The authors have read the journal's policy and have the following conflicts. Yonsei University to which the authors (Il Kwon, Sung-Yu Hong, Young Dae Kim, and Ji Hoe Heo) are affiliated has patent applications pending for saxatilin (Composition for Thrombolysis and Pharmaceutical Composition for Treating Diseases related to Blood Vessel Occlusion or Narrowness Comprising the Same; Korean Patent Application No. 10-2010-0107760, PCT/KR2011/008248, U.S. Patent Application No. 13/882,792, European Patent Application No. 11838212.6, Chinese Application for Invention No. 201180052931.0, and Japanese Patent Registration No. 51300921196). This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: IK SYH JHH. Performed the experiments: IK SYH SSK SHY. Analyzed the data: IK SYH YDK HSN SSK JHH. Contributed reagents/materials/analysis tools: IK SYH JHH. Wrote the manuscript: IK SYH JHH.
Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake
Intravenous (IV) administration of recombinant tissue plasminogen activator (rt-PA) is an effective treatment for ischemic strokes if administered within 4.5 hours of symptom onset [
Adhesion and aggregation of platelets are mediated by interactions of ligands with multiple integrins, including integrins α2bβ3 (glycoprotein [GP] IIb/IIIa), α2β1, α5β1, and αvβ3. Among these integrins, the GP IIb/IIIa receptor, which mediates the final common pathway of platelet aggregation by binding specifically to fibrinogen [
Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake
All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University College of Medicine (approval number: 2010-0268) and were performed in strict accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Eight-week-old male Institute of Cancer Research (ICR) mice weighing 32-34 g were used. Animals were housed in a temperature-controlled animal facility under a 12/12 hours reversed light and dark cycle, in a plastic cage with soft bedding, and given with a free access to food and water. For operative procedures, animals were anesthetized with 5% isoflurane in a mixture of 70% N2O and 30% O2. Anesthesia was maintained with 2% isoflurane. During operative procedures, body temperature was monitored continuously with a rectal probe and maintained at 37.0 ± 0.2°C using a homeothermic blanket control unit and a heating pad (Harvard Apparatus, Holliston, MA, USA). A FeCl3-induced carotid thrombosis model was used to test the thrombolytic activity of saxatilin
Consistency of the mouse model for thrombus formation and size was assessed. Ten minutes after complete occlusion, injured CCA segments were excised, immediately immersed in 4% paraformaldehyde for fixation, and embedded in paraffin for histological analysis. Paraffin blocks were consecutively sectioned longitudinally into 3 μm slices. Sectioned slices were mounted on glass slides and stained with hematoxylin and eosin. Thrombus size (longitudinal length and area) for each animal was determined using a light microscope (Axio Imager.D2; Carl Zeiss Microimaging, Oberkochen, Germany) and Zeiss AxioVision software (AxioVs40 V 4.8.1.0; Carl Zeiss imaging Solution) for a slice representing the largest part of the thrombus.
For transmission electron microscopy (TEM), arteries were immediately fixed with Karnovsky solution (pH 7.4, 2% glutaraldehyde, 2% paraformaldehyde, 0.5% CaCl2) overnight at 4°C, washed in 0.1 M phosphate buffer (pH 7.4) and post-fixed in 1% osmium tetroxide in the same buffer for 2 hours. Specimens were then dehydrated through a series of ascending ethanol concentrations, exchanged through propylene oxide, and incubated with a 1:1 mixture of EPON (EPON 812, MNA, DDSA, DMP 30) and propylene oxide for 18 hours, embedded in an EM oven, and trimmed. Subsequently, 0.25 μm semi-thin sections were stained with toluidine blue and observed under a light microscope to determine the FeCl3-damaged region. After retrimming, ultrathin sections (80 nm) were obtained by ultramicrotome (Ultracut UCT, Leica, Austria) with a diamond knife, double stained with uranyl acetate and lead citrate, and examined by TEM (JEOL-1011; JEOL, Japan) at 80 kV [
For scanning electron microscopy (SEM), specimens were fixed and dehydrated as described above, replaced with isoamyl acetate, dried in a critical point dryer (HCP-2; Hitachi Co., Tokyo, Japan), coated with a thin layer of gold (100 nm) in an ion coater (IB-3; Eiko engineering, Ibaraki, Japan), and examined with by field-emission SEM (S-800; Hitachi Co., Tokyo, Japan) at 20 kV.
To determine the optimal condition of the FeCl3-induced arterial thrombosis model for evaluation of thrombolytic effects, 10%, 20%, 30%, 40%, and 50% (w/v) concentrations of the FeCl3 were tested using five mice for each test concentration. Carotid blood flow was measured continuously for 150 minutes from complete occlusion.
Expression and mass production of saxatilin have been described [
To evaluate the dose response to saxatilin, animals were randomly divided into seven groups, with five mice in each group: normal saline (control group), 1, 1.75, 2.5, 3.75, 5.0, or 10.0 mg/kg saxatilin. Ten percent of the dose was administered by IV bolus, and the rest was infused continuously for 60 minutes.
The thrombolytic effects of saxatilin were assessed after different administration methods. A total dose of 5 mg/kg of saxatilin was used in each animal, and animals were divided into the following four groups with five mice in each group: 1) bolus injection of total dose (5 mg/kg) at 10 minutes after occlusion; 2) double bolus injection of saxatilin with a half dose (2.5 mg/kg) of saxatilin at 10 minutes after occlusion and 60 minutes after the first bolus injection; 3) half-dose bolus injection (2.5 mg/kg) at 10 minutes after occlusion, then continuous infusion for 60 minutes of the remaining dose; or 4) bolus injection of 10% of the total dose (0.5 mg/kg) at 10 minutes after occlusion, and then continuous infusion of the remaining dose (4.5 mg/kg) for 60 minutes.
The presence and degree of recanalization were assessed by measuring blood flow. Immediately after 2 hours of blood-flow monitoring, the CCA was removed from all mice, fixed with 4% paraformaldehyde solution, and embedded in paraffin for histological examination. Paraffin blocks were consecutively sectioned in a transverse direction into 3 μm slices, mounted on glass slides, and stained with hematoxylin and eosin.
Carotid blood flow was determined by calculating the area under time-flow curves. All measured values were standardized by the minimum blood flow of each animal to avoid differences from variations in physiological condition between animals. Thrombolytic effects were calculated as described below and expressed as percent of mean control baseline blood flow: (mean blood flow during 2 hours of monitoring/mean baseline blood flow) × 100. Mean values of each group in the dose-response study were calculated and graphed on a standard thrombolytic activity curve (mean ± SD).
Average blood flow was calculated for each animal each minute to achieve a representative time-dependent pattern during monitoring. Values for mean and standard deviation of all animals in each group were calculated, and temporal changes are shown as continuous bar graphs.
Time from administration of saxatilin to effective recanalization was assessed. Effective recanalization was defined as restoration of blood flow to at least 50% of the baseline level, maintained for longer than 30 minutes.
Blood (900 μl) was drawn by cardiac puncture from five mice anesthetized with isoflurane into a syringe containing 100 μl of 150 USP sodium heparin solution, resulting in a final heparin concentration of 15 USP/ml. A total of 500 μl of heparinized whole blood was mixed with the same volume of normal saline. Saxatilin concentrations were adjusted by dilution with normal saline, and 0.1, 1, or 2 μg of saxatilin against adenosine diphosphate (ADP) and 5, 50, or 100 μg of saxatilin against collagen were added into each test cuvette and preincubated with magnetic stirring at 37°C for 5 minutes. The same volume of normal saline was added as a control. Agonists for platelet aggregometry were 20 µM ADP or 5 μg/ml of collagen (Chronolog Corporation, Havertown, PA). Platelet aggregation activity was measured with an impedance method in a platelet aggregometer (Chronolog 700; Chronolog Corporation, Havertown, PA).
To test saxatilin effects on preformed platelet aggregates, 10, 50, and 250 μg of saxatilin or the same volume of normal saline as a control were added to cuvettes after maximal aggregation in response to 20 μM ADP or 5 μg/ml of collagen. Platelet disaggregation effects were calculated as percentage of restoration to baseline levels.
Fibrin/fibrinogen zymography was performed to determine fibrinolytic activity. Fibrinogen gels were prepared using 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels containing 1.2% fibrinogen (Hyphen Biomed, Neuville-sur-Oise, France) and 0.1 NIH unit/ml of thrombin (Hyphen Biomed, Neuville-sur-Oise, France). A total of 100 μg saxatilin or 3 ng rt-PA (Actilyse; Boehringer Ingelheim, Ingelheim, Germany) with an equal volume of sample buffer (80 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.01% bromophenol blue) was loaded into wells. Gels were rinsed in 150 ml of 2.5% Triton X-100 (15 minutes) and incubated with 250 ml reaction buffer (30 mM Tris, pH 7.4, 200 mM NaCl2, and 0.02% NaN3) for 12 hours at 37°C. Gels were stained with 0.1% amido black containing acetic acid, methanol, and distilled water (volume ratio 1:3:6) for 1 hour and destained by four washes with the same solution without amido black for 130 minutes. Gels were scanned using a flatbed scanner (ArtixScan F1; Microtek International Inc., Hsinchu, Taiwan) [
To investigate the interaction of saxatilin with integrins, Fc-tagged recombinant protein was generated because there was no available antibody against saxatilin. The vector encoded the Fc region of human IgG1 (~230 amino acids). Interaction with integrins was evaluated using enzyme-linked immunosorbent assay (ELISA). Integrins for α2bβ3, αvβ3, α5β1, αvβ1, αvβ5, α1β1, and α2β1 (7148-A2, 3050-AV, 3230-A5, 6579-AV, 2528-AV, 7064-AB, and 5698-A2; R&D Systems, Minneapolis, MN, USA) (100 ng) were immobilized in 96-well immunoplates for 16 hours at 4°C. Each well was blocked with skim milk and washed three times with 0.05% PBS-T. Saxatilin-Fc (~100 nM) was added to wells and incubated for 2 hours at room temperature. After washing three times with PBS-T, anti-HuFc antibody conjugated to HRP (31413; Thermo Fisher Scientific Inc., Rockford, IL, USA) (1:2000) was added to wells and incubated for 1 hour at room temperature. Nonadherent antibodies were removed by washing three times with PBS-T. Substrate was o-phenylenediamine tablets solubilized in phosphatidylcholine buffer; 100 μl was added to each well and incubated for 10 minutes. Absorbance at 490 nm was read on a spectrophotometer. We determined the Kd values from the titration curve using the simple linearization method [
We evaluated thrombolytic effects of other well-known plasminogen activators: rt-PA (Actilyse; Boehringer Ingelheim, Ingelheim, Germany), urokinase-type PA (u-PA) (Urokinase; Green Cross Corp., Yongin, Korea), and the GP IIb/IIIa receptor antagonists abciximab (ReoPro; Lilly Pharma Production GmbH & Co., Hamburg, Germany) and tirofiban (Aggrastat; Iroko Cardio Australia Pty Ltd, Sydney, Australia). Agents were administrated as 10% IV bolus injection with continuous infusion of the remaining 90% for 1 hour at 0.9, 1.8, 2.7, 4.8, 7.2, 9, or 18 mg/kg for rt-PA; 100, 500, 1000, 5000, 10,000, or 50,000 IU/kg for u-PA; 0.25, 0.5, 1, 2.5, 5, 10, 20, or 40 mg/kg for abciximab; and 0.5, 1.25, 2.5, 3.75, 5, or 10 mg/kg for tirofiban. An equal volume of normal saline was administered to control animals.
To evaluate half-life of saxatilin
Thrombosis was induced in the same manner as it was performed in the dose-response study. To avoid spontaneous recanalization, distal site of the CCA was ligated with Silkam 5-0 thread (Silkam, B Braun Aesculap, Tuttlingen, Germany) immediately after achieving complete occlusion. After 1, 3, 6, 12, or 24 hours, the CCA ligation was gently removed and then 5 mg/kg saxatilin was administered intravenously (10% bolus and then continuous infusion of the remaining dose for 60 minutes). Thrombus at a time of complete occlusion was used as control fresh thrombus. Randomly selected five mice were used in each group. To compare thrombolytic efficacy between saxatilin and rt-PA on aged thrombus, 9 mg/kg rt-PA was administered in the same way at 3 or 6 hours after complete occlusion.
To assess whether saxatilin administration causes thrombocytopenia or neutropenia, hematologic analyses were performed with blood collected from 5 mice after sham operation. Immediately after infusion of 5 mg/kg saxatilin or same volume of normal saline for 60 minutes, 1 ml of blood was collected in 10% EDTA via the lateral saphenous vein. Complete blood count (CBC) with differential counts was obtained using the automatic cell counter (MS9-5V; Melet Schloesing Laboratories, Cergy-Pontoise, France).
Randomly selected four mice were used in each group. Immediately after infusion of 5 mg/kg saxatilin or same volume of normal saline for 60 minutes, bleeding time was assessed by transection of the tail at 1 cm from the tail tip using a sharp scalpel. Blood was blotted every 15 seconds and the time period from tail transection to cessation of bleeding was defined as the bleeding time. Bleeding times exceeding 600 seconds were considered as 600 seconds.
Statistical analyses were performed using SPSS (version 20.0, SPSS Inc., Chicago, IL, USA). Normality of distributions was verified using the Kolmogorov-Smirnov test. Differences among groups in dose response studies were compared with a one-way ANOVA test, followed by a post-hoc Tukey method. Differences between groups in hematological assessment were compared with a Mann-Whitney U test. Values were presented as a mean ± standard deviation (SD). P < 0.05 was considered significant.
No complete occlusion was observed by 10% or 20% FeCl3. Two of the five mice by 30% and all of the five mice by 40% achieved complete occlusion, but all of them showed spontaneous recanalization during monitoring for 150 minutes except one mouse by 40%. All mice by 50% FeCl3 achieved complete occlusion, which was maintained without spontaneous recanalization during the monitoring (
After 5 minutes of FeCl3, CCA blood flow was rapidly and consistently reduced to nearly zero in all five animals examined (
The regions farthest from where FeCl3 was applied showed relatively normal vascular structure, with normally shaped endothelial cells, smooth muscle cells, erythrocytes, and discoid platelets (Figures 2Ca, Da). Endothelial cells appeared empty with a loss of organelles. Some platelets were activated and aggregated and adhered to the luminal surface in mildly damaged regions (Figures 2Cb, Db). At the thrombus border, activated and aggregated platelets adhered to the damaged luminal surface, and no intact endothelial cells were observed (Figures 2Cc, Dc). In severely damaged regions, platelet-rich thrombi containing erythrocytes completely occluded the vascular lumen (Figures 2Cd, Dd). FeCl3 treatment caused a loss of vascular undulation with a flattened and disconnected arrangement of smooth muscle cells. The internal elastic lamina remained intact even in severely damaged regions.
Saxatilin treatment did not cause any notable changes in blood flow at a dose of 1 mg/kg (2.36 ± 0.78%) or 1.75 mg/kg (5.14 ± 2.92%) compared to the normal saline group (2.42 ± 1.07%) (
Half-life of saxatilin, which was measured using saxatilin labeled with NHS-Rhodamine, was 4.1 minutes (
Fluorescence was measured from sera isolated at the indicated time-points (0, 5, 10, 20, 40, or 80 minutes) after administration of Rhodamine-labeled saxatilin. Half-life of saxatilin was 4.1 minutes in mice. Values are presented as a mean ± standard deviation.
Saxatilin at 5 mg/kg was effective in dose-response studies, so this dosage was used to determine the optimal method of intravenous administration of saxatilin (
Decreased thrombolytic effects were observed at different times according to administration method. Abrupt reocclusion was observed approximately 50 minutes after the first bolus injection in mice treated with double bolus injection, approximately 100 minutes after a total dose bolus injection, and approximately 110 minutes after a half-dose bolus injection with continuous infusion of the remaining half dose. Reocclusion was not observed with a bolus injection of 10% of the total dose and continuous infusion of the remaining dose (
Effective recanalization was not observed in mice treated with normal saline, 1 mg/kg saxatilin, or 1.75 mg/kg saxatilin. Only two of five mice treated with 2.5 mg/kg of saxatilin and three of five mice treated with 3.75 mg/kg of saxatilin showed effective recanalization. Effective recanalization was observed in all mice treated with saxatilin at 5 or 10 mg/kg (
Effective recanalization was defined as restoration of blood flow to at least 50% of baseline levels, maintained for longer than 30 minutes.
Effective recanalization was also evaluated according to saxatilin administration method. All mice treated with 5 mg/kg saxatilin achieved effective recanalization except for two mice administered a bolus injection of the total dose. Time to effective recanalization was 2.86 ± 0.22 minutes in mice treated with a bolus injection of the total dose, 13.44 ± 26.31 minutes in mice that received a double bolus injection, 19.48 ± 25.94 minutes in mice that received a half-dose bolus injection followed by continuous infusion of the other half dose, and 13.92 ± 6.02 minutes in mice treated with a bolus injection of 10% of the total dose with continuous infusion of the remaining dose (
Platelet aggregometry showed that saxatilin has a strong inhibitory effect on platelet aggregation (
Experiments using preformed thrombi showed dose-dependent disaggregating effects of saxatilin (
Both fibrin and fibrinogen zymograms showed clear bands of rt-PA. However, saxatilin did not demonstrate a clear fibrinolysis band, which suggested that saxatilin had no fibrinolytic activity (
We investigated the binding affinity of saxatilin-Fc to various integrins (
Dose-dependent effects of thrombolytic agents were determined by calculating the area under time-flow curves (
Dose-response curves of saxatilin (
Mean percentages of blood flow compared to baseline blood flow were 94.50 ± 20.47% in the group that received 5 mg/kg saxatilin at 1 hour; 17.71 ± 27.27% at 3 hours; 4.48 ± 1.37% at 6 hours; 10.67 ± 6.06% at 12 hours; and 4.51 ± 3.04% at 24 hours (
Thrombolytic effects of saxatilin were evaluated using 71 animals. Two animals had bleeding at the cervical incision site, and one died of bleeding approximately 90 minutes after a bolus injection of saxatilin. The two animals with bleeding complications had received a single bolus dose of 5 mg/kg. None of the mice in the other groups showed bleeding complications.
No significant differences in hematologic parameters were observed between normal saline-infusion group and 5 mg/kg saxatilin-infusion group (
Control | Saxatilin | P-value | |
---|---|---|---|
Red blood cell, ×106/mm3 | 6.97 ± 1.33 | 7.23 ± 0.53 | 0.465 |
Hemoglobin, g/dl | 11.15 ± 0.64 | 11.70 ± 0.57 | 0.346 |
Hematocrit, % | 46.20 ± 9.05 | 43.90 ± 5.37 | 0.917 |
Platelet, ×103/mm3 | 1114.00 ± 121.62 | 1132.00 ± 52.33 | 0.917 |
White blood cell, ×103/mm3 | 3.41 ± 0.90 | 1.40 ± 0.40 | 0.754 |
Neutrophil, % | 12.85 ± 14.35 | 13.25 ± 12.80 | 0.465 |
Lymphocyte, % | 80.80 ± 16.55 | 79.55 ± 15.77 | 0.465 |
Monocyte, % | 4.70 ± 3.11 | 4.85 ± 2.90 | 0.293 |
Eosinophil, % | 0.90 ± 0.85 | 2.00 ± 0.28 | 0.917 |
Basophil, % | 0.75 ± 0.07 | 0.35 ± 0.35 | 0.112 |
Values are presented as a mean ± standard deviation.
We demonstrated that saxatilin has strong thrombolytic effects
The findings of this study indicated that saxatilin had thrombolytic effects on platelets but not fibrin/fibrinogen. Saxatilin is an RGD-containing protein. RGD-containing proteins from snake venom are characterized by strong conservation of the tripeptide RGD and by disulfide bond arrangements. These structural features might be critical for potential inhibition of platelet adhesion/aggregation and antagonism of integrin-mediated adhesion [
Saxatilin appears to be safe because its effective dose was low and it rarely caused bleeding complications. In addition, saxatilin did not cause any notable changes in hematological parameters including platelet and neutrophil counts compared with control group. The most efficient dose in our study (5 mg/kg) was much lower than the known LD50 of saxatilin (400 mg/kg) in ICR mice [
We also determined the effect of saxatilin on aged thrombus. Thrombolytic effects of saxatilin decreased as thrombus age increased. Decreased thrombolytic effects in aged thrombi were also seen in rt-PA. While effects of thrombolytic drugs on aged thrombi have not well known, our findings suggest that effects of drugs acting on platelets as well as fibrinogen may be decreased as thrombus age increases and that reducing time from onset to treatment is important to enhance effects of the thrombolytic drugs.
Our methods had distinct features compared to other experimental models evaluating thrombolytic effects. First, to produce arterial thrombosis, we used a smaller filter paper saturated with a higher concentration (50%) of FeCl3 than previous studies (2.5–65%) [
Second, the assessment methods we used were different from previous studies in which recanalization is typically assessed to determine thrombolytic effects. Our study determined and calculated the degree of flow restoration on a minute-by-minute basis using ultrasound blood flow measurements. Histological examination showed that blood flow measurements were a good representation of the degree of recanalization and thrombus resolution. The assessment method we used provided quantitative data on degree of recanalization, time to effective recanalization, and real-time occurrence of reocclusion. Our method also enabled group comparisons as well as quantitative data on each animal. We found a dose-dependent thrombolytic effect for rt-PA. The effective dose of rt-PA in rodents is about 10-fold higher than for humans because of different PA systems [
Third, thrombolytic effects were determined using platelet aggregometry. Platelet aggregometry has been used to test inhibition of thrombus formation and is typically used to assess platelet anti-aggregating drugs. However, we used platelet aggregometry to assess platelet disaggregation after administering a thrombolytic drug to preformed thrombi. We found that platelet aggregometry could also be used to assess
In conclusion, disaggregation of platelets from fibrin is a potential approach to dissolving thrombi [
The authors thank our biostatistician Hye Sun Lee, MS For her statistical assistance.