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Anticoagulation factor I, a snaclec (snake C-type lectin) from Agkistrodon acutus venom binds to FIX as well as FX: Ca2+ induced binding data
Toxicon 59 (2012) 718–723
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Anticoagulation factor I, a snaclec (snake C-type lectin) from Agkistrodon acutus venom binds to FIX as well as FX: Ca2þ induced binding data Yan Zhang, Xiaolong Xu*, Dengke Shen, Jiajia Song, Mingchun Guo, Xincheng Yan Department of Chemistry, University of Science and Technology of China, No. 96, Jinzhai Road, Hefei, Anhui 230026, PR China
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a b s t r a c t
Article history: Received 27 October 2011 Received in revised form 23 February 2012 Accepted 6 March 2012 Available online 16 March 2012
Anticoagulation factor I (ACF I), a snake C-type lectin (snaclec) from the venom of Agkistrodon acutus binds specifically with activated factor X (FXa) in a Ca2þ-dependent manner and prolongs the blood-clotting time in vitro. In this study, the inhibition of the coagulation pathway by ACF I was measured in vivo by activated partial thromboplastin time and prothrombin time assays and the binding of ACF I to factor IX (FIX) was investigated by native PAGE and surface plasmon resonance. The results indicate that ACF I inhibits both intrinsic and extrinsic coagulation pathways, but does not inhibit thrombin activity. ACF I also binds FIX in a Ca2þ-dependent manner and their maximal binding occurs at 0.25 mM Ca2þ. ACF I has a higher binding-affinity to FIX than to FX. Ca2þ is required to maintain in vivo function of FIX Gla domain for its recognition of ACF I. However, Ca2þ at high concentrations (>0.25 mM) inhibits the binding of ACF I to FIX. Ca2þ functions as a switch for the binding between ACF I and FIX. The results suggest that the binding of ACF I with FIX may play a dominant role in the anticoagulation activity of ACF I in vivo. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Agkistrodon acutuss ACF I Snaclec Ca2þ Factor IX
1. Introduction Coagulation factors IX (FIX) and X (FX) are two critical plasma enzymes that play crucial roles in blood coagulation (Furie and Furie, 2008). They are all Ca2þ-binding proteins and require Ca2þ to express their physiological functions. Both proteins possess an N-terminal g-carboxyglutamic acid (Gla) domain that is essential for the amplification of the coagulation cascade. Snake venoms are rich in a large variety of proteins and enzymes (Biardi and Coss, 2011; Eble et al., 2011; Lane et al., 2011; Sarkar and El-Refael, 2009). A family of FIX and FX binding snake C-type lectins (FIX/X snaclecs) has been identified from various kinds of snake venoms (Atoda et al., 1995; Chen et al., 2011; Clemetson, 2010; Gopinath et al., 2007; Oliveira-Carvalho et al., 2008; Sekiya et al., 1993; Xu et al., 2000b). All FIX/X snaclecs have an anticoagulant activity and are highly homologous with each other. They bind to the Gla domain * Corresponding author. Fax: þ86 551 3603388. E-mail address: [email protected] (X. Xu). 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2012.03.006
of FIX or FX in a Ca2þ-dependent manner and block the amplification of the coagulation cascade (Ishikawa et al., 2009; Morita, 2005). Anticoagulation factor I (ACF I), a FX binding snaclec (FX snaclec) has been purified from the venom of Agkistrodon acutus (Xu et al., 2000b). ACF I forms a 1:1 complex with activated FX (FXa) in a Ca2þ-dependent fashion, and thereby shows a marked anticoagulant activity in vitro (Xu et al., 2000a). ACF I, as a naturally occurring anticoagulant, is devoid of hemorrhagic and lethal activities, which may be useful both as a basis for designing anticoagulant drugs and as a convenient tool in exploration of the complex mechanisms of the coagulation cascade. ACF I has a typical structure of FIX/X snaclecs (PDB code 1wt9) with a homologous sequence to other FIX/X snaclecs (Hu et al., 2005). Currently, it remains unclear whether ACF I also binds to FIX and whether ACF I also displays an anticoagulant activity in vivo. In this paper, the anticoagulant effect of ACF I was measured in vivo by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT)
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assays and the Ca2þ-induced binding of ACF I to FIX was investigated by native PAGE and surface plasmon resonance (SPR). Our results demonstrate that ACF I also has a marked anticoagulant activity in vivo and inhibits both intrinsic and extrinsic coagulation pathways and that ACF I also binds with FIX in the presence of 0.25 mM Ca2þ.
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administered through the caudal vein (Li et al., 2004). Ten min after administration of ACF I, two milliliter of arterial blood was drawn from the abdominal aorta into 3.8% citrate solution immediately and centrifuged. The plasma was collected for analysis of APTT, PT and TT. APTT, PT and TT were monitored by an Automated Coagulation Analyzer (Sysmex CA-1500, America Dade) (Wong et al., 2011).
2. Materials and methods 2.4. Electrophoresis 2.1. Materials Male Wistar–Imamichi rats (180–250 g, 7–8 weeks old, supplied by Animal Services Center of Anhui Medical University, China) were used in all experiments. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication 85-23, revised 1996). Lyophilized venom powder was provided by the TUN-XI Snakebite Institute (Anhui, P. R. China). Human FIX was purchased from MyBioSource (San Diego, CA, USA) and its concentration was calculated from its absorption coefficients (A1% 1cm ¼ 13:3) at 280 nm and its molecular weight (Mr ¼ 56 kD). Activated partial thromboplastin time reagent DadeÒ ActinÒ FS, prothrombin time reagent Thromboplastin C Plus and thrombin time reagent Test Thrombin Reagent were purchased from Siemens Healthcare Diagnostics (Deerfield, USA). Chelex-100 was purchased from Bio-Rad Laboratories (Richmond, CA, USA). All other reagents were of analytical reagent grade and produced by the Shanghai Institute of Biochemistry (Shanghai, P.R. China). Milli-Q purified water was used throughout. 2.2. Preparation of protein and solution ACF I was purified by a three-step chromatography procedure of anion-exchange chromatography, gel permeation chromatography and cation-exchange chromatography as described previously (Xu et al., 2000b). The molecular homogeneity of the purified ACF I was identified by polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of ACF I was calculated from its absorption coefficient (A1% 1cm ¼ 31) at 280 nm and its relative molecular weight (Mr ¼ 29.6 kD). The solution of Ca2þ was prepared from CaCl2 in Milli-Q water and standardized by titration with standard EDTA solution. Tris buffer used was freed from any possible contamination of multivalent cations by passage through a column (25 3 cm) of Chelex-100. Ca2þ-free ACF I (apo-ACF I) and Ca2þ-free FIX (apo-FIX) were prepared by dialysis of the purified ACF I and FIX against 2 mM EDTA in 0.02 M Tris– HCl (pH 7.4) for 12 h and then extensively against 0.02 M Tris–HCl (pH 7.4), respectively.
Native PAGE was used for analysis of Ca2þ-induced binding of apo-ACF I with apo-FIX. It was performed in 3% stacking gel at pH 6.8 and 8% separation gel at pH 8.8. Tris Gly solution (pH 8.8) was used as the electrolyte buffer solution. Both of the sample solution and the electrolyte buffer solution as well as polyacrylamide gel contained 0.25 mM Ca2þ to keep apo-ACF I and apo-FIX in the presence of 0.25 mM Ca2þ in the PAGE process. 2.5. Surface plasmon resonance SPR was used for analyzing the binding between apoFIX and apo-ACFI. Measurements were performed at 25 C using a Biacore 3000 instrument. Sensor surfaces were pretreated and then normalized by standard Biacore protocols (Manak and Ferl, 2007). Apo-FIX was diluted in immobilization buffer (1 mM, in 10 mM sodium acetate, pH 5.0) and immobilized onto one flow cell of a CM5 chip. For coupling of apo-FIX to CM5 sensor surfaces, the surfaces were activated by a 2-min pulse of N-ethyl-N-(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (10 ml/ min in PBS buffer), followed by injection of the protein, and then deactivated by a 2-min pulse of ethanolamine (pH 8.5, 10 ml/min). Apo-ACF I at various concentrations (0–4 mM) in 0.02 M Tris–HCl (pH 7.4) containing 0.25 mM Ca2þ was injected over the surface (10 ml/min, 30 ml injection with 300-s wash delay). The surface was regenerated between analyte injections with 1 M NaCl and 1 mM EDTA (50 ml at 20 ml/min). The data were transferred into BiaEvaluation 4.1 and the kinetic parameters, on-rate (kon) and off-rate (koff) were obtained for each interaction by a fit of the data to a 1:1 Langmuir model. The association and dissociation constants (KA and KD) were determined by the quotient kon/ koff and koff/kon, respectively (Scheme 1). 2.6. Steady-state fluorescence spectroscopy All fluorescence measurements were performed using a Shimadzu RF-5000 spectrophotometer using an excitation wavelength of 295 nm at 25 C. Emission spectra were recorded from 300 to 450 nm. The excitation and the emission bandwidths were both set at 5 nm. All spectra were corrected by subtracting the spectrum of the background, lacking the protein but otherwise identical to the
2.3. In vivo anticoagulation measurements
kon To determine the effect of ACF I on coagulation after i.v. administration, rats were anaesthetized using a mixture of 25% urethane and 1% a-chloralose (w/v) given intraperitoneally at a dose of 5 ml/kg body weight. ACF I was
ACF I + FIX
ACF I-FIX koff
Scheme 1. The binding of ACF I to immobilized FIX.
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sample. Each spectrum is the average of three consecutively acquired spectra. All the data were analyzed independently, and the fluorescent values obtained were averaged by three experiments. 3. Results 3.1. Effect of ACF I on APTT, AP and TT in vivo ACF I has been purified by a three-step chromatography procedure of anion-exchange chromatography, gel permeation chromatography and cation-exchange chromatography (Xu et al., 2000b) and is shown to be homogeneous determined by native PAGE and SDS-PAGE, respectively. ACF I shows a marked anticoagulant activity in vitro (Xu et al., 2000b). The anticoagulant effects of ACF I in vivo have been measured by APTT, PT and TT assays. The APTT, PT and TT of rats are 18.2 1.6 s, 14.6 1.3 s and 40.8 2.2 s (mean SD, n ¼ 5), respectively, for the saline group (control). As shown in Fig. 1(A, B), intravenous administration of ACF I prolongs APTT and PT in a dose-dependent manner. APTT is prolonged to more than seven-fold by ACF I at a dose of 1.65 mg/kg. APTT value is too high to be measured at a dose of 5 mg/kg of ACF I, since it exceeds the detecting limit of the instrument. PT is prolonged to more than two-fold by ACF I at a dose of 5 mg/kg. Prolongation of APTT suggests the inhibition of the intrinsic and/or common pathway in blood coagulation, while prolongation of PT suggests the inhibition of the extrinsic pathway (Park et al., 2010). The intrinsic and extrinsic pathways converge by the formation of FXa (Davie et al., 1991). FX is a critical coagulation factor for both intrinsic and extrinsic pathways, while FIX is an intrinsic coagulation factor. ACF I is a FXbinding protein, and the binding of ACF I to FX/FXa results in the prolongation of both APTT and PT, indicating that ACF I inhibits both intrinsic and extrinsic pathways in vivo. Interestingly however, ACF I extends APTT more strongly than PT, suggesting that besides binding to FX/FXa, ACF I probably also binds to FIX like other FIX/X snaclecs (Ishikawa et al., 2009; Morita, 2005), and enhances the inhibition of the intrinsic pathway. As shown in Fig. 1C, no obvious prolongation of TT was observed by ACF I at the dose up to 5 mg/kg, suggesting that ACF I does not inhibit thrombin activity. 3.2. Binding of ACF I with FIX assessed by PAGE Native PAGE was used to examine whether ACF I binds with FIX in the presence of Ca2þ. As shown in Fig. 2A, in the presence of 0.25 mM Ca2þ, the mixture of apo-ACF I and apo-FIX (molar ratio 1:1) produces a single band (lane 2) and the band corresponding to apo-ACF I disappears, suggesting that apo-ACF I should form a complex with apo-FIX in the presence of 0.25 mM Ca2þ. When apo-FIX was mixed with excessive apo-ACF I (1:2 mol/mol) in the presence of 0.25 mM Ca2þ, two bands for apo-ACF I and the complex of apo-FIX and apo-ACF I were observed on the gel (lane 3). These results together indicate that apo-ACF I and apo-FIX forms a 1:1 complex in the presence of 0.25 mM Ca2þ. As shown in Fig. 2B, in the absence of Ca2þ, the mixture of apoACF I and apo-FIX (1:1 mol/mol) produces two bands on the
Fig. 1. Effect of ACF I on APTT, PT and TT in vivo after i.v. administration of ACF I in anesthetized rats. ACF I was administered through the caudal vein in anaesthetized rats at selected doses. Blood samples were taken out 10 min after administration of ACF I, and then APTT, PT and TT were measured, respectively. Means S.E.M., n ¼ 5.
PAGE (lane 2), corresponding to the band of apo-ACF I and the band of apo-FIX, respectively. Obviously, apo-ACF I does not form a complex with apo-FIX in the absence of Ca2þ. These results indicate that ACF I binds with FIX in a Ca2þdependent manner. 3.3. Binding of ACF I with FIX assessed by SPR We also used SPR spectroscopy to investigate the Ca2þinduced binding between ACF I and FIX, using a CM5 chip to which apo-FIX was covalently attached. Fig. 3A shows the association and dissociation curves for 3 mM apo-ACF I
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Fig. 2. Native PAGE analysis of the binding of apo-ACF I to apo-FIX. (A) The samples were electrophoresed in the presence of 0.25 mM Ca2þ. Lane 1, 3 mL apo-FIX; Lane 2, the mixture of 3 mL apo-FIX and 3 mL apo-ACF I; Lane 3, the mixture of 6 mL apo-ACF I and 3 mL apo-FIX; Lane 4, 3 mL apo-ACF I. (B) The samples were electrophoresed in the absence of Ca2þ. Lane 1, 3 mL apo-FIX; Lane 2, the mixture of 3 mL apo-FIX and 3 mL apo-ACF I; Lane 3, 3 mL apo-ACF I. The concentrations of both apo-ACF I and apo-FIX are 15 mM.
interacting with apo-FIX in the absence and presence of 0.25 mM Ca2þ. Apo-ACF I obviously binds with apo-FIX in the presence of 0.25 mM Ca2þ. However, no obvious binding between apo-ACF I and apo-FIX has been observed in the
absence of Ca2þ. Fig. 3B shows the specific binding between apo-ACF I and apo-FIX in the presence of 0.25 mM Ca2þ. The kinetic parameters, kon, koff, KA, and KD, were obtained for the interaction by a fit of the data to a 1:1 Langmuir model. The values of kon, koff, KA and KD are (2.6 0.3) 104 M1s1, (4.9 0.5) 103 s1, (5.3 0.2) 106 M1 and (1.9 0.2) 107 M, respectively. The low KD value indicates that apo-ACF I binds apo-FIX with high affinity in the presence of 0.25 mM Ca2þ. In order to analyze the dependence on the Ca2þ concentration of the binding of apo-ACF I to apo-FIX, we studied the interaction between apo-ACF I to apo-FIX in the presence of Ca2þ of various concentrations. Fig. 4A shows the association and dissociation curves for 3 mM apo-ACF I interacting with apo-FIX in the presence of increasing concentrations of Ca2þ. The surface charge density oscillation of the CM5 chip markedly increases with the increase of Ca2þ at low concentrations of Ca2þ (<0.25 mM) and obviously decreases with the increase of Ca2þ at high concentrations of Ca2þ (>0.25 mM). This result indicates that the binding of apo-ACF I to apo-FIX is dependent on the concentration of Ca2þ and the maximal binding of apoACF I to apo-FIX occurs at 0.25 mM Ca2þ. However, Ca2þ at high concentrations (>0.25 mM) inhibits the binding between apo-ACF I to apo-FIX. 3.4. The effect of Ca2þ on the intrinsic fluorescence of FIX
Fig. 3. SPR kinetic analysis of Ca2þ-induced binding of apo-ACF I with apoFIX. (A) 3 mM apo-ACF I in 0.02 M Tris–HCl (pH 7.4) in the presence and absence of 0.25 mM Ca2þ was injected over an immobilized apo-FIX surface for 2 min, and dissociation was monitored for 3 min. (B) Apo-ACF I in 0.02 M Tris–HCl (pH 7.4) containing 0.25 mM Ca2þ was injected over a apo-FIXimmobilized surface at concentrations of 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM for 2 min, and dissociation was monitored for 3 min. A 1:1 Langmuir model was used to fit the data.
To examine the effect of Ca2þ on the conformation of FIX, the fluorescence measurements of apo-FIX in the absence and presence of 0.25 mM Ca2þ were performed at an excitation wavelength of 295 nm at 25 C. Upon excitation at 295 nm only the Trp residue emission is observed. As shown in Fig. 5A, the maximum emission of apo-FIX is at 333 nm. Addition of 0.25 mM Ca2þ to apo-FIX induces an obvious decrease (12%) of the Trp fluorescence of apo-FIX with no obvious shift in the emission maximum. The binding of Ca2þ to apo-FIX may result in a change of the microenvironment of relevant Trp residue(s) in apo-FIX. Although the change does not affect the hydrophobicity of the environment surrounding the relevant Trp residue(s), it probably causes some quenchers, such as the
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Fig. 4. SPR analysis of the effect of the Ca2þ concentration on the binding of apo-ACF I to apo-FIX. (A) 3 mM apo-ACF I in 0.02 M Tris–HCl (pH 7.4) containing 0, 0.06, 0.12, 0.25, 0.50, 0.75, 1.00, 1.50, 2.00 mM Ca2þ was injected over a apo-FIX-immobilized surface for 2 min, and dissociation was monitored for 3 min. (B) The effect of Ca2þ concentration on the surface charge density oscillation of the CM5 chip at the end of apo-ACF I injection. Each point represents the average of triplicate determinations.
charged carboxyl and/or amino groups, to shift near the relevant Trp residue(s). This shift probably results in the quenching of the fluorescence of the relevant Trp residue(s) of apo-FIX. As shown in Fig. 5B, the Ca2þ-induced quenching of the Trp fluorescence of apo-FIX is timedependent, suggesting that the Ca2þ induces a slow conformational transition of apo-FIX. The kinetics of Ca2þinduced fluorescence quenching was best fitted to a oneexponential term yielding a rate constant value of 0.073 0.001 s1, which corresponds to the process of Ca2þ-induced conformation transition. 4. Discussion Although FIX/X snaclecs from various snake venoms are highly homologous and have very similar structures, they have different binding affinities for FIX and FX (Atoda et al., 1995; Li et al., 2005; Xu et al., 2000b; Zang et al., 2003). According to their binding targets, these proteins can be divided into three groups: FIX binding snake C-type lectin (FIX snaclec), FX snaclec and FIX/X snaclecs. All FIX/X snaclecs are able to bind to the Gla domain of FIX or FX.
Fig. 5. Effect of Ca2þ on the fluorescence spectrum of FIX. (A) The intrinsic fluorescence spectra of 1 mM apo-FIX in 0.02 M Tris–HCl buffer (pH 7.4) in the absence and presence of 0.25 mM Ca2þ after excitation at 295 nm. (B) The fluorescence of 1 mM apo-FIX in 0.02 M Tris–HCl (pH 7.4) was measured at 333 nm by exciting at 295 nm in time-scanning mode. 0.25 mM Ca2þ was added at the down arrow.
Previously, ACF I from the venom of A. acutus has been identified as an FX snaclec in a Ca2þ-dependent manner (Xu et al., 2009). Present results firstly show that ACF I also binds FIX in a Ca2þ-dependent manner. Therefore, ACF I is a FIX/X snaclec that prolongs the clotting time in vivo through its binding with both FIX and FX. By using SPR technique, the KD value between apo-ACF I and apo-FIX in the presence of 0.25 mM Ca2þ has been determined to be (1.9 0.2) 107 M, which is lower than that between ACF I and FXa [(5.3 1.2) 107] (Xu et al., 2009). This result suggests that ACF I possesses a higher binding-affinity to FIX than to FXa. The binding of ACF I with FX/FXa results in the inhibition of both intrinsic and extrinsic coagulation pathways and the prolongation of both APTT and PT, while binding of ACF I with FIX results in the inhibition of the intrinsic coagulation pathway and the prolongation of APTT. ACF I extends APTT more strongly than PT, indicating that the binding of ACF I with FIX may play a dominant role in the anticoagulation activity of ACF I in vivo. ACF I has two Ca2þ-binding sites. Our previous studies showed that the binding of Ca2þ to ACF I increases the structural stability of ACF I, but the binding is not essential for the binding of ACF I with FXa and that the binding of Ca2þ to FXa is essential for the recognition between FXa and ACF I (Xu et al., 2009). FIX is a Ca2þ-binding protein
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with multiple Ca2þ-binding sites in its Gla domain (Sekiya et al., 1996; Shikamoto et al., 2003). The Gla domain is responsible for Ca2þ-dependent phospholipid membrane binding. Ca2þ is required to maintain in vivo function of FIX Gla domain during blood coagulation. Ca2þ can affect the conformation of FIX as determined by fluorescence (Fig. 5). Ca2þ may be required to maintain native conformation of FIX Gla domain. The binding of Ca2þ to FIX Gla domain and subsequent conformational rearrangement may be essential for its recognition of ACF I. In addition to the function of Ca2þ to maintain native conformation of FIX/FX Gla domains, Ca2þ has been found to form a bridge between habu IX-bp, a FIX snaclec from habu snake venom and FIX Gla domain as well as between the phosphatidylserine of membrane and FIX Gla domain (Sekiya et al., 1996). Although we cannot infer whether the interaction of FIX with ACF I also involves Ca2þ bridging between them from the present data, it is certain that Ca2þ is required to maintain in vivo function of FIX Gla domain for its recognition of ACF I. As shown in Fig. 4, Ca2þ at low concentrations (<0.25 mM) can induce the binding of apo-ACF I to apo-FIX. However, Ca2þ at high concentrations (>0.25 mM) inhibits the binding between apo-ACF I to apo-FIX. To our knowledge, Ca2þ has never been found to inhibit the binding between FIX/X snaclecs and FIX or FX. Therefore, Ca2þ has been found, for the first time, to function as a switch for the binding between ACF I and FIX. Further investigation is necessary to elucidate the complex role of Ca2þ in the anticoagulation activity of ACF I. In conclusion, ACF I not only binds FX but also binds FIX in a Ca2þ-dependent manner. ACF I inhibits both intrinsic and extrinsic coagulation pathways. Ca2þ is required to maintain in vivo function of FIX Gla domain for its recognition of ACF I. The binding of ACF I with FIX should play a dominant role in the anticoagulation activity of ACF I in vivo. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 21171157, 20871111, 20571069). Conflict of interest statement The authors declare that there is no conflict of interest. References Atoda, H., Ishikawa, M., Yoshihara, E., Sekiya, F., Morita, T., 1995. Bloodcoagulation factor IX-binding protein from the venom of Trimeresurus-Flavoviridis – purification and characterization. J. Biochem. 118, 965–973. Biardi, J.E., Coss, R.G., 2011. Rock squirrel (Spermophilus variegatus) blood sera affects proteolytic and hemolytic activities of rattlesnake venoms. Toxicon 57, 323–331. Chen, Z.M., Wu, J.B., Zhang, Y., Yu, G.Y., Lee, W.H., Lu, Q.M., Zhang, Y., 2011. Jerdonuxin, a novel snaclec (snake C-type lectin) with platelet aggregation activity from Trimeresurus jerdonii venom. Toxicon 57, 109–116.
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Anticoagulation factor I, a snaclec (snake C-type lectin) from Agkistrodon acutus venom binds to FIX as well as FX: Ca2þ induced binding data Yan Zhang, Xiaolong Xu*, Dengke Shen, Jiajia Song, Mingchun Guo, Xincheng Yan Department of Chemistry, University of Science and Technology of China, No. 96, Jinzhai Road, Hefei, Anhui 230026, PR China
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a b s t r a c t
Article history: Received 27 October 2011 Received in revised form 23 February 2012 Accepted 6 March 2012 Available online 16 March 2012
Anticoagulation factor I (ACF I), a snake C-type lectin (snaclec) from the venom of Agkistrodon acutus binds specifically with activated factor X (FXa) in a Ca2þ-dependent manner and prolongs the blood-clotting time in vitro. In this study, the inhibition of the coagulation pathway by ACF I was measured in vivo by activated partial thromboplastin time and prothrombin time assays and the binding of ACF I to factor IX (FIX) was investigated by native PAGE and surface plasmon resonance. The results indicate that ACF I inhibits both intrinsic and extrinsic coagulation pathways, but does not inhibit thrombin activity. ACF I also binds FIX in a Ca2þ-dependent manner and their maximal binding occurs at 0.25 mM Ca2þ. ACF I has a higher binding-affinity to FIX than to FX. Ca2þ is required to maintain in vivo function of FIX Gla domain for its recognition of ACF I. However, Ca2þ at high concentrations (>0.25 mM) inhibits the binding of ACF I to FIX. Ca2þ functions as a switch for the binding between ACF I and FIX. The results suggest that the binding of ACF I with FIX may play a dominant role in the anticoagulation activity of ACF I in vivo. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Agkistrodon acutuss ACF I Snaclec Ca2þ Factor IX
1. Introduction Coagulation factors IX (FIX) and X (FX) are two critical plasma enzymes that play crucial roles in blood coagulation (Furie and Furie, 2008). They are all Ca2þ-binding proteins and require Ca2þ to express their physiological functions. Both proteins possess an N-terminal g-carboxyglutamic acid (Gla) domain that is essential for the amplification of the coagulation cascade. Snake venoms are rich in a large variety of proteins and enzymes (Biardi and Coss, 2011; Eble et al., 2011; Lane et al., 2011; Sarkar and El-Refael, 2009). A family of FIX and FX binding snake C-type lectins (FIX/X snaclecs) has been identified from various kinds of snake venoms (Atoda et al., 1995; Chen et al., 2011; Clemetson, 2010; Gopinath et al., 2007; Oliveira-Carvalho et al., 2008; Sekiya et al., 1993; Xu et al., 2000b). All FIX/X snaclecs have an anticoagulant activity and are highly homologous with each other. They bind to the Gla domain * Corresponding author. Fax: þ86 551 3603388. E-mail address: [email protected] (X. Xu). 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2012.03.006
of FIX or FX in a Ca2þ-dependent manner and block the amplification of the coagulation cascade (Ishikawa et al., 2009; Morita, 2005). Anticoagulation factor I (ACF I), a FX binding snaclec (FX snaclec) has been purified from the venom of Agkistrodon acutus (Xu et al., 2000b). ACF I forms a 1:1 complex with activated FX (FXa) in a Ca2þ-dependent fashion, and thereby shows a marked anticoagulant activity in vitro (Xu et al., 2000a). ACF I, as a naturally occurring anticoagulant, is devoid of hemorrhagic and lethal activities, which may be useful both as a basis for designing anticoagulant drugs and as a convenient tool in exploration of the complex mechanisms of the coagulation cascade. ACF I has a typical structure of FIX/X snaclecs (PDB code 1wt9) with a homologous sequence to other FIX/X snaclecs (Hu et al., 2005). Currently, it remains unclear whether ACF I also binds to FIX and whether ACF I also displays an anticoagulant activity in vivo. In this paper, the anticoagulant effect of ACF I was measured in vivo by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT)
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assays and the Ca2þ-induced binding of ACF I to FIX was investigated by native PAGE and surface plasmon resonance (SPR). Our results demonstrate that ACF I also has a marked anticoagulant activity in vivo and inhibits both intrinsic and extrinsic coagulation pathways and that ACF I also binds with FIX in the presence of 0.25 mM Ca2þ.
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administered through the caudal vein (Li et al., 2004). Ten min after administration of ACF I, two milliliter of arterial blood was drawn from the abdominal aorta into 3.8% citrate solution immediately and centrifuged. The plasma was collected for analysis of APTT, PT and TT. APTT, PT and TT were monitored by an Automated Coagulation Analyzer (Sysmex CA-1500, America Dade) (Wong et al., 2011).
2. Materials and methods 2.4. Electrophoresis 2.1. Materials Male Wistar–Imamichi rats (180–250 g, 7–8 weeks old, supplied by Animal Services Center of Anhui Medical University, China) were used in all experiments. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication 85-23, revised 1996). Lyophilized venom powder was provided by the TUN-XI Snakebite Institute (Anhui, P. R. China). Human FIX was purchased from MyBioSource (San Diego, CA, USA) and its concentration was calculated from its absorption coefficients (A1% 1cm ¼ 13:3) at 280 nm and its molecular weight (Mr ¼ 56 kD). Activated partial thromboplastin time reagent DadeÒ ActinÒ FS, prothrombin time reagent Thromboplastin C Plus and thrombin time reagent Test Thrombin Reagent were purchased from Siemens Healthcare Diagnostics (Deerfield, USA). Chelex-100 was purchased from Bio-Rad Laboratories (Richmond, CA, USA). All other reagents were of analytical reagent grade and produced by the Shanghai Institute of Biochemistry (Shanghai, P.R. China). Milli-Q purified water was used throughout. 2.2. Preparation of protein and solution ACF I was purified by a three-step chromatography procedure of anion-exchange chromatography, gel permeation chromatography and cation-exchange chromatography as described previously (Xu et al., 2000b). The molecular homogeneity of the purified ACF I was identified by polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of ACF I was calculated from its absorption coefficient (A1% 1cm ¼ 31) at 280 nm and its relative molecular weight (Mr ¼ 29.6 kD). The solution of Ca2þ was prepared from CaCl2 in Milli-Q water and standardized by titration with standard EDTA solution. Tris buffer used was freed from any possible contamination of multivalent cations by passage through a column (25 3 cm) of Chelex-100. Ca2þ-free ACF I (apo-ACF I) and Ca2þ-free FIX (apo-FIX) were prepared by dialysis of the purified ACF I and FIX against 2 mM EDTA in 0.02 M Tris– HCl (pH 7.4) for 12 h and then extensively against 0.02 M Tris–HCl (pH 7.4), respectively.
Native PAGE was used for analysis of Ca2þ-induced binding of apo-ACF I with apo-FIX. It was performed in 3% stacking gel at pH 6.8 and 8% separation gel at pH 8.8. Tris Gly solution (pH 8.8) was used as the electrolyte buffer solution. Both of the sample solution and the electrolyte buffer solution as well as polyacrylamide gel contained 0.25 mM Ca2þ to keep apo-ACF I and apo-FIX in the presence of 0.25 mM Ca2þ in the PAGE process. 2.5. Surface plasmon resonance SPR was used for analyzing the binding between apoFIX and apo-ACFI. Measurements were performed at 25 C using a Biacore 3000 instrument. Sensor surfaces were pretreated and then normalized by standard Biacore protocols (Manak and Ferl, 2007). Apo-FIX was diluted in immobilization buffer (1 mM, in 10 mM sodium acetate, pH 5.0) and immobilized onto one flow cell of a CM5 chip. For coupling of apo-FIX to CM5 sensor surfaces, the surfaces were activated by a 2-min pulse of N-ethyl-N-(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (10 ml/ min in PBS buffer), followed by injection of the protein, and then deactivated by a 2-min pulse of ethanolamine (pH 8.5, 10 ml/min). Apo-ACF I at various concentrations (0–4 mM) in 0.02 M Tris–HCl (pH 7.4) containing 0.25 mM Ca2þ was injected over the surface (10 ml/min, 30 ml injection with 300-s wash delay). The surface was regenerated between analyte injections with 1 M NaCl and 1 mM EDTA (50 ml at 20 ml/min). The data were transferred into BiaEvaluation 4.1 and the kinetic parameters, on-rate (kon) and off-rate (koff) were obtained for each interaction by a fit of the data to a 1:1 Langmuir model. The association and dissociation constants (KA and KD) were determined by the quotient kon/ koff and koff/kon, respectively (Scheme 1). 2.6. Steady-state fluorescence spectroscopy All fluorescence measurements were performed using a Shimadzu RF-5000 spectrophotometer using an excitation wavelength of 295 nm at 25 C. Emission spectra were recorded from 300 to 450 nm. The excitation and the emission bandwidths were both set at 5 nm. All spectra were corrected by subtracting the spectrum of the background, lacking the protein but otherwise identical to the
2.3. In vivo anticoagulation measurements
kon To determine the effect of ACF I on coagulation after i.v. administration, rats were anaesthetized using a mixture of 25% urethane and 1% a-chloralose (w/v) given intraperitoneally at a dose of 5 ml/kg body weight. ACF I was
ACF I + FIX
ACF I-FIX koff
Scheme 1. The binding of ACF I to immobilized FIX.
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sample. Each spectrum is the average of three consecutively acquired spectra. All the data were analyzed independently, and the fluorescent values obtained were averaged by three experiments. 3. Results 3.1. Effect of ACF I on APTT, AP and TT in vivo ACF I has been purified by a three-step chromatography procedure of anion-exchange chromatography, gel permeation chromatography and cation-exchange chromatography (Xu et al., 2000b) and is shown to be homogeneous determined by native PAGE and SDS-PAGE, respectively. ACF I shows a marked anticoagulant activity in vitro (Xu et al., 2000b). The anticoagulant effects of ACF I in vivo have been measured by APTT, PT and TT assays. The APTT, PT and TT of rats are 18.2 1.6 s, 14.6 1.3 s and 40.8 2.2 s (mean SD, n ¼ 5), respectively, for the saline group (control). As shown in Fig. 1(A, B), intravenous administration of ACF I prolongs APTT and PT in a dose-dependent manner. APTT is prolonged to more than seven-fold by ACF I at a dose of 1.65 mg/kg. APTT value is too high to be measured at a dose of 5 mg/kg of ACF I, since it exceeds the detecting limit of the instrument. PT is prolonged to more than two-fold by ACF I at a dose of 5 mg/kg. Prolongation of APTT suggests the inhibition of the intrinsic and/or common pathway in blood coagulation, while prolongation of PT suggests the inhibition of the extrinsic pathway (Park et al., 2010). The intrinsic and extrinsic pathways converge by the formation of FXa (Davie et al., 1991). FX is a critical coagulation factor for both intrinsic and extrinsic pathways, while FIX is an intrinsic coagulation factor. ACF I is a FXbinding protein, and the binding of ACF I to FX/FXa results in the prolongation of both APTT and PT, indicating that ACF I inhibits both intrinsic and extrinsic pathways in vivo. Interestingly however, ACF I extends APTT more strongly than PT, suggesting that besides binding to FX/FXa, ACF I probably also binds to FIX like other FIX/X snaclecs (Ishikawa et al., 2009; Morita, 2005), and enhances the inhibition of the intrinsic pathway. As shown in Fig. 1C, no obvious prolongation of TT was observed by ACF I at the dose up to 5 mg/kg, suggesting that ACF I does not inhibit thrombin activity. 3.2. Binding of ACF I with FIX assessed by PAGE Native PAGE was used to examine whether ACF I binds with FIX in the presence of Ca2þ. As shown in Fig. 2A, in the presence of 0.25 mM Ca2þ, the mixture of apo-ACF I and apo-FIX (molar ratio 1:1) produces a single band (lane 2) and the band corresponding to apo-ACF I disappears, suggesting that apo-ACF I should form a complex with apo-FIX in the presence of 0.25 mM Ca2þ. When apo-FIX was mixed with excessive apo-ACF I (1:2 mol/mol) in the presence of 0.25 mM Ca2þ, two bands for apo-ACF I and the complex of apo-FIX and apo-ACF I were observed on the gel (lane 3). These results together indicate that apo-ACF I and apo-FIX forms a 1:1 complex in the presence of 0.25 mM Ca2þ. As shown in Fig. 2B, in the absence of Ca2þ, the mixture of apoACF I and apo-FIX (1:1 mol/mol) produces two bands on the
Fig. 1. Effect of ACF I on APTT, PT and TT in vivo after i.v. administration of ACF I in anesthetized rats. ACF I was administered through the caudal vein in anaesthetized rats at selected doses. Blood samples were taken out 10 min after administration of ACF I, and then APTT, PT and TT were measured, respectively. Means S.E.M., n ¼ 5.
PAGE (lane 2), corresponding to the band of apo-ACF I and the band of apo-FIX, respectively. Obviously, apo-ACF I does not form a complex with apo-FIX in the absence of Ca2þ. These results indicate that ACF I binds with FIX in a Ca2þdependent manner. 3.3. Binding of ACF I with FIX assessed by SPR We also used SPR spectroscopy to investigate the Ca2þinduced binding between ACF I and FIX, using a CM5 chip to which apo-FIX was covalently attached. Fig. 3A shows the association and dissociation curves for 3 mM apo-ACF I
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Fig. 2. Native PAGE analysis of the binding of apo-ACF I to apo-FIX. (A) The samples were electrophoresed in the presence of 0.25 mM Ca2þ. Lane 1, 3 mL apo-FIX; Lane 2, the mixture of 3 mL apo-FIX and 3 mL apo-ACF I; Lane 3, the mixture of 6 mL apo-ACF I and 3 mL apo-FIX; Lane 4, 3 mL apo-ACF I. (B) The samples were electrophoresed in the absence of Ca2þ. Lane 1, 3 mL apo-FIX; Lane 2, the mixture of 3 mL apo-FIX and 3 mL apo-ACF I; Lane 3, 3 mL apo-ACF I. The concentrations of both apo-ACF I and apo-FIX are 15 mM.
interacting with apo-FIX in the absence and presence of 0.25 mM Ca2þ. Apo-ACF I obviously binds with apo-FIX in the presence of 0.25 mM Ca2þ. However, no obvious binding between apo-ACF I and apo-FIX has been observed in the
absence of Ca2þ. Fig. 3B shows the specific binding between apo-ACF I and apo-FIX in the presence of 0.25 mM Ca2þ. The kinetic parameters, kon, koff, KA, and KD, were obtained for the interaction by a fit of the data to a 1:1 Langmuir model. The values of kon, koff, KA and KD are (2.6 0.3) 104 M1s1, (4.9 0.5) 103 s1, (5.3 0.2) 106 M1 and (1.9 0.2) 107 M, respectively. The low KD value indicates that apo-ACF I binds apo-FIX with high affinity in the presence of 0.25 mM Ca2þ. In order to analyze the dependence on the Ca2þ concentration of the binding of apo-ACF I to apo-FIX, we studied the interaction between apo-ACF I to apo-FIX in the presence of Ca2þ of various concentrations. Fig. 4A shows the association and dissociation curves for 3 mM apo-ACF I interacting with apo-FIX in the presence of increasing concentrations of Ca2þ. The surface charge density oscillation of the CM5 chip markedly increases with the increase of Ca2þ at low concentrations of Ca2þ (<0.25 mM) and obviously decreases with the increase of Ca2þ at high concentrations of Ca2þ (>0.25 mM). This result indicates that the binding of apo-ACF I to apo-FIX is dependent on the concentration of Ca2þ and the maximal binding of apoACF I to apo-FIX occurs at 0.25 mM Ca2þ. However, Ca2þ at high concentrations (>0.25 mM) inhibits the binding between apo-ACF I to apo-FIX. 3.4. The effect of Ca2þ on the intrinsic fluorescence of FIX
Fig. 3. SPR kinetic analysis of Ca2þ-induced binding of apo-ACF I with apoFIX. (A) 3 mM apo-ACF I in 0.02 M Tris–HCl (pH 7.4) in the presence and absence of 0.25 mM Ca2þ was injected over an immobilized apo-FIX surface for 2 min, and dissociation was monitored for 3 min. (B) Apo-ACF I in 0.02 M Tris–HCl (pH 7.4) containing 0.25 mM Ca2þ was injected over a apo-FIXimmobilized surface at concentrations of 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM for 2 min, and dissociation was monitored for 3 min. A 1:1 Langmuir model was used to fit the data.
To examine the effect of Ca2þ on the conformation of FIX, the fluorescence measurements of apo-FIX in the absence and presence of 0.25 mM Ca2þ were performed at an excitation wavelength of 295 nm at 25 C. Upon excitation at 295 nm only the Trp residue emission is observed. As shown in Fig. 5A, the maximum emission of apo-FIX is at 333 nm. Addition of 0.25 mM Ca2þ to apo-FIX induces an obvious decrease (12%) of the Trp fluorescence of apo-FIX with no obvious shift in the emission maximum. The binding of Ca2þ to apo-FIX may result in a change of the microenvironment of relevant Trp residue(s) in apo-FIX. Although the change does not affect the hydrophobicity of the environment surrounding the relevant Trp residue(s), it probably causes some quenchers, such as the
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Fig. 4. SPR analysis of the effect of the Ca2þ concentration on the binding of apo-ACF I to apo-FIX. (A) 3 mM apo-ACF I in 0.02 M Tris–HCl (pH 7.4) containing 0, 0.06, 0.12, 0.25, 0.50, 0.75, 1.00, 1.50, 2.00 mM Ca2þ was injected over a apo-FIX-immobilized surface for 2 min, and dissociation was monitored for 3 min. (B) The effect of Ca2þ concentration on the surface charge density oscillation of the CM5 chip at the end of apo-ACF I injection. Each point represents the average of triplicate determinations.
charged carboxyl and/or amino groups, to shift near the relevant Trp residue(s). This shift probably results in the quenching of the fluorescence of the relevant Trp residue(s) of apo-FIX. As shown in Fig. 5B, the Ca2þ-induced quenching of the Trp fluorescence of apo-FIX is timedependent, suggesting that the Ca2þ induces a slow conformational transition of apo-FIX. The kinetics of Ca2þinduced fluorescence quenching was best fitted to a oneexponential term yielding a rate constant value of 0.073 0.001 s1, which corresponds to the process of Ca2þ-induced conformation transition. 4. Discussion Although FIX/X snaclecs from various snake venoms are highly homologous and have very similar structures, they have different binding affinities for FIX and FX (Atoda et al., 1995; Li et al., 2005; Xu et al., 2000b; Zang et al., 2003). According to their binding targets, these proteins can be divided into three groups: FIX binding snake C-type lectin (FIX snaclec), FX snaclec and FIX/X snaclecs. All FIX/X snaclecs are able to bind to the Gla domain of FIX or FX.
Fig. 5. Effect of Ca2þ on the fluorescence spectrum of FIX. (A) The intrinsic fluorescence spectra of 1 mM apo-FIX in 0.02 M Tris–HCl buffer (pH 7.4) in the absence and presence of 0.25 mM Ca2þ after excitation at 295 nm. (B) The fluorescence of 1 mM apo-FIX in 0.02 M Tris–HCl (pH 7.4) was measured at 333 nm by exciting at 295 nm in time-scanning mode. 0.25 mM Ca2þ was added at the down arrow.
Previously, ACF I from the venom of A. acutus has been identified as an FX snaclec in a Ca2þ-dependent manner (Xu et al., 2009). Present results firstly show that ACF I also binds FIX in a Ca2þ-dependent manner. Therefore, ACF I is a FIX/X snaclec that prolongs the clotting time in vivo through its binding with both FIX and FX. By using SPR technique, the KD value between apo-ACF I and apo-FIX in the presence of 0.25 mM Ca2þ has been determined to be (1.9 0.2) 107 M, which is lower than that between ACF I and FXa [(5.3 1.2) 107] (Xu et al., 2009). This result suggests that ACF I possesses a higher binding-affinity to FIX than to FXa. The binding of ACF I with FX/FXa results in the inhibition of both intrinsic and extrinsic coagulation pathways and the prolongation of both APTT and PT, while binding of ACF I with FIX results in the inhibition of the intrinsic coagulation pathway and the prolongation of APTT. ACF I extends APTT more strongly than PT, indicating that the binding of ACF I with FIX may play a dominant role in the anticoagulation activity of ACF I in vivo. ACF I has two Ca2þ-binding sites. Our previous studies showed that the binding of Ca2þ to ACF I increases the structural stability of ACF I, but the binding is not essential for the binding of ACF I with FXa and that the binding of Ca2þ to FXa is essential for the recognition between FXa and ACF I (Xu et al., 2009). FIX is a Ca2þ-binding protein
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with multiple Ca2þ-binding sites in its Gla domain (Sekiya et al., 1996; Shikamoto et al., 2003). The Gla domain is responsible for Ca2þ-dependent phospholipid membrane binding. Ca2þ is required to maintain in vivo function of FIX Gla domain during blood coagulation. Ca2þ can affect the conformation of FIX as determined by fluorescence (Fig. 5). Ca2þ may be required to maintain native conformation of FIX Gla domain. The binding of Ca2þ to FIX Gla domain and subsequent conformational rearrangement may be essential for its recognition of ACF I. In addition to the function of Ca2þ to maintain native conformation of FIX/FX Gla domains, Ca2þ has been found to form a bridge between habu IX-bp, a FIX snaclec from habu snake venom and FIX Gla domain as well as between the phosphatidylserine of membrane and FIX Gla domain (Sekiya et al., 1996). Although we cannot infer whether the interaction of FIX with ACF I also involves Ca2þ bridging between them from the present data, it is certain that Ca2þ is required to maintain in vivo function of FIX Gla domain for its recognition of ACF I. As shown in Fig. 4, Ca2þ at low concentrations (<0.25 mM) can induce the binding of apo-ACF I to apo-FIX. However, Ca2þ at high concentrations (>0.25 mM) inhibits the binding between apo-ACF I to apo-FIX. To our knowledge, Ca2þ has never been found to inhibit the binding between FIX/X snaclecs and FIX or FX. Therefore, Ca2þ has been found, for the first time, to function as a switch for the binding between ACF I and FIX. Further investigation is necessary to elucidate the complex role of Ca2þ in the anticoagulation activity of ACF I. In conclusion, ACF I not only binds FX but also binds FIX in a Ca2þ-dependent manner. ACF I inhibits both intrinsic and extrinsic coagulation pathways. Ca2þ is required to maintain in vivo function of FIX Gla domain for its recognition of ACF I. The binding of ACF I with FIX should play a dominant role in the anticoagulation activity of ACF I in vivo. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 21171157, 20871111, 20571069). Conflict of interest statement The authors declare that there is no conflict of interest. References Atoda, H., Ishikawa, M., Yoshihara, E., Sekiya, F., Morita, T., 1995. Bloodcoagulation factor IX-binding protein from the venom of Trimeresurus-Flavoviridis – purification and characterization. J. Biochem. 118, 965–973. Biardi, J.E., Coss, R.G., 2011. Rock squirrel (Spermophilus variegatus) blood sera affects proteolytic and hemolytic activities of rattlesnake venoms. Toxicon 57, 323–331. Chen, Z.M., Wu, J.B., Zhang, Y., Yu, G.Y., Lee, W.H., Lu, Q.M., Zhang, Y., 2011. Jerdonuxin, a novel snaclec (snake C-type lectin) with platelet aggregation activity from Trimeresurus jerdonii venom. Toxicon 57, 109–116.
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