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YY-39, a tick anti-thrombosis peptide containing RGD domain
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YY-39, a tick anti-thrombosis peptide containing RGD domain Jing Tang a,1 , Yaqun Fang a,1 , Yajun Han a , Xuewei Bai a , Xiuwen Yan a , Yun Zhang b , Ren Lai a,b,∗ , Zhiye Zhang b,c,∗∗ a
Life Sciences College of Nanjing Agricultural University, 1st Weigang, Nanjing, Jiangsu 210095, China Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China c University of Chinese Academy of Sciences, Beijing 100009, China b
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Article history: Received 29 June 2014 Received in revised form 14 August 2014 Accepted 14 August 2014 Available online xxx Keywords: Tick Salivary gland Platelet inhibitor Anti-thrombosis Glycoprotein IIb/IIIa
a b s t r a c t Ticks are obligatory blood feeding ectoparasites, which continuously attach to their hosts for 1–2 weeks. There are many biologically active compounds in tick salivary glands interfering host haemostatic system and to successfully obtain blood meal. Several platelet aggregation inhibitors have been identified from ticks. A family of conserved peptides, which were identified from transcriptome analysis of many tick salivary glands, were found to contain unique primary structure including predicted mature peptides of 39–47 amino acid residues in length and a Pro/Glu(P/E)-Pro/His(P/H)-Lys-Gly-Asp(RGD) domain. Given their unique structure and RGD domain, they are considered a novel family of disintegrins that inhibit platelet aggregation. One of them (YY-39) was tested for its effects on platelets and thrombosis in vivo. YY-39 was found effectively to inhibit platelet aggregation induced by adenosine diphosphate (ADP), thrombin and thromboxane A2 (TXA2). Furthermore, YY-39 blocked platelet adhesion to soluble collagen and bound to purified GPIIb/IIIa in a dose-dependent manner. In in vivo experiments, YY-39 reduced thrombus weight effectively in a rat arteriovenous shunt model and inhibited thrombosis in a carrageenan-induced mouse tail thrombosis model. Combined with their prevalence in ticks and platelet inhibitory functions, this family of peptides might be conserved tick anti-haemostatic molecules. © 2014 Elsevier Inc. All rights reserved.
Introduction Blood-feeding arthropods have developed effective strategies to get blood meal. To combat vertebrate hemostasis, inflammation and other defense systems, ticks have evolved a variety of mechanisms, such as anti-hemostatic, anti-inflammatory, immunoregulation, vasodilation and other factors in their salivary glands [1,24,33]. Platelet aggregation plays key role in hemostatic responses, particularly in small wounds [30]. Several groups of compounds that inhibit platelet aggregation have been identified from tick salivary glands [9]. They include apyrases
∗ Corresponding author at: Life Sciences College of Nanjing Agricultural University, 1st Weigang, Nanjing, Jiangsu 210095, China. Tel.: +86 25 84396849; fax: +86 25 84396849. ∗∗ Corresponding author at: Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China. Tel.: +86 871 651 962 02; fax: +86 871 651 990 86. E-mail addresses: [email protected] (R. Lai), [email protected] (Z. Zhang). 1 These authors contributed equally to this work.
(ATP-diphosphohydrolase EC 3.6.1.5) [17,28], thromboxane A2 (TXA2)-binding proteins [15,20,32], thrombin inhibitors [16], and disintegrins [12,13,19,20,31]. At least three families of disintegrins have been identified and characterized from tick salivary glands. The first one is tick adhesion inhibitor (TAI), which is a 15-kDa sialogenin purified from the Ornithodoros moubata salivary gland, acting as an antagonist of integrin ␣2 1 [13]. It inhibits platelet adhesion to soluble collagen under static conditions with an IC50 of 8 nM. The second one is variabilin, which is identified from Dermacentor variabilis. Variabilin is a 5-kDa RGD-containing disintegrin containing 4 cysteine residues and can inhibit ADP-induced platelet aggregation with an IC50 of 150 nM [31]. The third one includes disagregin from O. moubata, which lacks RGD motifs, savignygrin from Ornithodorus savignyi [19], and monogrin from Argas monolakensis [20], which contain an RGD motif. Sequence alignment indicated that disagregin, savignygrin, and monogrin belong to the same family of peptides containing 6 conserved cysteines and they share conserved amino acid sequences [9]. Transcriptome analysis of the hard ticks Ixodes pacificus and Ixodes scapularis revealed a possible novel disintegrin family (ixodegrins), which displays sequence similarity to variabilin, including RGD position, but have two additional cysteines. The
http://dx.doi.org/10.1016/j.peptides.2014.08.008 0196-9781/© 2014 Elsevier Inc. All rights reserved.
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functions of ixodegrins remain to be studied [10]. In this study, one of ixodegrins (YY-39) was synthesized and refolded for further functional investigation. YY-39 was found to contain function to inhibit platelet aggregation by blocking glycoprotein IIb/IIIa.
Materials and methods Sequence identification of YY-39 Using the sequence of ixodegrin (YSFTCERIPCTNNSDCHGSDLCQCRPPRGDGFSYFCSEY) reported by Francischetti et al. [10]. BLAST search was performed. Similar sequences containing conserved RGD and cysteine motif were extracted.
Synthesis and re-folding of YY-39 YY-39 (YSSTCERIPCTNNSDCHGPDLCQCRPPRGDDFGYFCSEY, the sequence was obtained from GenBank: AAY66649.1) (Fig. 1) was synthesized by GL Biochem Ltd. (Shanghai, China) and analyzed by high performance liquid chromatography (HPLC) and mass spectrometry to confirm purity higher than 98%. The synthesized linear peptide was incubated in 0.1 M Tris–HCl, 0.1 M NaCl, 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione(GSSH), pH 9.0 for 24 h at 25 ◦ C to fold the peptide.
Platelet aggregation inhibition assays According to our previous method [18], human platelet-rich plasma (PRP) was used in platelet aggregation assays and prepared as follows: human blood from a healthy donor was centrifuged at 160 × g for 10 min at 22 ◦ C, and the PRP was decanted. After successive washes in Tyrode–protein buffer (137 mM NaCl, 2 mM KCl, 0.3 mM NaH2 PO4 , 12 mM NaHCO3 , 5.5 mM glucose, 0.35% BSA, 1 mM MgCl2 , and pH 7.4), the pelleted platelets were diluted to 2 × 105 platelets/l in Tyrode–protein buffer, and counted with a thrombocounter. Platelet aggregation was monitored by light transmission in an aggregometer (Plisen, Beijing, China) in which 300 l aliquots of PRP were incubated at 37 ◦ C with continuous stirring. Light transmission was recorded and ADP (5 M), thrombin (0.45 U/ml) or U46619 (a TXA2 analog, 10 M) were added to initiate platelet aggregation. The inhibition of platelet aggregation was measured at the maximum aggregation response.
Interaction assays between YY-39 and platelet GPIIb/IIIa by ELISA The assay was performed according to the following protocol: microtiter plates were coated with increasing concentrations of YY-39, fibrinogen and bovine serum albumin (BSA) diluted in 0.1 M sodium carbonate, pH 9.5 at 4 ◦ C overnight. After washing the microplate for 5 times with 300 l wash solution (0.1 M phosphate-buffered saline with 1% Tween-20), the microplate was then blocked by incubation with 200 l blocking solution (1% BSA in wash solution) for 1 h at 37 ◦ C. The microplate was washed, followed immediately by addition of purified GPIIb/IIIa (5 g/ml) in blocking solution. A mouse anti-GPIIIa (CD61) antibody (1:2000, Millipore Chemicon, USA) was used to detect GPIIb/IIIa, and a second anti-mouse IgG antibody (1: 2000, horseradish peroxidase (HRP) labeled, KPL, USA) was then used. A final wash was performed and color development was then carried out with 3,3 ,5, 5 -tetramethylbenzidine (TMB), the plates were incubated until color developed. The reaction was stopped with 0.5 M H2 SO4 and the absorbance at 450 nm was measured. Platelet adhesion assay under static conditions The inhibition of platelet adhesion assay was performed using a modification of a published method [4]. Briefly, coverslips were coated with 20 g/ml of soluble non-fibrillar collagen type I (BD Bioscience, San José, CA, USA) overnight at 4 ◦ C. After washing three times with 200 l of PBS, the coverslips were blocked with 200 l of PBS containing 2% BSA for 1 h. 200 l of platelets (2 × 105 /ml) with YY-39 (0–39.3 M) were then applied to coverslips and incubated for 45 min at room temperature. Non-adherent platelets were removed by washing the coverslips three times with PBS. Differential interference contrast images were recorded using a Nikon Eclipse 80i microscope (Nikon, Japan). Extent of platelet adhesion was expressed as percent area covered by platelets. All experiments were performed in quintuplicates. Rat arteriovenous shunt thrombosis model According to the method described previously by Sperzel [27], Wistar rats (180–220 g) were used for arteriovenous shunt thrombosis model. The animals were anesthetized by intraperitoneal (i.p.) administration of ketamine (50 mg/kg) and xylazine (15 mg/kg). The right common carotid artery and left jugular vein were isolated and cannulated with two 100-mm-long, salinefilled catheters (PE-60, Becton Dickinson, Sparks, MD, USA). A
Fig. 1. Structural characteristics comparison. (A) Comparison of the sequence of YY-39 with other similar sequences by BLAST search. The sequence of YY-39 was underlined, and the conserved RGD domain of each sequence was boxed. The corresponding GenBank Number was indicated. (B) Comparison of the sequence of YY-39 with other disintegrins from tick salivary glands. The conserved RGD domain in each sequence was boxed.
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30-mm-long polyethylene tube (PE-160, Sparks, USA) was used to connect the two PE-60 catheters, and a rough nylon thread (60 mm in length and 0.26 mm in diameter) contained in the PE-160 tube and folded into a double string to induce thrombosis. The femoral vein was exposed and cannulated with a saline-filled polyethylene catheter for sample administration (n = 9 per dose). After 15 min administration of sample, the shunt was opened for 5 min and then closed. The nylon strings were then withdrawn from the middle PE-160 catheter and the wet thrombi attached in the nylon strings were weighed.
male) were used in this assay. The samples or control saline were injected intravenously into the animals (n = 6). After the injection for 2 h, a 2 mm distal segment of the tail was severed with a razor blade. The tail was immediately immersed in 37 ◦ C saline with the tip of the tail 5 cm below the body. The bleeding time was recorded for the stream of blood to cease. All the experimental protocols of animal models were approved by the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences. The approval ID for this study was syxk2014-0007.
Carrageenan-induced mouse tail thrombosis model
Statistical analysis
The procedure was carried out according to the reported method [3] and male Kunming mice were used in this model. YY-39 or ozagrel were injected intravenously into the animals (n = 9, 18–20 g). 30 min later, the mice were injected intraperitoneally with 40 l (1%) -Carrageenan (type I, Sigma) dissolved in 0.9% NaCl to induce thrombosis in tail. Six hours after the injection of -Carrageenan, the tested samples were injected into mice by caudal vein again, and the length of thrombus in tails of mice was measured after 48 h treatment.
Data were assessed for statistical significance using Student’s (unpaired) t-test. Results were reported as mean ± SE with significance accepted at P < 0.05.
Bleeding evaluation Bleeding time was assessed following the protocol described in the tail transaction method [7,29], and Kunming mice (18–20 g,
Results Structural characteristics comparison Comparison of YY-39 with several other similar sequences extracted by BLAST search was illustrated in Fig. 1A, which displayed sequence similarities including a RGD sequence at the N-terminus of the whole peptide and 6 cysteines. There are 5 cysteine residues upstream of their RGD sequence and 1 downstream of the sequence. In addition, the distribution of these cysteines
Fig. 2. YY-39 inhibited human platelet aggregation induced by ADP (A), thrombin (B), and U46619 (C). The tracings represent a typical experiment, and the experiments were repeated 3 times.
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Fig. 3. Interactions of YY-39 with two platelet receptors GPIIb/IIIa and integrin ␣2 1 . (A) Binding of GPIIb/IIIa to immobilized YY-39. Fibrinogen and BSA were used as a binding control. Data represent mean ± SE of three individual experiments. (B) YY-39 prevented platelet adhesion to soluble collagen under static condition. (C) Dose-responses curve for YY-39-mediated inhibition of platelet adhesion to soluble collagen. Typical experiments were shown, data represent mean ± SE of three individual experiments.
in their sequences is similar, suggesting they may share a similar three-dimensional structure stabilized by disulfide bridges. The sequence comparison of YY-39 with other platelet aggregation inhibitors such as variabilin [10], disagregin, savignygrin [19], and monogrin [20] were also presented in Fig. 1B.
Inhibitory effect of YY-39 on platelet aggregation Three agonists were used to induce platelet aggregation for investigating the effects of YY-39 on platelet aggregation. As illustrated in Fig. 2, YY-39 dose-dependently inhibited platelet aggregation induced by ADP (Fig. 2A), thrombin (Fig. 2B), and U46619 (Fig. 2C), and the aggregations were completely inhibited at concentrations of 15 M, 3.76 M, and 12 M, respectively.
YY-37 bound to platelet GPIIb/IIIa The direct interaction of YY-39 with platelet GPIIb/IIIa was tested using a solid-phase assay. As illustrated in Fig. 3A, immobilized YY-39 dose-dependently bound to purified platelet GPIIb/IIIa. Additionally, at a dosage of 62.5 ng per well or above, the binding of YY-39 to GPIIb/IIIa was comparable to that of fibrinogen.
YY-37 inhibited platelet adhesion to soluble collagen under static condition Integrin ␣2 1 is known to mediate adhesion for platelet to collagen, to investigate whether YY-39 blocks integrin ␣2 1 –collagen interaction, platelets were added to coverslips coated with soluble collagen. As showed in Fig. 3B, YY-39 dose-dependently inhibited platelet deposition to soluble collagen with an IC50 ≈ 20 M (Fig. 3C).
YY-39 inhibited thrombus formation in vivo Anti-thrombosis ability of YY-39 was evaluated in rat arteriovenous shunt thrombosis model and carrageenan-induced mouse tail thrombosis model, and ozagrel, which is a clinical anti-thrombosis medicine acting as a selective thromboxane A2 synthetase inhibitor [14], was used as a positive control. As illustrated in Fig. 4A, both YY-39 and ozagrel significantly inhibited thrombosis in a dosedependent manner, while the anti-thrombosis ability of YY-39 was obviously better than ozagrel. The thrombus weight was 4.3 ± 0.4 in the control (0 nmol/kg), while after administration of YY-39 by 50 and 100 nmol/kg, the thrombus weight was reduced to 2.8 ± 0.3 and 1.6 ± 0.5 mg, respectively. However, when given the corresponding dosages of ozagrel, the thrombus weight was reduced to 3.5 ± 0.5 and 2.6 ± 0.4 mg, respectively. In carrageenan-induced mouse tail thrombosis model, YY-39 unambiguously inhibited thrombosis in a dose-dependent manner (Fig. 4B). At the dosages of 65 and 130 nmol/kg, thrombus formation was inhibited by 31.7% and 71.4%, respectively. When given the same dosages, ozagrel inhibited thrombus formation only by 18.7% and 46.9%, respectively. Bleeding evaluation As illustrated in Fig. 5, the average bleeding time was 2.5 min in the control group (saline), while the bleeding time was prolonged to 4 and 5.5 min when the animals were administered with YY39 at dosages of 0.17 and 0.6 mol/kg body weight. However, at dosage of 0.6 mol/kg body weight, ozagrel strikingly prolonged the bleeding time to 6.8 min. Discussion Hemostasis is a dynamic process of thrombus formation and fibrinolysis. The biological processes of platelet adhesion, activation and aggregation play pivotal role in the process of primary
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Fig. 4. Antithrombotic activities of YY-39 in vivo. (A) Antithrombotic effects of YY39 and ozagrel in the rat arteriovenous shunt thrombosis model. (B) Effects of YY-39 and ozagrel on thrombus formation induced by carrageenan in mouse tail. Data represent mean ± SE of nine individual experiments. The values for YY-39 or ozagrel-treated group were significant different from the values for the saline group (*P < 0.05, **P < 0.001).
hemostasis. At sites of vascular injury, collagen initiates recruitment of circulating platelets and triggers platelet activation cascade required to stimulate thrombus growth [8,22,23]. The contributions of two collagen platelet receptors-glycoprotein VI (GPVI) and integrin ␣2 1 to collagen-mediated platelet responses have been highlighted in platelet adhesion and activation, and several different models have been proposed in an attempt to explain how
Fig. 5. Bleeding time induced by intravenous injection of vasotab TY and ozagrel in vivo. Data represent of six individual experiments.
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platelets are activated by collagen [2,5,22]. Although integrin ␣2 1 was known to mediate platelet adhesion with collagen, it is now recognized that the type of collagen largely determines the requirement for ␣2 1 . Recent years studies demonstrate that platelets adhesion to fibrillar collagen is mediated mostly by GPVI, whereas adhesion to soluble collagen is exclusively dependent on integrin ␣2 1 [11,21,26]. In this study, a tick RGD containing peptide, YY39, was obtained by BLAST search and synthesized and refolded to investigate the platelet aggregation inhibition and anti-thrombosis abilities. It blocked platelet adhesion to soluble collagen with an IC50 ≈ 20 M (Fig. 3C). This result indicates that YY-39 is an antagonist of collagen receptor ␣2 1 on the surface of platelet and is a potential inhibitor of platelet aggregation. Platelet activation originates from the initial adhesive interactions and from the agonists released or generated at a site of vascular injury. The main inducers of platelet activation are thrombin, ADP, and thromboxane A2 . The serine protease ␣-thrombin activates platelets through G protein-linked protease-activated receptors (PARs) that convert an extracellular proteolytic cleavage event into an intracellular signal [6,25]. ADP is an agonist that induces both reversible platelet aggregation and synthesis of thromboxane A2 , and function as an amplifier for platelet aggregation responses induced by itself and other agonists. TXA2 activates platelets through specific G protein-coupled seventransmembrane-domain receptors. Accordingly, the three agonists as mentioned above were used to induce platelet aggregation for investigating the effects of YY-39 on platelet aggregation. As showed in Fig. 2, YY-39 potently inhibited platelet aggregation induced by the platelet agonists ADP, thrombin, and U46619. Platelet aggregation is the amplification step mediated by adhesive substrates bound to the membranes of activated platelets. In the process of platelet aggregation, it can be induced by a variety of agonists through several physiologic cascades, but the final common step of these cascades is the binding of fibrinogen to glycoprotein IIb/IIIa (GPIIb/IIIa) on the platelet surface [31]. Activated GPIIb/IIIa contributes to stable adhesion and mediates the immobilization of soluble adhesive proteins, notably von Willebrand factor (vWF), fibrinogen and fibronectin, onto the surface of adherent platelets [23]. Peptides with RGD motif (variabilin, ornatin, decorsin) are potential antihemostatic agents that function as antagonists of GPIIb/IIIa. In the present study, YY-39 bound to purified GPIIb/IIIa in a dose-dependent manner (Fig. 3A). This result indicates that YY-39 is an antagonist of GPIIb/IIIa on the surface of platelet. The RGD motif in YY-39 may facilitate the binding of it to GPIIb/IIIa. The anti-thrombosis effects of YY-39 were then evaluated by two animal models in vivo. Interestingly, YY-39 showed potent anti-thrombosis effects in arteriovenous shunt rat model and carrageenan-induced mouse tail thrombosis model (Fig. 4). Generally, the more potent an anti-thrombosis agent is, the more risk of bleeding complication comes, which is the major issue limiting clinical use of anti-thrombosis drugs. YY-39 showed little bleeding complication in a tail transaction model. In summary, the present study firstly demonstrated that the RGD-containing peptide YY-39, one of ixodegrins, effectively inhibited platelet aggregation and exerted potent anti-thrombosis activities. It blocked platelet adhesion to soluble collagen and bound to purified GPIIb/IIIa in a dose-dependent manner, suggesting that YY-39 is an antagonist of both collagen receptor ␣2 1 and GPIIb/IIIa on the surface of platelet. Further work is necessary to investigate other members of the ixodegrin family. The tertiary structure of YY-39 may help to illuminate the anti-thrombotic mechanism of the ixodegrins, which would facilitate our understanding of the blood feeding strategy of ticks.
Please cite this article in press as: Tang J, et al. YY-39, a tick anti-thrombosis peptide containing RGD domain. Peptides (2014), http://dx.doi.org/10.1016/j.peptides.2014.08.008
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Conflict of interest The authors have no conflicts of interest. Acknowledgments This work was supported by Ministry of Science and Technology (2010CB529800, 2013CB911300, 863 project 2012AA021801), National Natural Science Foundation (31025025, U1132601, U1302221, 31200590, 31260208), and CAS (KSZD-EW-Z-007, KJZDEW-L03), Jiangsu Province (BK2012365, BE2012748). References
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YY-39, a tick anti-thrombosis peptide containing RGD domain Jing Tang a,1 , Yaqun Fang a,1 , Yajun Han a , Xuewei Bai a , Xiuwen Yan a , Yun Zhang b , Ren Lai a,b,∗ , Zhiye Zhang b,c,∗∗ a
Life Sciences College of Nanjing Agricultural University, 1st Weigang, Nanjing, Jiangsu 210095, China Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China c University of Chinese Academy of Sciences, Beijing 100009, China b
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Article history: Received 29 June 2014 Received in revised form 14 August 2014 Accepted 14 August 2014 Available online xxx Keywords: Tick Salivary gland Platelet inhibitor Anti-thrombosis Glycoprotein IIb/IIIa
a b s t r a c t Ticks are obligatory blood feeding ectoparasites, which continuously attach to their hosts for 1–2 weeks. There are many biologically active compounds in tick salivary glands interfering host haemostatic system and to successfully obtain blood meal. Several platelet aggregation inhibitors have been identified from ticks. A family of conserved peptides, which were identified from transcriptome analysis of many tick salivary glands, were found to contain unique primary structure including predicted mature peptides of 39–47 amino acid residues in length and a Pro/Glu(P/E)-Pro/His(P/H)-Lys-Gly-Asp(RGD) domain. Given their unique structure and RGD domain, they are considered a novel family of disintegrins that inhibit platelet aggregation. One of them (YY-39) was tested for its effects on platelets and thrombosis in vivo. YY-39 was found effectively to inhibit platelet aggregation induced by adenosine diphosphate (ADP), thrombin and thromboxane A2 (TXA2). Furthermore, YY-39 blocked platelet adhesion to soluble collagen and bound to purified GPIIb/IIIa in a dose-dependent manner. In in vivo experiments, YY-39 reduced thrombus weight effectively in a rat arteriovenous shunt model and inhibited thrombosis in a carrageenan-induced mouse tail thrombosis model. Combined with their prevalence in ticks and platelet inhibitory functions, this family of peptides might be conserved tick anti-haemostatic molecules. © 2014 Elsevier Inc. All rights reserved.
Introduction Blood-feeding arthropods have developed effective strategies to get blood meal. To combat vertebrate hemostasis, inflammation and other defense systems, ticks have evolved a variety of mechanisms, such as anti-hemostatic, anti-inflammatory, immunoregulation, vasodilation and other factors in their salivary glands [1,24,33]. Platelet aggregation plays key role in hemostatic responses, particularly in small wounds [30]. Several groups of compounds that inhibit platelet aggregation have been identified from tick salivary glands [9]. They include apyrases
∗ Corresponding author at: Life Sciences College of Nanjing Agricultural University, 1st Weigang, Nanjing, Jiangsu 210095, China. Tel.: +86 25 84396849; fax: +86 25 84396849. ∗∗ Corresponding author at: Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China. Tel.: +86 871 651 962 02; fax: +86 871 651 990 86. E-mail addresses: [email protected] (R. Lai), [email protected] (Z. Zhang). 1 These authors contributed equally to this work.
(ATP-diphosphohydrolase EC 3.6.1.5) [17,28], thromboxane A2 (TXA2)-binding proteins [15,20,32], thrombin inhibitors [16], and disintegrins [12,13,19,20,31]. At least three families of disintegrins have been identified and characterized from tick salivary glands. The first one is tick adhesion inhibitor (TAI), which is a 15-kDa sialogenin purified from the Ornithodoros moubata salivary gland, acting as an antagonist of integrin ␣2 1 [13]. It inhibits platelet adhesion to soluble collagen under static conditions with an IC50 of 8 nM. The second one is variabilin, which is identified from Dermacentor variabilis. Variabilin is a 5-kDa RGD-containing disintegrin containing 4 cysteine residues and can inhibit ADP-induced platelet aggregation with an IC50 of 150 nM [31]. The third one includes disagregin from O. moubata, which lacks RGD motifs, savignygrin from Ornithodorus savignyi [19], and monogrin from Argas monolakensis [20], which contain an RGD motif. Sequence alignment indicated that disagregin, savignygrin, and monogrin belong to the same family of peptides containing 6 conserved cysteines and they share conserved amino acid sequences [9]. Transcriptome analysis of the hard ticks Ixodes pacificus and Ixodes scapularis revealed a possible novel disintegrin family (ixodegrins), which displays sequence similarity to variabilin, including RGD position, but have two additional cysteines. The
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Please cite this article in press as: Tang J, et al. YY-39, a tick anti-thrombosis peptide containing RGD domain. Peptides (2014), http://dx.doi.org/10.1016/j.peptides.2014.08.008
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functions of ixodegrins remain to be studied [10]. In this study, one of ixodegrins (YY-39) was synthesized and refolded for further functional investigation. YY-39 was found to contain function to inhibit platelet aggregation by blocking glycoprotein IIb/IIIa.
Materials and methods Sequence identification of YY-39 Using the sequence of ixodegrin (YSFTCERIPCTNNSDCHGSDLCQCRPPRGDGFSYFCSEY) reported by Francischetti et al. [10]. BLAST search was performed. Similar sequences containing conserved RGD and cysteine motif were extracted.
Synthesis and re-folding of YY-39 YY-39 (YSSTCERIPCTNNSDCHGPDLCQCRPPRGDDFGYFCSEY, the sequence was obtained from GenBank: AAY66649.1) (Fig. 1) was synthesized by GL Biochem Ltd. (Shanghai, China) and analyzed by high performance liquid chromatography (HPLC) and mass spectrometry to confirm purity higher than 98%. The synthesized linear peptide was incubated in 0.1 M Tris–HCl, 0.1 M NaCl, 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione(GSSH), pH 9.0 for 24 h at 25 ◦ C to fold the peptide.
Platelet aggregation inhibition assays According to our previous method [18], human platelet-rich plasma (PRP) was used in platelet aggregation assays and prepared as follows: human blood from a healthy donor was centrifuged at 160 × g for 10 min at 22 ◦ C, and the PRP was decanted. After successive washes in Tyrode–protein buffer (137 mM NaCl, 2 mM KCl, 0.3 mM NaH2 PO4 , 12 mM NaHCO3 , 5.5 mM glucose, 0.35% BSA, 1 mM MgCl2 , and pH 7.4), the pelleted platelets were diluted to 2 × 105 platelets/l in Tyrode–protein buffer, and counted with a thrombocounter. Platelet aggregation was monitored by light transmission in an aggregometer (Plisen, Beijing, China) in which 300 l aliquots of PRP were incubated at 37 ◦ C with continuous stirring. Light transmission was recorded and ADP (5 M), thrombin (0.45 U/ml) or U46619 (a TXA2 analog, 10 M) were added to initiate platelet aggregation. The inhibition of platelet aggregation was measured at the maximum aggregation response.
Interaction assays between YY-39 and platelet GPIIb/IIIa by ELISA The assay was performed according to the following protocol: microtiter plates were coated with increasing concentrations of YY-39, fibrinogen and bovine serum albumin (BSA) diluted in 0.1 M sodium carbonate, pH 9.5 at 4 ◦ C overnight. After washing the microplate for 5 times with 300 l wash solution (0.1 M phosphate-buffered saline with 1% Tween-20), the microplate was then blocked by incubation with 200 l blocking solution (1% BSA in wash solution) for 1 h at 37 ◦ C. The microplate was washed, followed immediately by addition of purified GPIIb/IIIa (5 g/ml) in blocking solution. A mouse anti-GPIIIa (CD61) antibody (1:2000, Millipore Chemicon, USA) was used to detect GPIIb/IIIa, and a second anti-mouse IgG antibody (1: 2000, horseradish peroxidase (HRP) labeled, KPL, USA) was then used. A final wash was performed and color development was then carried out with 3,3 ,5, 5 -tetramethylbenzidine (TMB), the plates were incubated until color developed. The reaction was stopped with 0.5 M H2 SO4 and the absorbance at 450 nm was measured. Platelet adhesion assay under static conditions The inhibition of platelet adhesion assay was performed using a modification of a published method [4]. Briefly, coverslips were coated with 20 g/ml of soluble non-fibrillar collagen type I (BD Bioscience, San José, CA, USA) overnight at 4 ◦ C. After washing three times with 200 l of PBS, the coverslips were blocked with 200 l of PBS containing 2% BSA for 1 h. 200 l of platelets (2 × 105 /ml) with YY-39 (0–39.3 M) were then applied to coverslips and incubated for 45 min at room temperature. Non-adherent platelets were removed by washing the coverslips three times with PBS. Differential interference contrast images were recorded using a Nikon Eclipse 80i microscope (Nikon, Japan). Extent of platelet adhesion was expressed as percent area covered by platelets. All experiments were performed in quintuplicates. Rat arteriovenous shunt thrombosis model According to the method described previously by Sperzel [27], Wistar rats (180–220 g) were used for arteriovenous shunt thrombosis model. The animals were anesthetized by intraperitoneal (i.p.) administration of ketamine (50 mg/kg) and xylazine (15 mg/kg). The right common carotid artery and left jugular vein were isolated and cannulated with two 100-mm-long, salinefilled catheters (PE-60, Becton Dickinson, Sparks, MD, USA). A
Fig. 1. Structural characteristics comparison. (A) Comparison of the sequence of YY-39 with other similar sequences by BLAST search. The sequence of YY-39 was underlined, and the conserved RGD domain of each sequence was boxed. The corresponding GenBank Number was indicated. (B) Comparison of the sequence of YY-39 with other disintegrins from tick salivary glands. The conserved RGD domain in each sequence was boxed.
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30-mm-long polyethylene tube (PE-160, Sparks, USA) was used to connect the two PE-60 catheters, and a rough nylon thread (60 mm in length and 0.26 mm in diameter) contained in the PE-160 tube and folded into a double string to induce thrombosis. The femoral vein was exposed and cannulated with a saline-filled polyethylene catheter for sample administration (n = 9 per dose). After 15 min administration of sample, the shunt was opened for 5 min and then closed. The nylon strings were then withdrawn from the middle PE-160 catheter and the wet thrombi attached in the nylon strings were weighed.
male) were used in this assay. The samples or control saline were injected intravenously into the animals (n = 6). After the injection for 2 h, a 2 mm distal segment of the tail was severed with a razor blade. The tail was immediately immersed in 37 ◦ C saline with the tip of the tail 5 cm below the body. The bleeding time was recorded for the stream of blood to cease. All the experimental protocols of animal models were approved by the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences. The approval ID for this study was syxk2014-0007.
Carrageenan-induced mouse tail thrombosis model
Statistical analysis
The procedure was carried out according to the reported method [3] and male Kunming mice were used in this model. YY-39 or ozagrel were injected intravenously into the animals (n = 9, 18–20 g). 30 min later, the mice were injected intraperitoneally with 40 l (1%) -Carrageenan (type I, Sigma) dissolved in 0.9% NaCl to induce thrombosis in tail. Six hours after the injection of -Carrageenan, the tested samples were injected into mice by caudal vein again, and the length of thrombus in tails of mice was measured after 48 h treatment.
Data were assessed for statistical significance using Student’s (unpaired) t-test. Results were reported as mean ± SE with significance accepted at P < 0.05.
Bleeding evaluation Bleeding time was assessed following the protocol described in the tail transaction method [7,29], and Kunming mice (18–20 g,
Results Structural characteristics comparison Comparison of YY-39 with several other similar sequences extracted by BLAST search was illustrated in Fig. 1A, which displayed sequence similarities including a RGD sequence at the N-terminus of the whole peptide and 6 cysteines. There are 5 cysteine residues upstream of their RGD sequence and 1 downstream of the sequence. In addition, the distribution of these cysteines
Fig. 2. YY-39 inhibited human platelet aggregation induced by ADP (A), thrombin (B), and U46619 (C). The tracings represent a typical experiment, and the experiments were repeated 3 times.
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Fig. 3. Interactions of YY-39 with two platelet receptors GPIIb/IIIa and integrin ␣2 1 . (A) Binding of GPIIb/IIIa to immobilized YY-39. Fibrinogen and BSA were used as a binding control. Data represent mean ± SE of three individual experiments. (B) YY-39 prevented platelet adhesion to soluble collagen under static condition. (C) Dose-responses curve for YY-39-mediated inhibition of platelet adhesion to soluble collagen. Typical experiments were shown, data represent mean ± SE of three individual experiments.
in their sequences is similar, suggesting they may share a similar three-dimensional structure stabilized by disulfide bridges. The sequence comparison of YY-39 with other platelet aggregation inhibitors such as variabilin [10], disagregin, savignygrin [19], and monogrin [20] were also presented in Fig. 1B.
Inhibitory effect of YY-39 on platelet aggregation Three agonists were used to induce platelet aggregation for investigating the effects of YY-39 on platelet aggregation. As illustrated in Fig. 2, YY-39 dose-dependently inhibited platelet aggregation induced by ADP (Fig. 2A), thrombin (Fig. 2B), and U46619 (Fig. 2C), and the aggregations were completely inhibited at concentrations of 15 M, 3.76 M, and 12 M, respectively.
YY-37 bound to platelet GPIIb/IIIa The direct interaction of YY-39 with platelet GPIIb/IIIa was tested using a solid-phase assay. As illustrated in Fig. 3A, immobilized YY-39 dose-dependently bound to purified platelet GPIIb/IIIa. Additionally, at a dosage of 62.5 ng per well or above, the binding of YY-39 to GPIIb/IIIa was comparable to that of fibrinogen.
YY-37 inhibited platelet adhesion to soluble collagen under static condition Integrin ␣2 1 is known to mediate adhesion for platelet to collagen, to investigate whether YY-39 blocks integrin ␣2 1 –collagen interaction, platelets were added to coverslips coated with soluble collagen. As showed in Fig. 3B, YY-39 dose-dependently inhibited platelet deposition to soluble collagen with an IC50 ≈ 20 M (Fig. 3C).
YY-39 inhibited thrombus formation in vivo Anti-thrombosis ability of YY-39 was evaluated in rat arteriovenous shunt thrombosis model and carrageenan-induced mouse tail thrombosis model, and ozagrel, which is a clinical anti-thrombosis medicine acting as a selective thromboxane A2 synthetase inhibitor [14], was used as a positive control. As illustrated in Fig. 4A, both YY-39 and ozagrel significantly inhibited thrombosis in a dosedependent manner, while the anti-thrombosis ability of YY-39 was obviously better than ozagrel. The thrombus weight was 4.3 ± 0.4 in the control (0 nmol/kg), while after administration of YY-39 by 50 and 100 nmol/kg, the thrombus weight was reduced to 2.8 ± 0.3 and 1.6 ± 0.5 mg, respectively. However, when given the corresponding dosages of ozagrel, the thrombus weight was reduced to 3.5 ± 0.5 and 2.6 ± 0.4 mg, respectively. In carrageenan-induced mouse tail thrombosis model, YY-39 unambiguously inhibited thrombosis in a dose-dependent manner (Fig. 4B). At the dosages of 65 and 130 nmol/kg, thrombus formation was inhibited by 31.7% and 71.4%, respectively. When given the same dosages, ozagrel inhibited thrombus formation only by 18.7% and 46.9%, respectively. Bleeding evaluation As illustrated in Fig. 5, the average bleeding time was 2.5 min in the control group (saline), while the bleeding time was prolonged to 4 and 5.5 min when the animals were administered with YY39 at dosages of 0.17 and 0.6 mol/kg body weight. However, at dosage of 0.6 mol/kg body weight, ozagrel strikingly prolonged the bleeding time to 6.8 min. Discussion Hemostasis is a dynamic process of thrombus formation and fibrinolysis. The biological processes of platelet adhesion, activation and aggregation play pivotal role in the process of primary
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Fig. 4. Antithrombotic activities of YY-39 in vivo. (A) Antithrombotic effects of YY39 and ozagrel in the rat arteriovenous shunt thrombosis model. (B) Effects of YY-39 and ozagrel on thrombus formation induced by carrageenan in mouse tail. Data represent mean ± SE of nine individual experiments. The values for YY-39 or ozagrel-treated group were significant different from the values for the saline group (*P < 0.05, **P < 0.001).
hemostasis. At sites of vascular injury, collagen initiates recruitment of circulating platelets and triggers platelet activation cascade required to stimulate thrombus growth [8,22,23]. The contributions of two collagen platelet receptors-glycoprotein VI (GPVI) and integrin ␣2 1 to collagen-mediated platelet responses have been highlighted in platelet adhesion and activation, and several different models have been proposed in an attempt to explain how
Fig. 5. Bleeding time induced by intravenous injection of vasotab TY and ozagrel in vivo. Data represent of six individual experiments.
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platelets are activated by collagen [2,5,22]. Although integrin ␣2 1 was known to mediate platelet adhesion with collagen, it is now recognized that the type of collagen largely determines the requirement for ␣2 1 . Recent years studies demonstrate that platelets adhesion to fibrillar collagen is mediated mostly by GPVI, whereas adhesion to soluble collagen is exclusively dependent on integrin ␣2 1 [11,21,26]. In this study, a tick RGD containing peptide, YY39, was obtained by BLAST search and synthesized and refolded to investigate the platelet aggregation inhibition and anti-thrombosis abilities. It blocked platelet adhesion to soluble collagen with an IC50 ≈ 20 M (Fig. 3C). This result indicates that YY-39 is an antagonist of collagen receptor ␣2 1 on the surface of platelet and is a potential inhibitor of platelet aggregation. Platelet activation originates from the initial adhesive interactions and from the agonists released or generated at a site of vascular injury. The main inducers of platelet activation are thrombin, ADP, and thromboxane A2 . The serine protease ␣-thrombin activates platelets through G protein-linked protease-activated receptors (PARs) that convert an extracellular proteolytic cleavage event into an intracellular signal [6,25]. ADP is an agonist that induces both reversible platelet aggregation and synthesis of thromboxane A2 , and function as an amplifier for platelet aggregation responses induced by itself and other agonists. TXA2 activates platelets through specific G protein-coupled seventransmembrane-domain receptors. Accordingly, the three agonists as mentioned above were used to induce platelet aggregation for investigating the effects of YY-39 on platelet aggregation. As showed in Fig. 2, YY-39 potently inhibited platelet aggregation induced by the platelet agonists ADP, thrombin, and U46619. Platelet aggregation is the amplification step mediated by adhesive substrates bound to the membranes of activated platelets. In the process of platelet aggregation, it can be induced by a variety of agonists through several physiologic cascades, but the final common step of these cascades is the binding of fibrinogen to glycoprotein IIb/IIIa (GPIIb/IIIa) on the platelet surface [31]. Activated GPIIb/IIIa contributes to stable adhesion and mediates the immobilization of soluble adhesive proteins, notably von Willebrand factor (vWF), fibrinogen and fibronectin, onto the surface of adherent platelets [23]. Peptides with RGD motif (variabilin, ornatin, decorsin) are potential antihemostatic agents that function as antagonists of GPIIb/IIIa. In the present study, YY-39 bound to purified GPIIb/IIIa in a dose-dependent manner (Fig. 3A). This result indicates that YY-39 is an antagonist of GPIIb/IIIa on the surface of platelet. The RGD motif in YY-39 may facilitate the binding of it to GPIIb/IIIa. The anti-thrombosis effects of YY-39 were then evaluated by two animal models in vivo. Interestingly, YY-39 showed potent anti-thrombosis effects in arteriovenous shunt rat model and carrageenan-induced mouse tail thrombosis model (Fig. 4). Generally, the more potent an anti-thrombosis agent is, the more risk of bleeding complication comes, which is the major issue limiting clinical use of anti-thrombosis drugs. YY-39 showed little bleeding complication in a tail transaction model. In summary, the present study firstly demonstrated that the RGD-containing peptide YY-39, one of ixodegrins, effectively inhibited platelet aggregation and exerted potent anti-thrombosis activities. It blocked platelet adhesion to soluble collagen and bound to purified GPIIb/IIIa in a dose-dependent manner, suggesting that YY-39 is an antagonist of both collagen receptor ␣2 1 and GPIIb/IIIa on the surface of platelet. Further work is necessary to investigate other members of the ixodegrin family. The tertiary structure of YY-39 may help to illuminate the anti-thrombotic mechanism of the ixodegrins, which would facilitate our understanding of the blood feeding strategy of ticks.
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Conflict of interest The authors have no conflicts of interest. Acknowledgments This work was supported by Ministry of Science and Technology (2010CB529800, 2013CB911300, 863 project 2012AA021801), National Natural Science Foundation (31025025, U1132601, U1302221, 31200590, 31260208), and CAS (KSZD-EW-Z-007, KJZDEW-L03), Jiangsu Province (BK2012365, BE2012748). References
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