Super‐high procoagulant activity of gecko thrombin: A gift from sky dragon

Abstract Aims Gecko, the “sky dragon” named by Traditional Chinese Medicine, undergoes rapid coagulation and scarless regeneration following tail amputation in the natural ecology, providing a perfect opportunity to develop the efficient and safe drug for blood clotting. Here, gecko thrombin (gthrombin) was recombinantly prepared and comparatively studied on its procoagulant activity. Methods The 3D structure of gthrombin was constructed using the homology modeling method of I‐TASSER. The active gthrombin was prepared by the expression of gecko prethrombin‐2 in 293 T cells, followed by purification with Ni2+‐chelating column chromatography prior to activation by snake venom‐derived Ecarin. The enzymatic activities of gthrombin were assayed by hydrolysis of synthetic substrate S‐2238 and the fibrinogen clotting. The vulnerable nerve cells were used to evaluate the toxicity of gthrombin at molecular and cellular levels. Results The active recombinant gthrombin showed super‐high catalytic and fibrinogenolytic efficiency than those of human under different temperatures and pH conditions. In addition, gthrombin made nontoxic effects on the central nerve cells including neurons, contrary to those of mammalian counterparts, which contribute to neuronal damage, astrogliosis, and demyelination. Conclusions A super‐high activity but safe procoagulant candidate drug was identified from reptiles, which provided a promising perspective for clinical application in rapid blood clotting.


| INTRODUC TI ON
High efficient procoagulant drug is urgent for immediate blood clotting, as excessive hemorrhage caused by natural disaster, war, or several diseases often results in the death of the subjects. Several species of reptiles, if not all, have evolved a rapid coagulation mechanism that prevents them from excessive bleeding and infection, providing an alternative opportunity for the development of the efficient procoagulant drug. As lower amniotes, reptiles occupy an important evolutionary position in the phylogeny. It is estimated that more than 10,000 reptile species live in the world, being classified into four orders, i.e., Crocodilia, Testudines, Squamata, and Sphenodontia. 1 Some of them (Crocodilia) are in huge size that often fights for territory, preys, or even mating rights, while a part of them (Squamata) in tiny size becomes preys of other predators. The rapid blood clotting following injury makes them escape from the frequent danger of death. The blood clotting in vertebrates is carried out by cells (thrombocytes or platelets) and a series of thrombin-mediated protease reactions. Thrombin, a multifunctional serine protease, plays key roles in procoagulant pathways of hemostasis. 2 It cleaves fibrinogen into fibrin, which is stabilized by factor XIIIa-catalyzed cross-links. 3 Also, thrombin can accelerate coagulation by activation of factor V and factor VIII. 4,5 Such procoagulant mechanism of thrombin has been shown to be phylogenetically conserved across the vertebrates, though several factors such as FXI and/or FXII are absent from fish and chicken. 6,7 Conversely, thrombin is able to activate an anticoagulant pathway by binding to thrombomodulin on vascular endothelial cells to promote the activation of protein C. 2 The relevant enzymatic properties and physiological function of thrombin have been extensively documented in several vertebrates including fish, amphibian, birds, and mammals. 6,8 However, relevant evidences from reptiles are lacking.
Thrombin is proteolytically generated from its zymogen, prothrombin, by cleavage of factor Xa in the presence of factor Va, calcium ions, and phospholipid. 9 Prothrombin is biosynthesized by hepatocytes and circulates in the blood, containing a Gla domain, two kringle domains, and a protease domain. 10 Removal of the Gla domain and two kringle domains from prothrombin by factor Xa will result in the smallest single-chain precursor to α-thrombin, the prethrombin-2. This intermediate product can be prokaryotic and eukaryotic expressed in high yield for preparing active thrombin.
However, the catalytic efficacy of prothrombinase for prethrombin-2 is much lower than for prothrombin. 9 Ecarin, a snake venomderived protease isolated from Echis carinatus, has been found to have a specific and high activity in cleavage of prethrombin-2 to form thrombin. 9,11 In addition to facilitating hemostasis, thrombin is involved in the regulation of multiple physiological and pathological processes, such as embryonic development, wound healing, inflammation, atherosclerosis, sepsis, and cancer. 12 However, the protease is currently highlighted for its roles in the central nervous system (CNS), in which it mediates distinct neuronal and glial responses (cytoprotective or cytotoxic) through activation of G-protein coupled protease-activated receptors (PARs). 13,14 High concentration of thrombin is proved to be neurotoxic that can cause vascular disruption, 15 microglial activation, 16 synaptic dysfunction, 17 neuronal damage, 18 astrogliosis, and demyelination of CNS in pathology. 19 Many neurological diseases, such as acute ischemic stroke, 20 intracerebral hemorrhage, 21 Alzheimer's disease, 22 Parkinson's disease, 23 and multiple sclerosis, are linked with aberrant activation of thrombin.
Elevated expression of thrombin has been observed in the traumatic spinal cord of mice and contributes to functional decline. 19 While knockout of PAR-1 receptor in mice displays improved locomotor recovery and reduces signatures of inflammation and astrogliosis, suggesting the detrimental action of the serine protease in the damaged spinal cord. 19 However, the pathophysiological functions of reptilian thrombin following CNS insults remain elucidated.
The amniotic gecko, also named as sky dragon in Traditional Chinese Medicine, is able to autotomize its tail once being captured by the predators. The biopsy site rapidly develops a clot of blood for minimal bleeding, which will be lost in 8-14 days following wounding. The animal proceeds to regenerate the lost part of the tail by regrowth of new dermis, skeletal muscle, cartilage, and nerves. [24][25][26] Notably, wounding-induced activation of thrombin has no deleterious effects on the regenerating nerve tissues, contrary to those observed in mammals. Such distinct physiological property of gecko thrombin (gthrombin) has provided a promising perspective for developing a safe and efficient procoagulant drug in control of multiple types of acute and chronic hemorrhage, especially for the incidence of CNS bleeding. To quantify the enzymatic activity and assess the neurotoxic effects of gthrombin, we analyzed the characteristics of gecko prothrombin and then prepared the prethrombin-2 recombinant protein. Subsequently, we acquired the active gthrombin by Ecarin cleavage of prethrombin-2, followed by measurement of the enzymatic activity, as well as evaluation of its toxic effects on the vulnerable central nerve cells. Our study has revealed a super-high procoagulant candidate drug with nontoxicity, which has never been characterized before as far as we know.

| Gecko model
Adult Gekko japonicus was obtained from the Experimental Animal Center of Nantong University. They were fed mealworms ad libitum and were housed in an air-conditioned room with a controlled temperature (25-28°C) and saturated humidity. Anesthesia was induced by cooling the animals on ice prior to tail amputation. Amputation was performed at the sixth caudal vertebra, based on the special tissue structure present at that position, 25

| Cell culture and treatment
PC12 cells were grown in RPMI 1640 media (Invitrogen, Shanghai, China) supplemented with 5% horse serum, 10% (v/v) fetal bovine serum, 50 units/mL penicillin, and 50 μg/mL streptomycin at 37°C in a humidified incubator with 5% CO 2 . PC12 cells were switched to differentiating media (DM; RPMI 1640 with L-glutamine, 0.2% horse serum, and 100 unit penicillin/100 mg streptomycin) after 24 h. After serum starvation (in DM) for 18 h, cells were exposed to 10 μg/mL thrombin, and alternatively followed by treatment with 50 ng/mL NGF to induce differentiation. The culture of gecko oligodendrocyte cell line Gsn3, and astrocyte cell line Gsn1, was referred to the methods by Wang et al. 27 The cells were grown in DMEM supplemented with 10% fetal bovine serum at 30°C supplied with 5% CO 2 . The cells with 95% confluency were changed to serum-free DMEM and stimulated with 0, 5, and 10 μg/mL gthrombin for 24 h, respectively.

| Cloning and analysis of gecko prothrombin
Sequence of gPTM was annotated from genome sequence, which was deposited in the GenBank. 28 To obtain the full length of gPTM, the primers were designed according to the genome sequences. 28 Both 5′-RACE and 3′-RACE were performed using the SMARTer RACE 5′/3' Kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions.

| Sequence analysis and homology modeling
Comparison against the protein database was performed using the PSI-BLAST network server at the National Center for Biotechnology Information. Multiple protein sequences were aligned using the MegAlign program by the CLUSTAL method in the DNASTAR software package. Phylogenetic tree was constructed using the PHYML implementation of Maximum-Likelihood, with the GTR (CDS sequences) and JTT (protein sequences) substitution model.
The three-dimensional structure of gthrombin was generated by the I-TASSER suite based on the amino acid sequence (XP_015262498.1). 29 A total of five predicted models were produced by the I-TASSER, and the one with the highest C-score was chosen as the final model. The electrostatic potential maps of the proteins were computed using the APBS (Version 3.0). 30 The results were visualized using the PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC) and VMD (Version 1.9.3). 31

| Preparation of gecko prethrombin-2 recombinant protein
The open reading frame of gPre2 was amplified from cDNA using the elution buffer (20 mM Tris-HCl, 500 mM NaCl, and 500 mM imidazole, pH 7.9) following the manufacturer's instructions. The eluted fusion gPre2 was stored in the stock solution (20 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.5) at −20°C before before converted into thrombin. gPre2. The reaction mixture was applied to the Ni 2+ -chelating column for affinity chromatography, and the gthrombin was purified as per above description.

| Enzyme kinetics and fibrinogen-clotting assay of gthrombin and hthrombin
The enzymatic activity of gthrombin or human thrombin (hthrombin, Sigma, USA) was assayed by using synthetic substrate S-2238
After centrifugation at 13,000 r/min for 30 min at 4°C, 20 μg of total protein of each sample was loaded into a 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore Sigma, USA). The membrane was then blocked with 5% nonfat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h, followed by incubation with primary antibodies at 4°C overnight. A further reaction with the second antibody was performed at room temperature for 2 h, and the HRP activity was detected using enhanced chemiluminescence. The membrane was scanned with a ChemiDOC XRS+ Imager (Bio-Rad, Hercules, CA, USA). The data were analyzed using PDQuest 7.2.0 software (Bio-Rad). GAPDH was used as an internal control.

| Wound healing assay
Gsn1 cells were seeded into each well of a 12-well plate and grown to confluent monolayers. The cells were then starved in DMEM supplemented with 0.15 mg/mL of mitomycin C (Sigma, USA) for 12 h, followed by scratching to generate a standardized 500-mm wound.
The cells were incubated at 0-10 μg/mL gthrombin and allowed for further culture at 24 h. Closure of the wound was monitored and photographed at multiple sites. Representative images were captured and analyzed with Wimscratch Quantitative Wound Healing Image Analysis (Wimasis GmbH, Munich, Germany).

| Measurements of intracellular calcium concentration [Ca 2+ ] i
Calcium imaging was prepared as described previously. 20

| Statistical analysis
The statistical significance of the differences between groups was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc comparison test with SPSS 15.0 (SPSS, Chicago, IL, USA). Prior to statistical analyses, the data sets for each group were tested for normality of distribution using the Kolmogorov-Smirnov test. Statistical significance was set at p < 0.05.

| Characterization of gecko prothrombin (gPTH) and construction of three-dimensional modeling of gthrombin
To understand the procoagulant role of gthrombin, a gross observation was made to compare the natural hemostatic time between gecko and rat following their tail amputation. The preliminary timing of stop bleeding at wounding site demonstrated that gecko cost less time for hemostasis than the rat did ( Figure 1A). Therefore, the characteristics of gPTH sequences were analyzed by bioinformat- with that of snake, evolved a distinct characteristic by lack of thrombin cleavage site ( Figure S1).
As was previously reported, the absolute conservation of the residues that constitute the catalytic apparatus, His91, Asp147, and Ser251 (numbering from the amino terminus of the light chain), was seen in this alignment. 32 Structurally, these functional catalytic residues are located in an equatorial cleft, which is involved in the specificity of the binding substrates. 33 Several lines of evidence illustrate that thrombin is allosterically regulated by Na + , and the binding of Na + exhibits increased catalytic properties relative to the Na + -free form due to subtle structural changes. 34 To shed light on the flexibility of gthrombin in the attraction of Na + that is enzymatic activity-relevant, we compared three-dimensional structure (3D) and electrostatic potentials of gecko and human thrombin (hthrombin). The 3D modeling of gthrombin was constructed using the homology modeling method according to the procedure of I-TASSER. The hthrombin structures, PDB codes 4HZH, 35 1JWT, 36 and 1MKW, 37 were chosen as templates from the PDB library based on the structural similarity. Among the five predicted models, the one with a C-score value of 0.24 was selected as the final model, where the C-score was a confidence score for estimating the quality of predicted models by I-TASSER. The C-score was in a range between −5 and 2, and generally, a higher C-score value signifies a model with higher confidence. The predicted gthrombin structure was thus in good quality for further analysis. To address the electrostatic potentials of gthrombin and hthrombin, they were further computed and mapped on the solvent-accessible surfaces. By using the same range, the electrostatic potential distributions of the two proteins were investigated. As shown in Figure 2A,B, the red regions in the two proteins, corresponding to the negative electrostatic potentials, were around the Glu residues (Glu248 and Glu251 for gecko and human, respectively). Intriguingly, gthrombin showed a lower electrostatic potential than that of the hthrombin, which may lead to more potent activity.
To gain an insight into the evolutionary relationships of reptile thrombin, we analyzed sequences of prethrombin-2 from representative reptiles including turtles, crocodiles, snakes, and lizards. The phylogenetic tree demonstrates that gthrombin is evolutionarily primitive comparing with those of other reptiles ( Figure 2C), indicating an ancient physiological role of gthrombin during the evolution of reptiles.

| Preparation of recombinant gthrombin
The purified recombinant gPre2 was subsequently subjected to activation by the addition of different concentrations of snake venomderived Ecarin. At the ratio of 0.05 U of Ecarin to 3 μg of gPre2, the gPre2 was completely cleaved ( Figure 2H). The gthrombin produced F I G U R E 1 Gross observation of gecko wounding hemostasis and analysis of amino acid sequence of gecko prothrombin. (A) Comparison of gecko and rat natural hemostatic time following tail amputation. The tail of gecko was amputated above 6th, while the rat at 9-11th caudal vertebra. (B) Multiple alignment of amino acid sequences of gecko prothrombin with those of human and mouse. Each residue in the alignment is assigned a color if the amino acid profile of the alignment at that position meets some minimum criteria specific for the residue type. Gaps introduced into sequences to optimize alignment are represented by dashes. The Gla domain, kringle I domain, kringle II domain, and trypsin domain are indicated by dot line, line, or double line, respectively. The potential cleavage sites by coagulation factor Xa and thrombin are boxed and indicated by the black arrowhead. The conserved catalytic residues, His91, Asp147, and Ser251 (numbering from the amino terminus of the light chain), are indicated by the red arrowhead. The Glu248 for gecko and Glu251 for human, around which the negative electrostatic potentials are analyzed, are indicated by the asterisks. Prothrombin sequences of gecko (XP_015262498), human (AAC63054), and mouse (NP_034298) are obtained from GenBank.
in an enlarged scale was highly purified by Ni 2+ -chelating affinity chromatography, and was preserved in the 0.01 M PBS at a final concentration of 0.284 mg/mL ( Figure 2I). An unknown product was simultaneously detected in the samples, which was electrophoretically collected for mass spectrometry analysis ( Figure 2I). Results demonstrated that the unidentified band was derived from degradation of gthrombin ( Figure S2).

| The gthrombin exhibits higher enzymatic activities than those of hthrombin
To analyze the enzyme kinetics of gthrombin, the enzymatic activities of gthrombin at different pH values or temperatures were assayed by hydrolysis of synthetic substrate S-2238. As the pH value of tissue fluid following gecko tail amputation is near neutral ( Figure 3A), the enzymatic parameters of gthrombin at 4, 30, and 37°C were firstly determined at pH 7.0. As shown in Figure 3B, the maximal enzymatic activity (kcat/Km) of gthrombin at pH 7.0 was observed at 37°C, which was significantly higher than that of human.
However, the highest catalytic efficiency of gthrombin was detected at alkalescency condition ( Figure 3B). The results indicate that the optimal condition for gthrombin action is in weak alkalinity at 37°C. maximum turbidity. Results showed that the clotting time of fibrinogen by gthrombin was significantly shorter than that by hthrombin ( Figure 3C-E). The data indicate that gthrombin has higher enzymatic activities in clot formation than those of hthrombin.

| Nontoxic effects of gthrombin on neuronal differentiation and elongation of neurites
Thrombin has been shown to affect neuronal growth and intracellular Ca 2+ homeostasis. 38 To address the potential effects of gthrombin on the cellular events of neurons, we cultured PC12 cells, the cell line derived from rat pheochromocytoma with physiological and biochemical functions close to those of neurons, and examined their differentiation and elongation of neurites following cell treatment with gthrombin. Results showed that the addition of 10 μg/mL gthrombin in the serum-free medium did not significantly induce the differentiation of PC12 cells ( Figure 4A,B). When the cells were stimulated with 10 μg/mL gthrombin for 24 h, followed by inducing cell differentiation with 50 ng/mL NGF for 48 h, the total length of neurites showed an undetectable difference comparing with the control, indicating that gthrombin is inefficient in mediating the differentiation of neurons ( Figure 4C,D,G). Similarly, treatment of 10 μg/mL gthrombin on differentiated neurons for 24 h did not impact the elongation of neurites ( Figure 4E,F,H). However, the parallel experiments on equivalent hthrombin demonstrated that the human-derived serine protease was able to significantly inhibit the differentiation of PC12 cells induced by NGF, though had undetectable effects on the neurite length of the differentiated neurons ( Figure 4I-P). The data indicate nontoxic effects of gthrombin on neurons in different developmental stages.
Thrombin induces a transitory dose-dependent increase in intracellular free calcium concentration that influences neuronal function. 39 To understand the effects of gthrombin on the concentration of intracellular Ca 2+ , the differentiated PC12 cells were treated with 5 μM Fluo4-AM for 30 min, followed by exposure to 10 μg/mL gthrombin. The amplitude of calcium response was measured spectrophotometrically at every 5 s. Results showed that gthrombin failed to change the intracellular Ca 2+ concentration at all times recorded ( Figure 4Q,I).

| The gthrombin cannot induce the astroglial reaction of gecko
Thrombin has been found to induce astroglial reaction via activation of PARs that are either cytoprotective or cytotoxic. 13,40 To examine the effects of gthrombin on the cell events of astrocytes from gecko spinal cord, the astrocyte cell line (Gsn1) was treated with 0-10 μg/mL gthrombin for 24 h. The results demonstrated that the gthrombin neither influenced the cell proliferation measured by EdU assay ( Figure 5A,B), nor the migration of the astrocytes detected by wound scratching (Figure 5C,D). Western blot analysis revealed that GFAP protein levels in astrocytes were unaffected by the stimulation of gthrombin ( Figure 5E). Contrarily, the hthrombin was able to promote the proliferative response of astrocytes ( Figure 5F,G), in consistency with those of other mammals. 40 The data indicate that the gthrombin is not able to induce the astroglial reaction of gecko. F I G U R E 3 Measurement of enzymatic activity of gthrombin and hthrombin. (A) Examination of tissue fluid pH value following gecko tail amputation. The pH value of the sample was indicated at pH 7.0. (B) The enzymatic activity (kcat/ Km) of gthrombin and hthrombin was assayed using synthetic substrate S-2238 at 4, 30, and 37°C at pH 7.0, and pH 6.0, 7.0, and 8.0 at 37°C, respectively. (C and D) Turbidity assays on human fibrin clots at 10 mg/mL fibrinogen with 1 μg gthrombin (C) and hthrombin (D) in every 2 min following initiation. (E) Comparative analysis of clotting time between gthrombin and hthrombin. The clotting time was determined from the end of the clot lag period to 90% maximum turbidity. All assays were carried out in triplicate. Data are represented as mean ± SEM (p < 0.01).

| High dose of gthrombin has unfavorable effects on oligodendrocyte function through inhibiting phosphorylation of ERK1/2
Thrombin receptor PAR-1 has been assumed to be a key suppressor of developmental myelination through regulation of ERK1/2 and AKT signaling. 41 To test the effects of gthrombin on the oligodendrocytes, gecko oligodendrocyte cell line Gsn3 was incubated in the serum-free medium containing 0-10 μg/mL of gthrombin for 24 h. An assay of CCK-8 revealed that gthrombin at a dose of 10 μg/mL significantly decreased the cell viability ( Figure S3). The serine protease at a dose of 10 μg/mL also reduced the proliferation of Gsn3, as well as the elongation of processes ( Figure 6A-D

| DISCUSS ION
Several species of reptiles undergo many times of appendage loss or body injury over their lifetimes and exhibit differential regenerative ability. 42,43 They have evolved a self-defense mechanism to protect them from lethal infection by rapid hemostasis and wound healing. A high activity of thrombin is therefore essential for blood coagulation in diverse ecological niches. Many species of lizard including geckos are able to repeatedly regenerate the lost tail in order to adapt their ecological environment. 43 Unveiling the unique physiological properties of thrombin from these amazing animals will speed up the progress in the development of the procoagulant drugs applicable in chronic hemorrhage. In the present study, we demonstrated that the gthrombin manifested a higher enzymatic activity comparing with that of hthrombin under various conditions, suggesting an adaptive evolution of the reptile in response to the predation pressures. Interestingly, thrombin of the poikilothermal gecko was able to maintain high enzymatic activity at different temperatures, indicating the biological significance of the gthrombin in blood clotting at diverse ecology. Construction of 3D modeling predicted that gthrombin had a lower electrostatic potential around Glu251 than that of the hthrombin, a property favorable for enhanced catalytic activity by binding of Na + . However, the conclusion remains further clarified by overall screening those of other reptilian thrombin proteins.
In addition to the central role in hemostasis, thrombin also contributes to the neuropathological progression of CNS, especially in the brain. 44 The serine protease is produced immediately after hemorrhage or breakdown of blood-brain (spinal cord) barrier.
Meanwhile, the parenchymal cells in the CNS are also important producers in response to the injury. [45][46][47] A low concentration of thrombin (from 50 pM to 100 nM) has been proposed to be neuroprotective by inducing intracellular Ca 2+ spikes. 45,48 However, the serine protease will cause neuronal cell death by inducing a sustained Ca 2+ elevation at the high concentration. 47,49 In the present study, we determined the viability of PC12 cells incubated at 0-20 μg/mL (equivalent to 584 nM) of gthrombin, which showed nontoxic effects on the cells.
The 10 μg/mL of gthrombin also did not impact on the neuronal differentiation, elongation of neurites, and alteration of intracellular Ca 2+ , suggesting a unique physiological property of gthrombin in the balance of hemostasis and regeneration of CNS.
Astrocytes are one of the predominant cell types in the CNS that express PAR-1, PAR-3, and PAR-4 receptors. 50 Thrombin induces morphological changes and proliferative reaction of astrocytes through proteolytic activation of PAR-1 signaling. 13,51,52 These astroglial reactions responding to thrombin stimulation are associated with the formation of glial scar, which will impede functional recovery of the injured CNS in the mammals. 13 It is generally recognized that the "real" stellate astrocytes are only present in F I G U R E 5 Effects of gthrombin on the cellular events of gecko astrocytes. (A) EdU assay of gecko astrocyte cell line Gsn1 following treatment with 0-10 μg/ mL gthrombin for 24 h. (B) Statistical analysis of (A). (C) Wound healing assay of Gsn1 following 0-10 μg/mL of gthrombin treatment for 24 h. The cells were pretreated with 0.15 mg/mL of mitomycin C for 12 h before wound scratching. (D) Statistical analysis of (C). (E) Western blot analysis of GFAP after Gsn1 was treated with 0-10 μg/mL gthrombin for 24 h. (F) EdU assay of Gsn1 following treatment with 0-10 μg/mL hthrombin for 24 h. (G) Statistical analysis of (F). Data are represented as mean ± SEM (p < 0.05). Scale bars, 750 μm in (A); 200 μm in (F).
amniotes, 53 despite several debates. 54 We have previously demonstrated that gecko can regenerate the spinal cord following injury without evoking astrocytic responses. 55,56 Here, we also displayed that gthrombin was inefficient in regulating cellular events of astrocytes, recapitulating the in vivo observation in the severed spinal cord of gecko.
Thrombin-conveyed activation of PAR-1 generates profound effects on myelination of the CNS. Deletion of PAR-1 has been found to promote differentiation of oligodendrocyte progenitor cells (OPCs), onset of axon ensheathment, and myelin thickness in adult mouse, 41,57 indicating that thrombin is a negative regulator of myelination following CNS injury. We demonstrated that 10 μg/mL of gthrombin significantly decreased the proliferation and elongation of oligodendrocyte processes through ERK signaling, suggesting the conserved function of the serine protease in negative regulation of the myelination during the amniotic evolution. It is noteworthy that gecko can spontaneously regenerate the spinal cord composed of ependymal lining and descending axons without associated DRGs following tail amputation. 24,26,58 Amputation-induced activation of the gthrombin does not influence the regenerating cord of the gecko. The most possibility attributes to limited bleeding controlled by muscle contracting at a unique fracture plane. 59 It is estimated that 1 mL of whole blood can only produce about 1-2 nM of thrombin, 48 which is insufficient in affecting myelination of the oligodendrocytes. There are several concerns regarding the adverse effects of gthrombin on the CNS myelin, which impede its potential application as a procoagulant drug. In the present study, we showed that gthrombin at a dose of 5 μg/mL (146 nM) equivalent to at least 70 mL of whole blood, 48 made no effects on the cell events of oligodendrocyte, suggesting the less toxicity of the serine protease on the CNS myelin. However, a long-term in vivo experiment of gthrombin is indispensable for evaluating its exact impact on myelination under physiological and pathological conditions.
In conclusion, the active thrombin has successfully been prepared from gecko, which exhibits super-high procoagulant activity under various temperature and pH conditions. The gthrombin has evolved to the low neurotoxicity on the vulnerable central nerve cells so as to promote hemostasis with safety.

AUTH O R CO NTR I B UTI O N S
Yongjun Wang designed this work. Yongjun Wang wrote the paper.