α-Conotoxin Bt1.8 from Conus betulinus selectively inhibits α6/α3ß2ß3 and α3ß2 nicotinic acetylcholine receptor subtypes.

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3ß2ß3 and rα3ß2 nAChRs with an IC50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3ß2ß3 and hα3ß2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3ß2ß3 subtype compared to rα3ß2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3ß2ß3 and α3ß2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3ß2ß3 nAChRs.

A Conantokin Peptide Con-T[M8Q] Inhibits Morphine Dependence with High Potency and Low Side Effects.

N-methyl-D-aspartate receptor (NMDAR) antagonists have been found to be effective to inhibit morphine dependence. However, the discovery of the selective antagonist for NMDAR GluN2B with low side-effects still remains challenging. In the present study, we report a selective NMDAR GluN2B antagonist con-T[M8Q](a conantokin-T variant) that potently inhibits the naloxone-induced jumping and conditioned place preference of morphine-dependent mice at nmol/kg level, 100-fold higher than ifenprodil, a classical NMDAR NR2B antagonist. Con-T[M8Q] displays no significant impacts on coordinated locomotion function, spontaneous locomotor activity, and spatial memory mice motor function at the dose used. Further molecular mechanism experiments demonstrate that con-T[M8Q] effectively inhibited the transcription and expression levels of signaling molecules related to NMDAR NR2B subunit in hippocampus, including NR2B, p-NR2B, CaMKII-α, CaMKII-ß, CaMKIV, pERK, and c-fos. The high efficacy and low side effects of con-T[M8Q] make it a good lead compound for the treatment of opiate dependence and for the reduction of morphine usage.

Structure-Activity Analysis of N-Type Calcium Channel Inhibitor SO-3.

As an ω-conopeptide originally discovered from Conus striatus, SO-3 contains 25 amino acid residues and three disulfide bridges. Our previous study has shown that this peptide possesses potent analgesic activity in rodent pain models (mouse and rat), and it specifically inhibits an N-type calcium ion channel (Cav2.2). In the study presented here, we investigated the key amino acid residues for their inhibitory activity against Cav2.2 expressed in HEK 293 cells and analgesic activity in mice. To improve the inhibitory activity of SO-3, we also evaluated the effects of some amino acid residues derived from the corresponding residues of ω-peptide MVIIA, CVID, or GVIA. Our data reveal that Lys6, Ile11, and Asn14 are the important functional amino acid residues for SO-3. The replacement of some amino acid residues of SO-3 in loop 1 with the corresponding residues of CVID and GVIA improved the inhibitory activity of SO-3. The binding mode of Cav2.2 with SO-3 amino acids in loop 1 and loop 2 may be somewhat different from that of MVIIA. This study expanded our knowledge of the structure-activity relationship of ω-peptides and provided a new strategy for improving the potency of Cav2.2 inhibitors.

Cloning, Synthesis and Functional Characterization of a Novel α-Conotoxin Lt1.3.

α-Conotoxins (α-CTxs) are small peptides composed of 11 to 20 amino acid residues with two disulfide bridges. Most of them potently and selectively target nicotinic acetylcholine receptor (nAChR) subtypes, and a few were found to inhibit the GABAB receptor (GABABR)-coupled N-type calcium channels (Cav2.2). However, in all of α-CTxs targeting both receptors, the disulfide connectivity arrangement "C¹-C³, C²-C4" is present. In this work, a novel α4/7-CTx named Lt1.3 (GCCSHPACSGNNPYFC-NH2) was cloned from the venom ducts of Conus litteratus (C. litteratus) in the South China Sea. Lt1.3 was then chemically synthesized and two isomers with disulfide bridges "C¹-C³, C²-C4" and "C¹-C4, C²-C³" were found and functionally characterized. Electrophysiological experiments showed that Lt1.3 containing the common disulfide bridges "C¹-C³, C²-C4" potently and selectively inhibited α3ß2 nAChRs and not GABABR-coupled Cav2.2. Surprisingly, but the isomer with the disulfide bridges "C¹-C4, C²-C³" showed exactly the opposite inhibitory activity, inhibiting only GABABR-coupled Cav2.2 and not α3ß2 nAChRs. These findings expand the knowledge of the targets and selectivity of α-CTxs and provide a new structural motif to inhibit the GABABR-coupled Cav2.2.

A novel 4/6-type alpha-conotoxin ViIA selectively inhibits nAchR α3ß2 subtype.

Conotoxins (CTxs) are typically small peptides composed of 12-50 amino acid residues with 2-5 disulfide bridges. Most of them potently and selectively target a wide variety of ion channels and membrane receptors. They are highly valued as neuropharmacological probes and in pharmaceutical development. In this work, a novel α4/6-CTx named ViIA (RDCCSNPPCAHNNPDC-NH2) was identified from a cDNA library of the venom ducts of Conus virgo (C. virgo). ViIA was then synthesized chemically and its disulfide connectivity was identified as 'C(1)-C(3), C(2)-C(4)'. Its molecular targets were further assessed using two-electrode voltage clamping. The results indicated that ViIA selectively inhibited nicotinic acetylcholine receptor (nAChR) α3ß2 subtype with an IC50 of 845.5 nM, but did not target dorsal root ganglion sodium (Na(+))-, potassium (K(+))- or calcium (Ca(2+))-ion channels. Further structure-activity relationship analysis demonstrated that Arg(1) and His(11) but not Asp(2) were the functional residues. To the best of our knowledge, ViIA is the first 4/6 α-CTx that selectively inhibits nAChR α3ß2 subtype. This finding expands the knowledge of targets of α4/6-family CTxs.

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3ß2, α9α10 and α6/α3ß2ß3 Subtypes.

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I ß-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3ß2, α9α10 and α6/α3ß2ß3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3ß2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3ß2, α3ß4, α2ß4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

Characterization of a T-superfamily conotoxin TxVC from Conus textile that selectively targets neuronal nAChR subtypes.

T-superfamily conotoxins have a typical cysteine pattern of "CC-CC", and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH2) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes α4ß2 and α3ß2, with IC50 values of 343.4 and 1047.2nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in LßT2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR α4ß2, all demonstrated that the residues Ile(6) and Leu(14) were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to α4ß2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors.

A 4/8 Subtype α-Conotoxin Vt1.27 Inhibits N-Type Calcium Channels With Potent Anti-Allodynic Effect.

A novel 4/8 subtype α-conotoxin, Vt1.27 (NCCMFHTCPIDYSRFNC-NH2), was identified from Conus vitulinus in the South China Sea by RACE methods. The peptide was synthesized and structurally characterized. Similar to other α-conotoxins that target neuronal nicotinic acetylcholine receptor (nAChR) subtypes, Vt1.27 inhibited the rat α3ß2 nAChR subtype (IC50 = 1160 nM) and was inactive at voltage-gated sodium and potassium channels in rat sensory neurons. However, Vt1.27 inhibited high voltage-activated N-type (CaV2.2) calcium channels expressed in HEK293T cells with an IC50 of 398 nM. An alanine scan of the peptide showed that residues Phe5, Pro9, Ile10, and Ser13 contribute significantly to the inhibitory activity of Vt1.27. The molecular dockings indicate that Vt1.27 inhibits the transmembrane region of CaV2.2, which is different from that of ω-conotoxins. Furthermore, Vt1.27 exhibited potent anti-allodynic effect in rat partial sciatic nerve injury (PNL) and chronic constriction injury (CCI) pain models at 10 nmol/kg level with the intramuscular injection. The pain threshold elevation of Vt1.27 groups was higher than that of α-conotoxin Vc1.1 in CCI rat models. These findings expand our knowledge of targets of α-conotoxins and potentially provide a potent, anti-allodynic peptide for the treatment of neuropathic pain.

cDNA cloning of conotoxins with framework XII from several Conus species.

In our efforts for cloning novel I(2)-superfamily conotoxins using the signal peptide sequence, we identified a novel conotoxin Lt12.4 from Conus litteratus. This gene has a framework XII (-C-C-C-C-CC-C-C-), which is distinct from the cysteine pattern I(2)-superfamily conotoxin (-C-C-CC-CC-C-C-). Subsequently, we found the signal peptide sequence of Lt12.4 by 5'-RACE. Using this new sequence, we identified another five novel conotoxins with this cysteine pattern from four Conus species (Conus eburneus, Conus imperialis, Conus marmoreus, and C. litteratus). These novel conotoxins have the same cysteine pattern as the reported Gla-TxX and Gla-MII, and may contain Gla residues. Furthermore, they have the highly conserved signal peptide and hypervariable mature peptide sequences, and widely exist in Conus species. Therefore, it could be defined as a new superfamily of E-conotoxins.

Characterization and potential function of a novel pre-implantation embryo-specific RING finger protein: TRIML1.

Members of the super-class of zinc finger proteins are key regulators in early embryogenesis. Utilizing in silico mining of EST Databases for pre-implantation Embryo-Specific Zinc Finger Protein Genes, we characterized a novel zygotic mouse gene-tripartite motif family-like 1 (TRIML1), which expresses in embryo before implantation. Knocking down of TRIML1 resulted in the fewer cell number of blastocysts and failture to give rise to neonates after embryo transfer. The binding partner of TRIML1, Ubiquitin-specific protease 5 (USP5), was identified by yeast two-hybrid screening assay. The interaction was confirmed by GST pull-down and coimmunoprecipitation analysis. The role of TRIML1 in ubiquitin pathway during the development stage of mouse blastocyst was further discussed.

A mWAP-hLF hybrid gene locus gave extremely high level expression of human lactoferrin in the milk of transgenic mice.

The production of pharmaceuticals by current mammary gland bioreactor techniques is limited by the low expression level of foreign proteins. We describe here a novel method that solves this problem. A successive three-step gap-repair strategy was developed to replace the genomic coding sequence in mouse whey acidic protein (mWAP) gene locus with that of human lactoferrin (hLF) precisely from the start code to the end code. A 50-kb mWAP-hLF hybrid gene locus was constructed, and corresponding transgenic mice were generated. An extremely high-level expression of rhLF in the milk was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The expression level ranged from 16.7 to 29.8 g/l among five transgenic lines, as indicated by the ELISA assay. Importantly, the expressed rhLF maintained the same antibacterial activity as the native hLF. Our strategy can very likely also be used for the efficient expression of other valuable pharmaceutical proteins.

Targeting of N-Type Calcium Channels via GABAB-Receptor Activation by α-Conotoxin Vc1.1 Variants Displaying Improved Analgesic Activity.

α-Conotoxins exhibiting analgesic activity, such as Vc1.1, have been shown to inhibit α9α10 nicotinic acetylcholine receptors (nAChRs) and GABAB-receptor (GABABR) coupled N-type (CaV2.2) calcium channels. Here, we report two Vc1.1 variants, Vc1.1[N9R] and benzoyl-Vc1.1[N9R], that selectively inhibit CaV2.2 channels via GABABR activation but exhibit reduced inhibitory activity at α9α10 and other neuronal nAChR subtypes compared with Vc1.1. Surprisingly, the analgesic activity of Vc1.1[N9R] and benzoyl-Vc1.1[N9R] was more potent than that of Vc1.1 when tested in partial sciatic nerve ligation injury and chronic constriction injury models. Vc1.1[N9R] and benzoyl-Vc1.1[N9R] exhibited either similar or tenfold higher activity of GABABR-mediated CaV2.2 inhibition but no activity at CaV2.2 alone; however, the mechanism of increased analgesic activity is unknown. The effects on analgesic activity and α9α10 nAChR of other Vc1.1 variations at position 9 and the N-terminus were also determined. Our findings provide new insights for designing potent inhibitors for GABABR-coupled N-type (CaV2.2) calcium channels.

A novel α-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity.

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3ß4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5-5.0 µg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

Cloning, expression and functional characterization of a D-superfamily conotoxin Lt28.1 with previously undescribed cysteine pattern.

As a class of peptides with 10 cysteine residues (-C-CC-C-CC-C-C-C-C-), D-superfamily conotoxins (D-conotoxins) can specifically act on nicotinic acetylcholine receptors (nAChRs). According to the conserved signal peptides of D-conotoxins, seven D-conotoxin precursor sequences with a previously undescribed Cys arrangement (-C-C-C-CC-C-C-C-C-C-) were identified by PCR-RACE methodology in the present study. The alignment of sequences revealed that signal peptide regions were same as D-VxXXA from Conus vexillum, and their mature peptides were almost different from the D-conotoxins. Analyses of the evolutionary tree demonstrated that they had low homology to those reported conotoxins with 10 cysteine residues (less than 35%) and lied in a separate branch in the evolutionary tree. Furthermore, a previously undescribed D-superfamily conotoxin Lt28.1 was further expressed in Pichia pastoris and then functionally characterized. The results showed that the recombinant Lt28.1 targeted α9α10 nAChRs but not other nAChRs subtypes. These findings defined a new branch of D-superfamily and expanded our knowledge of targets and potential application of D-conotoxins.

[Characteristics and Influencing factors of Sperm Interaction with Exogenous DNA].

Exogenous DNA localization and the frequency of spermatozoa carrying exogenous DNA after sperm/DNA co-culture are key to a successful sperm mediated-gene transfer (SMGT). In the study, the characteristics and influencing factors of exogenous DNA uptake by spermatozoa were tested using digoxigenin (DIG) labeled DNA as trace. Results showed that goat spermatozoa could spontaneously take up exogenous DNA. The exogenous DNA was initially bound to the outer sperm membrane at postacrosomal region; subsequently party of the bound DNA was internalized into nucleus. There were considerable differences in the capability of spermatozoa from different donors to bind and internalize exogenous DNA. In 35 samples, binding rates (before DNase I digestion) and internalization rates (the positive rate after DNase I digestion) varied between 4.6%-62.4% and 2.1%-53.8%, respectively. For the spermatozoa from the same goat, the binding and internalization capacities were mostly inhibited by the seminal fluid. Compared to ejaculate sperm, the binding rate and internalization rate were increased three and five times in washed sperm cells, respectively. At the same time, capacitated spermatozoa also had lower exogenous DNA uptake (P<0.01). Dead spermatozoa did not complete the internalization process. The highest positive rate (before DNase I digestion) was found in membrane-broken spermatozoa as a result of freeze-thawing and this was independent of the sperm donors. These results suggest that selection of appropriate sperm donors and optimization of sperm processing procedures are the key steps for successful SMGT.

Molecular basis of toxicity of N-type calcium channel inhibitor MVIIA.

MVIIA (ziconotide) is a specific inhibitor of N-type calcium channel, Cav2.2. It is derived from Cone snail and currently used for the treatment of severe chronic pains in patients unresponsive to opioid therapy. However, MVIIA produces severe side-effects, including dizziness, nystagmus, somnolence, abnormal gait, and ataxia, that limit its wider application. We previously identified a novel inhibitor of Cav2.2, ω-conopeptide SO-3, which possesses similar structure and analgesic activity to MVIIA's. To investigate the key residues for MVIIA toxicity, MVIIA/SO-3 hybrids and MVIIA variants carrying mutations in its loop 2 were synthesized. The substitution of MVIIA's loop 1 with the loop 1 of SO-3 resulted in significantly reduced Cav2.2 binding activity in vitro; the replacement of MVIIA loop 2 by the loop 2 of SO-3 not only enhanced the peptide/Cav2.2 binding but also decreased its toxicity on goldfish, attenuated mouse tremor symptom, spontaneous locomotor activity, and coordinated locomotion function. Further mutation analysis and molecular calculation revealed that the toxicity of MVIIA mainly arose from Met(12) in the loop 2, and this residue inserts into a hydrophobic hole (Ile(300), Phe(302) and Leu(305)) located between repeats II and III of Cav2.2. The combinative mutations of the loop 2 of MVIIA or other ω-conopeptides may be used for future development of more effective Cav2.2 inhibitors with lower side effects.

Im10A, a short conopeptide isolated from Conus imperialis and possesses two highly concentrated disulfide bridges and analgesic activity.

In the present study, we isolated, synthesized and NMR structurally characterized a novel conopeptide Im10A consisting of 11 amino acids (NTICCEGCMCY-NH2) from Conus imperialis. Unlike other conopeptides with four cysteine residues, Im10A had only two residues in loop 1 and one residue in loop 2 (CC-loop1-C-loop2-C), which formed a stable disulfide connectivity "I-IV, II- III" (framework X) with a type I ß-turn. Interestingly, Im10A exhibited 50.7% analgesic activity on rat partial sciatic nerve ligation (PNL) at 2h after Im10A administration. However, 10µM Im10A exhibited no apparent effect on neuronal nicotinic acetylcholine receptor, and it did not target DRG voltage-dependent sodium, potassium and calcium ion channels and opioid receptor. To our knowledge, Im10A had the most concentrated disulfide bridges among conopeptides with four cysteine residues. This finding provided a new motif for the future development of biomimetic compounds.

Con-T[M8Q] potently attenuates the expression and development of morphine tolerance in mice.

As a variant of peptide conantokin-T (con-T), con-T[M8Q] is derived from the venom of Conus tulipa. Our previous study has demonstrated that con-T[M8Q] selectively targets N-methyl-d-aspartate receptor (NMDAR) NR2B subunit. In the present study, we determined the effects of con-T[M8Q] on the expression and development of morphine tolerance using hot plate test and acetic acid writhing test. Our results demonstrated that con-T[M8Q] could efficiently attenuate the expression and development of morphine analgesic tolerance in mice at low doses (5-20nmol/kg), and it exhibited more potent effects compared with ifenprodil, a typical small-molecule antagonist of NMDAR. In addition, low doses of con-T[M8Q] (5-20nmol/kg) did not cause drug resistance and apparent analgesic activity compared with morphine. Taken together, con-T[M8Q] could be a promising new candidate in attenuating morphine tolerance.

High-throughput mapping of brain-wide activity in awake and drug-responsive vertebrates.

The reconstruction of neural activity across complete neural circuits, or brain activity mapping, has great potential in both fundamental and translational neuroscience research. Larval zebrafish, a vertebrate model, has recently been demonstrated to be amenable to whole brain activity mapping in behaving animals. Here we demonstrate a microfluidic array system ("Fish-Trap") that enables high-throughput mapping of brain-wide activity in awake larval zebrafish. Unlike the commonly practiced larva-processing methods using a rigid gel or a capillary tube, which are laborious and time-consuming, the hydrodynamic design of our microfluidic chip allows automatic, gel-free, and anesthetic-free processing of tens of larvae for microscopic imaging with single-cell resolution. Notably, this system provides the capability to directly couple pharmaceutical stimuli with real-time recording of neural activity in a large number of animals, and the local and global effects of pharmacoactive drugs on the nervous system can be directly visualized and evaluated by analyzing drug-induced functional perturbation within or across different brain regions. Using this technology, we tested a set of neurotoxin peptides and obtained new insights into how to exploit neurotoxin derivatives as therapeutic agents. The novel and versatile "Fish-Trap" technology can be readily unitized to study other stimulus (optical, acoustic, or physical) associated functional brain circuits using similar experimental strategies.

A novel stearic acid-modified hirudin peptidomimetic with improved pharmacokinetic properties and anticoagulant activity.

A novel hirudin isoform 3 mimetic peptide, named peptide S2, has been prepared by introduction of a stearic acid modification. Peptide S2 exhibited superior inhibitory activity to hirulog-1 (Bivariludin) and showed significantly higher anticoagulant potency in vivo. Peptide S2 elevated the thrombin time, prothrombin time and activated partial thromboplastin time of rat and human plasma more efficiently than hirulog-1 and the unmodified form of peptide S2 (peptide 1). Furthermore, peptide S2 inhibited arterial thrombosis and inferior vena cava in rat model 8 h after administration, and was 10-fold more potent than hirulog-1 300 min after administration of 0.1 µmol/kg peptide. The enhanced antithrombotic activity could be attributed to its long half-life (T1/2 = 212.2 ± 58.4 min), which was 13.1 and 14.7-fold longer than those of hirulog-1 (T1/2 = 15.1 ± 1.3 min) and peptide 1 (T1/2 = 13.5 ± 2.6 min), respectively. Further enzymatic degradation and binding assay with human serum albumin (HSA) demonstrated that the longer duration time should be originated from the slowing of trypsin or thrombin-mediated degradation, as well as its binding to HSA. The improved pharmacokinetic properties observed for peptide S2 has made it a promising therapeutic agent for the treatment of thrombi-related diseases.