. After dehydration with anhydrous ethanol, purified DNA was subjected to depolymerization.
1045-1056/$36.00 Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biologicals.2012.12.003
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2.2. Preparation of porcine intestinal genomic DNA Total genomic DNA was extracted via enzymatic hydrolysis and ultrafiltration as previously described, with a minor adjustment [3]. Fresh porcine intestinal mucosa was ground and subjected to complete proteolysis by papain. The filtered solution of proteolysate was passed through a Mr 100,000 cut-off membrane (Pellicon 0.5 m2, Millipore), and CaCl2 was added to the retentate at a concentration of 0.3 mol/L, pH 3.0. The precipitation was collected and dissolved in 1.5 mol/L NaAc, pH 8.0, and ethanol was added to the resulting solution to precipitate the DNA sodium salt. 2.3. Controlled thermal depolymerization of DNA Porcine intestinal mucosal DNA and salmon sperm DNA were dissolved in pH 4.2 HAc-NaAc buffer (3 mol/L) at a concentration of 3% and 5%, respectively. Depolymerization was done at 751 C and monitored by HPLC. For HPLC sample preparation, at 15e30 min intervals, the reaction solution was diluted in 0.05 mol/L NaOH to disrupt hydrogen bonds stabilizing double-stranded DNA. The depolymerization process was stopped until the degradation product had similar molecular weight distribution to defibrotide (Gentium SpA, Italy). After thermal denaturation, insoluble particulates were removed by a filtration membrane (0.22 mm, Millipore). The products were finally precipitated with alcohol and dried under vacuum. 2.4. Size exclusion HPLC Polymeric substances can be separated according to their molecular weights via size exclusion chromatography. HPLC analyses were performed with a Shimadzu Corporation (Tokyo, Japan) liquid chromatograph system, equipped with a TSK G4000SW column (used for high molecular weight DNA analysis) and a TSK G3000SW column (for oligonucleotide analysis), both obtained from Tosoh Corporation (Tokyo, Japan). The mobile phase was isocratic and consisted of the following: 50 mM sodium phosphate buffer and 100 mM NaCl pH 7.0, at a flow rate of 0.7 ml/min. Nucleic acid in the eluate was determined by spectrophotometry at 260 nm. 2.5. Anion exchange HPLC Oligodeoxyribonucleotide is a polyanion and has a particular net negative charge based on the number of phosphodiester groups in the molecule. Anion-exchange chromatography could be another choice for analysis of such biochemical materials. Depolymerization products were performed on a Gen-PakÔ FAX column, 4.6 100 mm (Waters; WAT015490). The eluents were as follows: buffer A, 10 mM TriseHCl, 1 mM EDTA, pH 8.0; buffer B, 1 M NaCl in buffer A. The gradient pattern for separation was a 30e85% linear gradient of eluent B for 35 min, and the flow rate was held constant at 0.5 ml/min. Elution was monitored by absorbance at 260 nm. 2.6. Chromogenic microtitre plate assay The biological activities of oligonucleotides derived from different animal origins were compared by using a chromogenic microtitre plate assay [4]. The kinetics of human plasmin (Sigmae Aldrich Corp., St. Louis, USA) activity was measured in the presence of oligonucleotides (1e8 mg/ml). A 96-well plate containing oligonucleotides and 0.6 mmol/L of the chromogenic substrate HD-Val-Leu-Lys-pNA (S-2251, Chromogenix, Sweden) was incubated at 37 C. Plasmin was added to each well to a final concentration 8 mU/ml. The plate was then incubated at 37 C for 100 min, and the rate of change of A405 was monitored with a spectrophotometer.
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The plasmin activity levels were expressed as the slopes of absorbance versus time curves. 2.7. Human umbilical vein endothelial cell fibrinolytic parameters To evaluate the effect of oligodeoxyribonucleotides on fibrinolytic properties of endothelial cells, human umbilical vein endothelial cells (HUVECs) were cultured and treated as previously described [5]. Confluent monolayers of HUVECs grown in a 24-well plate were incubated with various oligonucleotides (200 mg/ml) or the vehicle (control). After 48 h of incubation at 37 C, conditioned media (CM) were harvested and filtered through a 0.22 mm filter. Then the levels of tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen and t-PA activity in CM were measured. A human t-PA total antigen assay kit (Boster, Cat No. EK0897, China) and t-PA activity assay kit (Gentaur, Cat No. CT1001, Belgium) were used to detect the total t-PA antigen and active t-PA level in CM, respectively. PAI-1 was also measured in CM by ELISA (Boster, Cat No. EK0859, China). All assays were performed strictly as recommended by the manufactures. Results for the antigens are expressed as ng/105 cells, and IU/105 cells for activity. 2.8. Euglobulin clot lysis time assay The study was performed on male New Zealand white rabbits, weighing 1.5e2.0 kg, receiving a standard diet and water. Sterility and absence of pyrogens contamination was verified for all agents. Various oligonucleotides were dissolved in saline and injected intravenously into rabbits at 60 mg/kg. The control group is given linear polyanion polystyrene sulfonate (PSS, 6e15 kDa, Shanghai Institute of Materia Medica, China) at 60 mg/kg. Blood was collected from the marginal ear vein before and 2 h after administration. The euglobulin clot lysis time (ELT) was assessed, based on the method described previously [6,7]. Briefly, 400 ml of plasma was diluted with distilled water up to 4 ml. pH was adjusted to 5.6 with 0.25% acetic acid and incubated in an ice bath for 20 min. After 1 min centrifugation at 14,900 g, the euglobulin precipitate was dissolved in 400 ml buffer (13.4 mM KH2PO4, 53.6 mM Na2HPO4). The clot formation started when 0.2 IU thrombin was added. The fraction was put at 37 C and the time for complete lysis of the clot (ELT) was recorded. Results were finally expressed as a percentage of ELT obtained before treatment [100 (ELT at 2 h after administration/ELT before treatment)]. 3. Results 3.1. Ethanol fractionation of salmon sperm DNA Salmon testes, as DNA-rich waste, are abundant in the food processing industry. Salmon sperm DNA is commercially available as powder containing some oligonucleotides with a molecular weight lower than defibrotide (Fig. 1a). After utilization of the ethanol fractionation process, oligonucleotides were completely removed (Fig. 1b). Otherwise, residual oligonucleotides will change the molecular weight distribution of the depolymerization products, the chromatogram of which is a non-normal distribution curve (data not shown). 3.2. High molecular weight genomic DNA was extracted from porcine intestinal mucosa Genomic DNA, using the modified method, was successfully isolated from porcine intestinal mucosa. After tissue homogenization, the porcine intestine was completely digested. The resultant proteolysate was mainly composed of genomic DNA (Fig. 2a, peak 1),
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Fig. 1. Chromatogram of salmon sperm DNA and of defibrotide, produced by Gentium SpA. Molecular size analysis of salmon sperm DNA was carried out by gel filtration (TSK G4000SW, with the detection of UV absorbance at 260 nm). Commercial available salmon sperm DNA (a) usually contained deoxyribonucleic acid of both higher and lower molecular weight than defibrotide (c). High molecular weight DNA (b), isolated by fractional precipitation from an aqueous solution of salmon sperm DNA in the presence of salt by addition of ethanol, was then subjected to depolymerization. Combining (a), (b) and (c) to obtain (d).
oligopeptides and amino acids (Fig. 2a, peak 2). Protein hydrolysis products were effectively removed by ultrafiltration (Fig. 2b). Due to its polydispersity and polyanionic nature, intestinal polysaccharides are very difficult to separate from DNA. Heparin, the main fraction of intestinal oligosaccharides, can increase the risk of bleeding. Therefore its residue should be strictly controlled in the final product. Watersoluble heparin calcium can be effectively separated from DNA calcium salt, as the latter is poorly soluble inwater. It is also demonstrated that different extraction methods greatly affect the molecular weight of DNA (Fig. 2c). In order to obtain high molecular weight DNA, mechanical shear should be avoided as much as possible. 3.3. Fragments derived by chemical depolymerization of DNA Depolymerization of DNA sodium salts in acidic buffer was performed at 75 C. The salt concentration and pH conditions of the aqueous buffer affect the molecular weight distribution of the product, and the rate of depolymerization, respectively. Therefore, these key parameters have been optimized firstly. Depolymerization was controlled by monitoring the polymer molecular weight at fixed intervals, and carried out until the molecular weight fell within the limit of defibrotide (Fig. 3a). Finally, Oligodeoxyribonucleotides were formed by removing the hydrogen bonds in the double-stranded DNA fragments. The time required for depolymerization mainly depends on the molecular weight of the starting material. Reaction time required for intestinal mucosal DNA depolymerization is about 4 h (Fig. 3c), in contrast to 1 h for salmon sperm DNA (Fig. 3b).
Fig. 2. Preparation of genomic DNA from porcine intestinal mucosa. Fractions taken from each step were analyzed by TSK G4000SW. The filtered solution of proteolysate contained two molecular species (a). The identity of peak 1 (genomic DNA) and peak 2 (oligopeptides and amino acid) were confirmed by amino acid composition analysis. Genomic DNA was fully retained by the ultrafiltration membrane, with a nominal molecular weight cut-off of 100,000 Da (b). (c) 2% agar electrophoresis analysis of DNA samples. Markers were run in lane 1, and their molecular mass values are indicated. Lane 2: calf thymus DNA (SigmaeAldrich); lane 3: porcine intestinal mucosal genomic DNA extracted in this paper; lane 4: salmon sperm DNA.
3.4. Quality control of oligonucleotides from different biological origins Despite the polydisperse nature of the mixture, defibrotide still has well defined chemico-physical properties. In this study, we compared the chemical properties of oligonucleotides derived from salmon sperm DNA (ONT-S) and porcine intestinal mucosal genomic DNA (ONT-P). The molecular weight distribution of them was nearly identical to defibrotide from Gentium SpA (Fig. 3d). In addition, they all share similar chemical properties, as shown in Table 1. Besides chain length, the polyanionic nature is another important property of defibrotide, as it is associated with biological activities. Oligonucleotides prepared in this study were also
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Fig. 3. Preparation of chemically defined and reproducible single-stranded oligodeoxyribonucleotides. Polymerized DNA degradation was analyzed with a TSK G3000SW gel permeation column, and the eluate was monitored at 260 nm. Controlling the depolymerization through the measurement of the variation of the polymer molecular weight until it achieves the value of defibrotide, which as control (a). Depolymerization of highly polymerized DNA derived from salmon sperm (b) and intestinal mucosa (c) was compared. Combining the chromatograms of defibrotide from Gentium SpA (a), depolymerization products obtained from salmon sperm DNA (b) and intestinal mucosal DNA (c) to obtain (d).
characterized by anion-exchange chromatography (Fig. 4). Both of them showed similar elution curve.
(control versus treatment group, P < 0.05; Fig. 6c). The difference between treatment groups was not statistically significant.
3.5. Effect of oligonucleotides on plasmin activity
3.7. Effect of oligonucleotides on euglobulin lysis time
Although ONT-S was chemically identical to oligonucleotide extracted from mammalian organs, a biological activity assay was necessary due to its intrinsic biopolymeric nature. Defibrotide was demonstrated to have a stimulatory effect on plasmin activity, which led to the development of in vitro activity assay of defibrotide [4]. As we expected, oligonucleotides derived from different origins all produced a concentration-dependent enhancement of plasmin activity (Fig. 5).
Determination of clot lysis time on euglobulin fraction was used to assess the overall activity of the fibrinolytic system. After a single intravenous dose of defibrotide, shortening of ELT was observed in both healthy volunteers and peripheral arterial disease patients [9]. Defibrotide administered intravenously was also reported to enhance the fibrinolytic activity of the euglobulin fraction in many animal species [6,9]. Although the fibrinolytic activity assay is related only to one among the activities of defibrotide, it is a biological calibration usually done by manufacturer (Gentium SpA, Italy) [4,9]. Intravenous administration of ONT-P or ONT-S (60 mg/ kg) to rabbits all caused a significant decrease in ELT measured ex vivo. Similar to the defibrotide group, the decrease of ELT was about 14e18% (Fig. 7). Polyanionic molecules such as sulfated polyvinylacrylates have been suggested to be associated with plasminogen activation and plasmin activity [10]. However, polyanion PSS did not have significant effect on ELT, as shown in Fig. 7.
3.6. Effect of oligonucleotides on the fibrinolytic properties of HUVECs Endothelium is believed to be the major target for defibrotide activity, and in vitro studies have demonstrated that defibrotide significantly influenced the fibrinolytic properties of both macrovascular and microvascular endothelial cells [5,8]. HUVECs were incubated for 48 h with 200 mg/ml defibrotide, ONT-P and ONT-S. All oligonucleotides did not significantly affect PAI-1 antigen secretion compared with control cells (Fig. 6a), while a remarkable increase of t-PA antigen was found in the CM of HUVECs stimulated with all agents (control:0.520 0.036, defibrotide: 0.807 0.038, ONT-P: 0.830 0.031, ONT-S: 0.733 0.043 ng/105 cells; P < 0.05 versus control; Fig. 6b). A progressive increase in the level of t-PA activity was also observed after oligonucleotides stimulation
4. Discussion In the present study, Oligonucleotides derived from salmon sperm DNA were successfully prepared. Its chemical and physical properties, especially the molecular weight and the negative charge property, were similar to those of defibrotide. More importantly, oligonucleotides derived from non-mammalian origins have shown
Table 1 Chemical characteristics of the oligonucleotide preparations. Item
Oligodeoxyribonucleotides
Appearance DNA content Phosphorus Nitrogen (A þ G)/(C þ T) mol P/(A þ G þ C þ T) mol Hyperchromicity
Light yellow, hygroscopic, amorphous powder 91.2% 90.6% 8.65% 8.54% 13.91% 13.20% 0.96 0.94 1.07 1.04 13.9% 14.1%
(Gentium SpA)
Method (Porcine intestine)
(Salmon sperm) 91.7% 8.66% 13.73% 0.95 1.06 13.6%
Calculation from a phosphorus content Fiske-Subbarow method Kjeldahl method HPLC Alkaline denaturation
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Fig. 4. Elution profiles of various oligonucleotides on a DEAE-resin column. Gentium’s defibrotide (a), ONT-S (b) and ONT-P (c) were injected onto an anion-exchange column equilibrated with 25 mM TriseHCl (pH 8.0) containing 1 mM EDTA, and eluted at a speed of 0.5 ml/min with a 17.5-ml linear NaCl gradient (from 0.3 to 0.85 M) in the same buffer. The eluate was monitored at 260 nm. Combining (a), (b) and (c) to obtain (d).
promising activity as an alternative to defibrotide for further development. Salmon sperm is a liquid with a very high density of DNA that is considered a waste product of the fishing industry. Thus, salmon sperm DNA is an affordable and abundant alternative to mammalian genomic DNA. In addition, the GC content of salmon sperm genome is 44.4% [11], similar to that of swine genome (38.2%) [12]. Oligonucleotides sequence features and base compositions are at least partially related to GC content. Oligonucleotides derived from other materials, such as microbial genomic DNA with similar GC
Fig. 5. Effect of various oligonucleotides on plasmin activity. Increasing concentrations of oligonucleotides were added to the standard assay of plasmin activity with substrate S-2251. Defibrotides from Gentium SpA, ONT-P and ONT-S could increase the activity of plasmin to degrade the S-2251 substrate, which contains the preferred cleavage site for plasmin. Upon cleavage, the release of p-nitroaniline was followed spectrophotometrically at 405 nm. Levels of activity of plasmin were calculated as the slopes of the linear portion of the absorbance versus time curves in 5 min steps for a period of 20e 60 min. The test was performed three times in at least triplicate, and the figure shows data from one representative experiment.
content, are also under investigation. Defibrotide is actually a mixture of oligodeoxyribonucleotides with different molecular chain lengths. The reversible hyperchromicity is a peculiar optical property of oligonucleotides [3]. This property is very obvious in the double helix DNA, whereas in the case of a 15-mer oligonucleotide, this value is almost zero. The hyperchromicity is a measure of the capacity of oligonucleotides rearrangement (DNA base pairing) in the solution, which makes oligonucleotides different from other polyanionic biologicals, such as heparin and pentosan polysulfate. The hyperchromicity is mainly determined by oligonucleotide length and base composition. There is evidence that oligonucleotide chain length (the optimum value of hyperchromicity) is important for the biological activity. The high molecular weight fractions of defibrotide were responsible for the endotheliumdependent fibrinolytic effect [7]. However, a hyperchromicity higher than 20% involved risks of undesirable side effects [3]. Therefore, the molecular weight distribution is crucial for quality control of defibrotide and its oligonucleotide analog. The mammalian plasminogen (Plg)-plasmin system has a central role in fibrinolysis. The proenzyme Plg circulates in plasma at high concentration and can be converted to plasmin, a potent and nonspecific serine protease [13]. Previous studies have shown that defibrotide exerts a length-dependent stimulatory effect on plasmin activity in vitro, that is reflected in an increase in the kcat of plasmin [4]. The stimulation of plasmin activity by oligonucleotides is closely associated with their mean molecular weights. The stimulatory effect increases as the oligonucleotides chain length increases. It is believed that oligonucleotides interact with a conserved lysine binding region of the kringle domains, producing a structural change that enhances its catalytic ability [4]. This is different from systemic anticoagulants and thrombolitycs, including t-PA and heparin, which are always associated with excessive bleeding complications [14,15]. Defibrotide exerts no obvious
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Fig. 7. Oligonucleotides increased overall plasma fibrinolytic activity in rabbits. New Zealand white male rabbits (4 or 5 rabbits per treatment group) were given various oligonucleotides or polyanion PSS intravenously in a dose of 60 mg/kg. Blood samples were collected before and 2 h after the injection, and ELT was then determined. Results are presented as percentage of ELT after treatment relative to ELT before treatment. The error bars represent the standard deviation of the mean, *P < 0.05 by unpaired t test, significantly different from PSS group. There was no statistically significant difference between oligonucleotides groups (P > 0.05).
Fig. 6. Effect of oligonucleotides on fibrinolytic protein expression by HUVECs. HUVECs were treated with 200 mg/ml defibrotide, ONT-P and ONT-S for 48 h, and subsequently CM was collected and tested for PAI-1 and t-PA antigens and for t-PA activity. Various oligonucleotides didn’t significantly affect the expression of PAI-1, but they all significantly increased t-PA antigen and activity. Each value represents mean of three independent assays done in triplicate SD. The asterisks indicate significant differences compared to the control (*P < 0.05). P values are determined by paired t-test.
systemic anticoagulant effects, and it has emerged as a safe treatment option for deep-venous thrombosis and hepatic venoocclusive disease [2,16]. In the preclinical research and development, defibrotide was reported to enhance the fibrinolytic activity of the euglobulin fraction in some animal species [6,17]. Following intravenous administration of defibrotide to Wistar rats, a dose-dependent shorten of the plasma ELT was observed [18]. In an early clinical trial, defibrotide administration was associated with shortening of the ELT both in healthy volunteers and peripheral obliterative arterial disease patients, in whom an increase in t-PA level was also verificated [19]. Numerous studies in human and in animal model show that defibrotide affects the expression of plasma proteins with antithrombotic activities, such as t-PA and its inhibitor, tissue factor pathway inhibitor, prostacyclin, P-selectin and thrombomodulin, and reduces platelet and neutrophil functions [16,20].
Defibrotide appears to have a more substantial effect on t-PA activity. Defibrotide dose-dependently counteracted the proinflammatory stimulus induced decrease in t-PA activity expression, and significantly increased t-PA antigen expression in resting endothelial cells [5]. ONT-S and ONT-P treatment also significantly increased t-PA expression in HUVEC, but didn’t significantly affect PAI-1 release. The activation of the fibrinolytic system is another important antithrombotic mechanism induced by oligonucleotides, which is mainly attributed to the increase of t-PA in the arterial wall [1]. Multiple myeloma alters the profibrinolytic potential of endothelial cells decreasing t-PA and increasing PAI levels, which is potentiated by thalidomide, Defibrotide was able to counteract these effects [8]. These studies suggested that endothelium may be the major target of oligonucleotides. Adenosine receptors A1 and A2 were demonstrated to be their receptors in vascular endothelium. They belong to a nucleotide receptors family that is involved in endothelial cell regulation and response to injury [21]. Urokinase (u-PA) is the main PA in eukaryotic cell migration processes, but tissue-type plasminogen activator (t-PA) appears to be the primary PA in fibrinolysis [22]. Heparin is known to bind various components of the fibrinolytic system, especially t-PA, u-PA and Plg. The activation of Plg by t-PA and u-PA can be increased by heparin [23]. Similarly, in an in vitro study conducted by Echart et al., t-PA or u-PA was added to a mixture of Plg and S-2251, and then incubated with and without defibrotide. Defibrotide was demonstrated to stimulate t-PA and u-PA mediated plasminogen activation [4]. Oligonucleotides may contain various aptamers responsible for their diverse biological activities. The three aptamers identified from defibrotide happened to be potent inhibitors of thrombin [24]. Oligonucleotide aptamers inhibit Toll-like receptor liganddependent dendritic cells activation and is blocked by adenosine receptor antagonist [25]. For the time being, only limited aptamers have been identified in defibrotide. Oligonucleotides derived from animal genome seem to be an aptamer database. Various potent oligomers and second-generation derivative oligomers may offer a more effective alternative to defibrotide and its analog in the future.
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