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[email protected] Protein & Peptide Letters, 2014, 21, 69-74
69
Construction and Characterization of Novel Hirulog Variants with Antithrombin and Antiplatelet Activities Zheng Yu, Yuanyuan Huang, Yu Wang, Chen Dai, Mingxin Dong, Zhuguo Liu, Shuo Yu, Jie Hu and Qiuyun Dai* Beijing Institute of Biotechnology, Beijing 100071, People’s Republic of China Abstract: The RGD sequence was used to design potent hirudin isoform 3 mimetic peptides with both antithrombin activity and antiplatelet aggregation activity. The RGD and proline were inserted between the catalytic active binding domain (D-Phe-Pro-Arg-Pro) on the N-terminus and the anion-binding exosite binding domain (QGDFEPIPEDAYDE) on the Cterminus. Thrombin titration assay and ATP-induced platelet aggregation test revealed that the peptide with the linker RGDWP or RGDGP possessed potent antithrombin and antiplatelet activities, while other peptides without the Pro residue in the linker only showed antithrombin activity. Similar results were obtained in the RGD-containing hirulog-1 variants. Our study indicates that the inserted Pro residue facilitates the exposure of RGD and the binding of the peptide to glycoprotein IIb/IIIa (GPIIb/IIIa). The strategy of combining the RGD sequence and the Pro residue may be used for future designs of bifunctional antithrombotic agents.
Keywords: Antiplatelet aggregation, antithrombin, hirudin, mimetic peptide, RGD-motif,. INTRODUCTION Thrombin and platelets play pivotal roles in thrombogenic diseases that are frequently treated with a combination of various anticoagulants and antiaggregants in current clinical practices [1-3]. Previous studies have tried to design more effective antithrombotic agents with both antithrombin and antiplatelet activities [4-10]. The Arg-Gly-Asp (RGD) sequence, a key motif of fibrinogen for its binding to the glycoprotein IIb/IIIa (GPIIb/IIIa) on the surface of platelets [11], has been used to design such bifunctional antithrombotic agents. One of the strategies was to introduce the RGD motif to hirudin [6,7,10], a natural thrombin inhibitor with 65-amino acids. The replacement of the Ser-Asp-Gly-Glu sequence at position 32-35 by RGDS rendered an antiplatelet aggregation activity, in addition to the original antithrombin activity [6]. Insertions of RGD into other peptide inhibitors of thrombin have also been reported. Church et al. linked RGD to the N-terminus of hirudin to form hybrid RGDhirudin that showed both antithrombin and antiplatelet activities [4]. However, the antithrombin activity of the RGDhirudin was low, because it lacked the N-terminal sequence of hirudin that is responsible for the binding and inhibiting of the active site of thrombin. Recently, Ilas et al. added RGD to the C-terminus of hirudin by the sequence D-Phe–Pro– Arg, a thrombin active binding motif, which also rendered hirudin antiplatelet aggregation activity but weak thrombin inhibitory activity [8]. To date, the flexibility of RGD has been an obstacle for the design of effective bifunctional antithrombotic peptides, and currently available bifunctional *Address correspondence to this author at Beijing Institute of Biotechnology, Beijing 100071, China; Tel: +86-10-66948897; Fax: +86-10-63833521; E-mail:
[email protected] -5/14 $58.00+.00
peptides have either lower antiplatelet aggregation activity or lower antithrombin activity. In this study, we report the design of a novel anticoagulant (fPRP-RGDX1X2-QGDFEPIPEDAYDE; f=D-Phe) (Table 1) possessing the ability to inhibit both thrombin and platelet aggregations, composed of a catalytic active binding domain (D-Phe-Pro-Arg-Pro) of hirulog-1 (Bivalirudin), an anion-binding exosite binding domain (QGDFEPIPEDAYDE) from hirudin isoform 3 [12], and a RGDcontaining linker (RGDX1X2, X represents W, S, G or P). The Trp, Ser, Gly, and Pro residues were selected for the investigation of the effects of hydrophobic, hydrophilic, and conformation-constrained residues on the antiplatelet aggregation activity. The results showed that the peptides with the linker RGD with P exhibited antithrombin and antiplatelet activities. Our study may promote the design of potent antithrombotic agents with or without antiplatelet activity. MATERIALS AND METHODS Reagents Bovine, porcine, and human thrombins and human fibrinogen were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Bovine and porcine fibrinogens were purchased from Sigma (St. Louis, USA). Chromozym TH (Tos-Gly-Arg-ProPNA) was from Roche (Indianapolis, USA). Eptifibatide was obtained from GL Biochem Ltd. (Shanghai, China). The kits for testing activated partial thromboplastin time (APTT) and thrombin time (TT) were purchased from TECO GmbH Corp (Neufahrn, Germany). ADP (super pure) was from Amresco(Solon, USA).
© 2014 Bentham Science Publishers
70 Protein & Peptide Letters, 2014, Vol. 21, No. 1
Table 1.
Constants for the peptide inhibition of the hydrolysis of Chromozym TH catalyzed by bovine and human thrombin（n=3）
Peptide
a
Yu et al.
Ki (nM)a
Sequence Bovine
Human
Hirulog-1
D-FPRP-GGGG-QGDFEEIPEEYL
23.97± 0.53
11.77 ± 0.09
Peptide 1
D-FPRP-GRGDWP-DFEEIPEEYL
61.6 ± 4.05
10.9±0.04
Peptide 2
D-FPRP-RGDWPG-DFEEIPEEYL
44.19 ± 1.2
17.71 ± 0.04
Peptide 3
D-FPRP-GGGG- QGDFEPIPEDAYDE-NH 2
10.06 ± 0.56
5.8 ± 0.55
Peptide 4
D-FPRP-GRGDS- QGDFEPIPEDAYDE-NH2
16.17 ± 0.51
5.32 ± 0.26
Peptide 5
D-FPRP-RGDWP-QGDFEPIPEDAYDE-NH2
15.65 ± 0.71
7.61 ± 0.25
Peptide 6
D-FPRP-RGDGP- QGDFEPIPEDAYDE-NH2
>100
>100
Peptide 7
D-FPRP-RGDWG-QGDFEPIPEDAYDE-NH2
39.37 ± 0.29
16.28 ± 0.33
Peptide 8
D-FPRP-RGDGG- QGDFEPIPEDAYDE-NH2
76.56 ± 1.34
17.24 ± 0.48
The inhibition constants (Ki) were determined by the following equation for competitive inhibition: 1/v=Km/Vmax(1+[I]/Ki) + 1/V max .
Peptide Synthesis [13, 14] All peptides were synthesized by using the solid-phase method on an Applied Biosystem 433A Peptide Synthesizer (Foster City, USA) and were then purified by semipreparative reversed-phase HPLC using a 9.4250 mm Zorbax C18 column equilibrated in 0.1% TFA (95%) and 0.1% TFA/CH3CN (5%) at a flow rate of 3.0 ml/min. A 25 min gradient of 15–75% of 0.1%TFA/CH3CN was implemented to elute the peptides. After lyophilization, the purity of the peptides was assessed by analytical reversed-phase HPLC. The final products were >98% pure. Confirmation of the correct molecular mass was ascertained by Ultraflex III TOF/TOF mass spectrometry (Bruker). Animals Male Sprague-Dawley rats (280-320 g), purchased from the Beijing Animal Center, were used in the activated partial thromboplastin time (APTT) and bleeding time (BT) tests. The rats were housed in plastic boxes with a 12 hour light/dark cycle, constant temperature of 24 ± 2 oC, and a relative humidity of 50%. Food pellets and water were available ad libitum. All experiments were conducted in accordance with the guidelines of the Beijing Institutes for Biological Science Animal Research Advisory Committee and conformed to the European Community Directives for the care and use of laboratory animals. Inhibition of Bovine and Human Thrombin by Peptides 0.05 mL peptide (the concentration was 10, 25, 50, 100, 150, 500 and 1000 nM, respectively) was added to 0.4 mL of reaction mixture containing Hepes buffer (10 mM Hepes, 10 mM Tris, 0.1M NaCl, 0.1% PEG 6000, pH 7.4) and bovine or human thrombin (0.25 NIH) for incubation at 25 oC for 2 min, followed by the addition of 0.05 mL Chromozym TH (the concentration was 25, 33, 40, 50, 100, 125, 200, 330, 500, 1000 nM, respectively ). The absorbance at 405 nm was
immediately measured for 12 min using a Beckman DU640 spectrometer. The inhibition constants (Ki) were then determined by the following equation for competitive inhibition [15]:
Where S is the initial substrate concentration; I is the inhibitor concentration; v is the initial steady-state velocity; Vmax is the limiting maximal velocity; and Km is the dissociation constant of the complex. Thrombin Titration Assay The anticoagulative activity was determined by using the thrombin titration assay with minor modifications [16]. Briefly, tubes (0.75 10 cm) containing 200 L 0.5% bovine, porcine, or human fibrinogen and 50 L peptide in Tris buffer (0.05 M Tris, 0.05 M NaCl, pH 7.4) were incubated at 37 oC for 5 min. Five L of thrombin at varied concentrations (0.1, 0.2, 0.5, 1, 2, 4 NIH) was added, mixed, and incubated at 37 oC for 1 min. If the mixture did not coagulate in 1 min, another 5 L of thrombin solution (same or smaller concentration) was added, until the mixture was able to coagulate in 1 min. The total thrombin titration volume and the anticoagulative activity of peptides (ATU/nmol) were then calculated. The titration of every sample was repeated three times. APTT Tests in Rat Plasma Antithrombotic peptides were administrated to male SD rats (280-320 g) via their tail veins. At different time points (0, 20, 40, 60, 90, 120, 180, and 240 min; or 0, 1, 2, 3, 4, 5, 8, and 12 h) after peptide injection, blood samples were taken via retroorbital vein plexus, mixed with 3.2% sodium citrate in a ratio of 9:1 (v/v), and centrifuged for 15 min at 2,000g at 4 oC. The APTT of rat plasma was determined on a
Novel Bifunctional Antithrombotic Peptides
coagulation analyser (Coatron M1, TECO, Germany) following the manufacturer’s manual. Briefly, a cuvette with 0.05 mL rat plasma was warmed to 37 oC for 2 min, and the warmed APTT reagent (0.05 mL) was added and incubated for another 3 min at 37 oC. Afterwards, 50 L of a calcium chloride solution (0.25 M) was added into the cuvette, the coagulation analyser immediately started the optic and recorded the clotting time of the test plasma (APTT). Bleeding Time [17] Thirty minutes after peptide injection, bleeding was induced by tail transection of the anesthetized rats 2 mm from the tail tip with a size 21 disposable scalpel blade, and the bleeding tail was immediately placed in a cuvette filled with 37 oC saline. Bleeding was monitored visually until the blood flow stopped for a complete 30 s interval; if the bleeding did not stop, the bleeding time was recorded over 30 min. Platelet Aggregation Assay Platelet aggregation assay was performed in a SC-2000 platelet aggregation analyzer (Successder, Beijing, China) with human platelet-rich plasma (PRP) as previously reported [18]. The blood sample was taken from a volunteer, who had not taken any aspirin or related products, and mixed with 3.2% sodium citrate in a ratio of 9:1 (v/v). The upper platelet-rich plasma was prepared by centrifugation at 800 g for 10 min at room temperature. The platelet-poor plasma (PPP) in the remaining part was prepared by centrifugation at 2,000 g for 25 min at room temperature. For the platelet aggregation assay, the PRP was diluted to 250,000 platelets/L with the PPP. The diluted PRP (285 L) was mixed with 10 L of peptide or saline and incubated in an aggregometer at 37 oC for 5 min. 5 L of the aggregating agent ADP (1.0 mM) was added, and the light transmission was recorded for 5 min. The transmission of PPP without PRP was set at 100%, the final concentration of peptide was 10~80 M.
Protein & Peptide Letters, 2014, Vol. 21, No. 1
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replacing the C-teminus of hirulog-1 with the corresponding fragment of hirudin HV3 (QGDFEPIPEDAYDE), exhibited significantly higher inhibitory activity (Ki = 10.06±0.56 nM) to bovine thrombin than hirulog-1 did (Ki = 23.97±0.53 nM). The peptides 4 and 5, which were derived by replacing the 4Gly linker in the peptide 3 with GRGDS and RGDWP, also showed higher inhibitory activity (Ki = 16.17±0.51 nM and 15.65±0.71 nM for peptides 4 and 5, respectively) than that of hirulog-1 but slightly lower inhibitory activity than that of the peptide 3. However, other peptides, such as peptides 7 and 8, showed lower inhibitory activity to bovine thrombin than that of hirulog-1. Similar results were observed in the inhibition kinetics of human thrombin. The Anticoagulative Activity In Vitro The anticoagulative activities of peptides to porcine, bovine and human thrombins were determined by using the thrombin titration assay (Table 2). For the hirulog-1 mutants, the replacements of the linker GGGG by GRGDWP (peptide 1) and RGDWPG (peptide 2) resulted in reduced anticoagulative activity to thrombin from all the three species. However, peptide 3 exhibited significantly higher anticoagulative activity for human and bovine thrombins than hirulog-1 did, which is consistent with the results in bovine thrombin– catalyzed hydrolysis of Chromozym TH. The analogs with the replacement of the linker containing RGD showed significantly lower anticoagulative activity to bovine and human thrombins except for peptide 4, which was similar to peptide 3. However, all peptide 3 analogs with the linker RGD, except for peptide 5, significantly increased the anticoagulative activity of porcine thrombin. These results suggest that the RGD linkers between the catalytic active binding domain and the anion-binding exosite binding domain significantly affect the anticoagulative activity to thrombin in vitro. Effects on APTT In Vivo
All data are expressed as mean ± SD. Unpaired t-test or one-way analysis of variance (ANOVA) was used to determine the significance of differences. A difference with a pvalue of less than 0.05 was considered statistically significant.
The effects of peptides 3 to 5 and hirulog-1 on the activated partial thromboplastin time (APTT) of rat plasma were determined, and the peptides 4 and 5 showed significantly lower APTT compared with the peptide 3 (Fig. 1). The high APTT values of peptide 3 may be derived from its higher inhibitory activity and anti-enzymatic hydrolysis since it lacks easily hydrolytic residues RGD.
RESULTS
Inhibition of ADP-induced Platelet Aggregation
A series of RGD-containing antithrombotic peptides (DFPRP-RGDX1X2-QGDFEPIPEDAYDE), X represents W, S, G or P) (Table 1) were designed and synthesized. The Trp, Ser, Gly, and Pro residues were selected for the investigation of the effects of hydrophobic, hydrophilic, and conformation-constrained residues on the antiplatelet aggregation activity. In comparison with similar hirulog variants, the RGDcontaining hirulog-1 analogs were also synthesized (Table 1).
For the hirulog-1 mutants, the replacement of the linker GGGG by GRGDWP or RGDWPD resulted in increased inhibitory activity to the ADP-induced human platelet aggregation, compared with the non-active hiruolog-1 (Fig. 2A). Similarly, peptide 5 with the linker RGDWP exhibited potent inhibitory activity to the ADP-induced human platelet aggregation, and peptide 6 remained active even after the replacement of the Trp residue in the linker RGDWP with G (Fig. 2B). However, peptides 7 and 8, in which the linker is RGDWG or RGDGG, did not show apparent inhibitory activity to the ADP-induced human platelet aggregation. These results indicate the important role of the linker RGDWP,
Statistical Analysis
The inhibitory activity of peptides against the bovine and human thrombin was determined using the competitive inhibition model (Table 1). Peptide 3, which was derived by
72 Protein & Peptide Letters, 2014, Vol. 21, No. 1
Table 2.
Yu et al.
The anticoagulative activity of peptides to thrombins determined by thrombin titration assay (n=3) Activity (ATU/nmol )a Peptide
a
Porcine thrombin
Bovine thrombin
Human thrombin
Hirulog-1
0.52 ± 0.02
2.16 ± 0.08
12.40 ± 0.40
Peptide 1
0.42 ± 0.02
1.68 ± 0.08
8.40 ± 0.14
Peptide 2
0.22 ± 0.01
0.88 ± 0.04
2.40 ± 0.20
Peptide 3
0.66 ± 0.05
17.60 ± 0.56
22.40 ± 0.78
Peptide 4
1.82 ± 0.11
18.40 ± 0.66
18.40 ± 0.64
Peptide 5
0.44 ± 0.02
2.32 ± 0.08
12.40 ± 0.41
Peptide 6
12.60 ± 0.62
0.94 ± 0.05
15.60 ± 0.55
Peptide 7
16.40 ± 0.32
1.12 ± 0.06
17.60 ± 0.70
Peptide 8
8.20 ± 0.16
0.26 ± 0.02
1.20 ± 0.07
The potency of peptide anticoagulant activity is expressed in antithrombin unit (ATU) where one ATU is the amount of peptide neutralizes one NIH Unit of thrombin [16].
Table 3.
Effects of peptides on rat bleeding time Bleeding Time (s)a Peptides
Hirulog-1
0.3 mol/k
0.6 mol/kg
1.0 mol/kg
821.34±112.81
1130.58±143.30
1418.57±193.12
Peptide 1
n.d
n.d.
1513.58±189.47
Peptide 2
1105.64±153.85
1397.18±172.71
1483.26±183.54
Peptide 3
994.74±121.53
1281.17±149.26
1521.32±187.36
Peptide 4
861.89±147.78
1208.26±173.36
1485.39±196.33
Peptide 5
1089.33±139.19
1399.26±162.99
1753.08±194.35
Peptide 6
1173.94±137.32
1225.73±140.21
1331.82±148.43
Peptide 7
1023.62±131.51
1136.47±139.27
1257.70±145.15
Peptide 8
819.24±113.67
1004.73±129.62
1115.91±132.38
a
The peptides (0.3, 0.6, and 1.0 mol/kg) were administered to rats for 30 min before tail transection. The ANOVA tests revealed significant differences in bleeding times between peptide 5 and other peptides. Similar results were obtained for the peptides 1~2 and hirulog-1. As control, the bleeding time after saline administration was 685.6±108.35 s.
especially the Pro residue, on the inhibition of the ADPinduced platelet aggregation. In addition, both hirulog-1 and the peptide 3 analogs were less active than the eptifibatide. Bleeding Time
Figure 1. Effects of the peptides on APTT. Hirulog-1 (), peptide 3 (), peptide 4 (), and peptide 5 () were administered at doses of 1 mol/kg.
To further investigate the anticoagulative activities of the RGD-modified peptides, the bleeding time of rats was recorded 30 min after the administration of varied amounts of peptides (Table 3). Peptide 3 provided slightly longer bleeding times than hirulog-1. The bleeding times of peptide 5 were 1089.33±139.19, 1399.26±162.99, and 1753.08±194.35 s at the doses of 0.3, 0.6 and 1.0 mol/kg, respectively, which were 9.5%, 9.2%, and 15.2% longer than those of peptide 3. These results are consistent with the higher antithrombin and antiplatelet aggregation activities. On the contrary, peptides 4, 6, 7 and 8 had shorter bleeding times due to their low inhibitory activity to thrombin. The two hirulog-1 analogs peptide 1 and 2 resulted in longer bleeding times than hirulog-1, despite their low inhibitory activity of porcine, bovine and human thrombins in vitro (Table 2), due to
Novel Bifunctional Antithrombotic Peptides
Protein & Peptide Letters, 2014, Vol. 21, No. 1
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Figure 2. Inhibition of ADP-induced platelet aggregation by the designed peptides. Inhibition of platelet aggregation was examined in the presence of peptides (10~80 M), and 285 L PRP and 5 L ADP were mixed and incubated for 5 min at 37 oC. The ADP induced platelet aggregation for the control NS was 58.75±3.01%.
the introduction of RGDWP linker with its inhibition of antiplatelet aggregation. DISCUSSION Evidence from structural studies demonstrates that the RGD sequence of fibrinogen usually inserts into the type II-turn between the two -sheets extending from the core of the glycoprotein GPIIb/IIIa on the surface of platelet, which induces platelet aggregation [19]. The naturally-occurring RGD-containing GP IIB/IIIa antagonists, such as decorsin [20], tablysin [21], and snake venom [22,23], show potent antiplatelet aggregation activity and some selectivity. These results demonstrate that this interaction requires specific conformations of the RGD epitope. Some peptides with cyclic, rigid RGD sequences, such as eptifibatide [24], Cyclic (RGDfV) [25], and some hirulog-1 variants [26], possessed potent activity of antiplatelet aggregation. However, cyclization is not only technically challenging but also costly for the synthesis of bifunctional antithrombin and antiplatelet agents. In this study, proline was used for the design of bifunctional antithrombotic agents, due to its structural restriction. The peptides 5 and 6, which contain the linkers RGDWP and RGDGP, respectively, exhibited potent activity of antiplatelet aggregation (Fig. 2B). However, peptides 4, 7 and 8, which also contain the RGD sequence but no Pro residues, did not show apparent antiplatelet activity (Fig. 2A). Similar results were obtained in the RGD-containing hirulog-1 variants. These results suggest that Pro insertion significantly enhances the antiplatelet aggregation activity. In addition, the antiplatelet aggregation activity of peptide 5 was higher than that of peptide 6, suggesting that the Trp residue may also help to increase the activity. The introduction of RGD linker resulted in slight decreases in the inhibitory activity to human thrombin (Table 2). However, some other peptides with the RGD linker, such as peptides 6~8, exhibited potent inhibitory activity to porcine thrombin, suggesting that the RGD linker, possibly with
other residues, affects the peptide binding to thrombin’s catalytic active site and anion-binding exosite. In summary, we obtained a potent anticoagulant with both antithrombin activity and antiplatelet aggregation activity through the introduction of the linker RGDWP in the hirudin isoform 3 peptidomimetic. The combination of the RGD sequence with the Pro residue may be used for future designs of more effective bifunctional antithrombotic agents. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS This work was supported by grants from the Basic Research Program of China (No. 2010CB529802), the China Specific Project for Developing New Drugs during the Eleventh Five-Year Plan Period (No. 2009ZX09103-628), and the China Natural Science Foundation (No. 81072676). REFERENCES [1] [2] [3] [4]
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Received: September 7, 2012
Revised: July 16, 2013
Accepted: August 4, 2013
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