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Functional roles of the two distinct domains of halysase, a snake venom metalloprotease, to inhibit human platelet aggregation
BBRC Biochemical and Biophysical Research Communications 339 (2006) 964–970 www.elsevier.com/locate/ybbrc
Functional roles of the two distinct domains of halysase, a snake venom metalloprotease, to inhibit human platelet aggregation q Weon-Kyoo You a, Yoon-Jung Jang b, Kwang-Hoe Chung c, Ok-Hee Jeon b, Doo-Sik Kim a
b,*
Cardiovascular Research Institute, Comprehensive Cancer Center, and Department of Anatomy, University of California, San Francisco, CA, USA b Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Republic of Korea c Cardiovascular Biology Laboratory, BioBud Research Center, Seoul 120-110, Republic of Korea Received 9 November 2005 Available online 28 November 2005
Abstract Halysase, a hemorrhagic metalloprotease, has an apparent molecular weight of 66 kDa and belongs to the class P-III snake venom metalloprotease. Class P-III snake venom metalloproteases have multifunctional domains including a protease domain and a disintegrinlike domain. Halysase was able to preferentially hydrolyze the a-chain of fibrinogen. Proteolytic activity of the enzyme was completely inhibited by metal chelating agents but not by other typical protease inhibitors. The enzyme principally cleaves X-Leu, X-Tyr, X-Phe, and X-Ala peptide bonds of the oxidized insulin B-chain. Halysase strongly suppresses collagen-induced human platelet aggregation in a dose-dependent manner. Apohalysase that is devoid of its metalloprotease activity was also able to inhibit the platelet aggregation to a certain extent. Experimental evidence clearly indicates that each of the two distinct domains of halysase, the metalloprotease and the disintegrin-like domains, plays its characteristic role to inhibit human platelet aggregation. 2005 Elsevier Inc. All rights reserved. Keywords: Hemorrhagic metalloprotease; Snake venom; Platelet aggregation; Disintegrin-like domain; Fibrinogen; Collagen
Snake venoms contain various metalloproteases that are highly toxic, resulting in a severe bleeding by interfering with the blood coagulation and by degrading the basement membrane or extracellular matrix (ECM) components [1]. Most of the venom metalloproteases are classified into four major groups by their domain structures of proteins or q Abbreviations: BSA, bovine serum albumin; CD, Circular dichroism; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethylether)tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; GP IIb–IIIa, glycoprotein IIb–IIIa; HPLC, high-performance liquid chromatography; MALDI-MS, matrixassisted laser desorption and ionization mass spectrometry; MDC, metalloprotease/disintegrin/cysteine-rich; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; RGD, Arg-Gly-Asp; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SECD, Ser-Glu-Cys-Asp; TACTS, 20 mM Tris–HCl (pH 7.5), 0.02% sodium azide, 2 mM calcium chloride, 0.05% Tween 20, 150 mM sodium chloride. * Corresponding author. Fax: +82 2 312 6027. E-mail address: [email protected] (D.-S. Kim).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.083
their cDNAs [2,3]. Low molecular weight hemorrhagic metalloproteases (class P-I) consist of only the protease domain. Class P-II metalloproteases have a protease and a disintegrin domains. And it has been proposed that disintegrins are generated from class P-II metalloprotease by proteolytic processing [4]. High molecular weight hemorrhagic metalloproteases (class P-III) contain a disintegrinlike and a cysteine-rich domain following the protease domain. The disintegrin-like domain contains a disulfidebonded XXCD sequence in place of RGD or KGD sequence that is the characteristic integrin-binding motif of disintegrins. Class P-IV venom metalloproteases have an additional disulfide-linked C-type lectin domain. The most intensively studied snake venom hemorrhagic proteins are hemorrhagic metalloprotease and disintegrin families. These metalloproteases contain a conserved characteristic Zn2+-binding sequence, HEXXHXXGXXH. It has been suggested that hemorrhagic metalloproteases interact in a specific way with platelet surface proteins
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resulting in an alteration of platelet function [5]. Disintegrins are another class of venom proteins that also obstruct blood homeostasis as inhibitors of platelet aggregation by binding to the platelet glycoprotein IIb–IIIa (GP IIb–IIIa) complex [6]. However, the exact mechanism of venom-induced hemorrhage is not fully understood yet. Snake venom-induced hemorrhage is closely related with degradation of ECM proteins by metalloproteases [7,8]. Proteolytic degradation of these proteins may lead to breakdown of the structural integrity of the ECM resulting in functional disruption. The proteolytic attack of these enzymes on ECM proteins is very slow, while the in vivo hemorrhagic effect of the venom occurs within minutes [8,9]. This indicates that the mechanism of metalloprotease-induced hemorrhage may be more complex. In this study, we report the functional characteristics of halysase on platelet aggregation and adhesion. In our previous report, we have found that halysase, a high molecular weight hemorrhagic metalloprotease from Gloydius halys venom, induces apoptosis of vascular endothelial cells [10]. Experimental results in this work demonstrate that the metalloprotease domain and the disintegrin-like-domain of halysase cooperatively contribute to inhibit collagen-induced human platelet aggregation. Materials and methods Materials. Fresh venom of G. halys was obtained from the local snake farm in Korea. Human fibrinogen, trypsin, oxidized insulin B-chain, PMSF, pepstatin, antipain, and iodoacetamide were purchased from Sigma Chemical (St. Louis, MO). SDS–PAGE gel and molecular weight markers were from Invitrogen (Carlsbad, CA). Collagen was obtained from Chron-log (Havertown, PA). GP IIb–IIIa was from Calbiochem (La Jolla, CA). Q-Sepharose (fast flow), Mono-Q HR 5/5 column, and Superdex 75 H/R 10/30 column were products of Amersham Biosciences (Uppsala, Sweden). Purification of halysase. Halysase was purified from G. halys venom as described in our previous report [10]. Briefly, 1 g of crude venom diluted with 50 ml of 20 mM Tris–HCl (pH 8.0) was loaded onto a Q-Sepharose column pre-equilibrated in the same buffer, sufficiently washed, and then eluted with the buffer containing 50 mM NaCl. Metalloprotease activity was assayed by analyzing the degradation pattern of fibrinogen on SDS– PAGE. Active fractions were pooled and concentrated, followed by separating in Superdex 75 HR 10/30 gel filtration column equilibrated with PBS. Fractions that hydrolyze fibrinogen were collected and dialyzed against 20 mM Tris–HCl (pH 8.0). Dialyzed sample was loaded onto a Mono-Q HR 5/5 column and eluted with a linear gradient of 0–1 M NaCl. Purified halysase was analyzed on SDS–PAGE. CD spectra analysis. CD spectra of halysase and apohalysase were measured at room temperature on J-720 spectrophotometer (Jasco International, Tokyo, Japan) in the wavelength range from 190 to 250 nm. The protein sample concentration was 0.2 mg/ml in PBS. The spectra were obtained by average of 10 scans with a scan speed of 10 nm/min. Degradation of fibrinogen by halysase. Fibrinogenolytic activity was measured according to the method as described in the previous report [11]. Human fibrinogen (10 lg) was reacted with 15 nM halysase in 20 mM Tris–HCl (pH 8.0) containing 150 mM NaCl and 2.5 mM CaCl2. The enzyme reaction was carried out at 37 C for 2 h. Inhibition of its proteolytic activity was examined with various protease inhibitors such as EDTA, EGTA, iodoacetamide, PMSF, antipain, and pepstatin. The reaction was performed at 37 C for 1 h in the above reaction buffer containing each inhibitor (5 mM EDTA, EGTA, iodoacetamide, PMSF, and 10 lM of antipain, pepstatin). Apohalysase was prepared by pre-in-
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cubating halysase with 10 mM EDTA in 20 mM Tris–HCl (pH 7.5) at 4 C for 24 h. Then, the buffer was changed three times with 20 mM Tris–HCl (pH 7.5) using Microcon-10 (Millipore, Bedford, MA). The proteolytic activity of apohalysase was also measured by using fibrinogen as a substrate in the above reaction condition in the presence or absence of metal ions containing 2.5 mM CaCl2 and 1 mM ZnCl2. The reaction products were analyzed by SDS–PAGE. Proteolysis of oxidized insulin B-chain by halysase. Oxidized insulin Bchain (250 lM) was incubated with 0.2 lM halysase at 37 C for 48 h in 20 mM ammonium bicarbonate buffer (pH 8.0) containing 5 mM CaCl2. The proteolytic degradation of oxidized B-chain was confirmed with reverse phase HPLC using Delta Pak C18 column (Waters, Milford, USA). The reaction mixture was lyophilized and then analyzed with MALDI-MS spectroscopy. Platelet aggregation assay. Platelet aggregation assay was performed with human whole blood as described in previous report [12]. Fresh whole human blood was diluted with an equal volume of PBS and then pre-incubated with either purified halysase or PBS alone in a Chrono-log Aggregometer (Chrono-log Co.) at 37 C. Platelet aggregation was initiated by the addition of collagen (2 lg/ml). Light transmittance was recorded and the inhibition of platelet aggregation was measured at the maximum aggregation response. Integrin-binding assay. Fibrinogen/GP IIb–IIIa ELISA was performed with the modified method of the previous report [13]. Microtiter plate (96well) was coated with 0.2 ml of human fibrinogen (20 lg/well) in 0.1 M sodium bicarbonate (pH 7.4) buffer at 4 C for 16 h. After blocking with 1% (w/v) bovine serum albumin (BSA) in TACTS for 1 h, the plate was washed sufficiently with TACTS followed by addition of each protein sample with GP IIb–IIIa (40 lg/ml) in TACTS containing 0.5% BSA. After 2 h incubation, mouse anti-GP IIb monoclonal antibody was added. Following additional 1 h incubation, goat anti-mouse Ig-G conjugated to horseradish peroxidase (Bio-Rad) was added. A final wash was performed, and developing solution, 1-Step ABTS (Pierce, Rockford, IL), was added. Then the plate was incubated until color developed. The reaction was stopped with 1% (w/v) SDS solution followed by absorbance measurement at 405 nm. Platelet adhesion assay. Platelet adhesion assay was carried out according to the previous report [14]. Microtiter plate (96-well) was coated with 0.2 ml of human fibrinogen (40 lg/well) or type I collagen (20 lg/ well) for 1 h at 37 C. Platelets were pre-incubated in TyrodeÕs buffer (1 · 108 platelets/ml) for 10 min with 50 nM each protein sample or 10 lg/ ml anti-integrin b1 monoclonal antibody (Chemicon). TyrodeÕs buffer contains 12 mM NaHCO3, 0.3 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 5.5 mM glucose, 2 mM KCl, and 1 mM MgCl2. After washing with PBS, platelets (0.1 ml) were then added to the coated wells followed by incubation of 90 min at 37 C. After washing the wells with PBS, the adherent platelets were reacted for 60 min at 37 C with 0.1 ml lysis buffer containing 0.1 M sodium citrate, 0.1% Triton X-100, and 5 mM p-nitrophenyl phosphate (Sigma), a chromogenic substrate for platelet acid phosphatase. After the reaction was stopped by the addition of 2 N NaOH (0.1 ml), the adherent platelets were estimated by measuring the absorbance at 405 nm.
Results Purification of halysase A hemorrhagic metalloprotease, designated as halysase, was isolated from the G. halys snake venom as described in our previous report [10]. The crude venom was diluted and initially fractionated by anion-exchange chromatography in a column of Q-Sepharose (Fig. 1, lanes 1 and 2). The proteolytic activity in each fraction was assayed by using fibrinogen as a substrate in the process of purification. When the partially purified protein sample containing the proteolytic activity was further fractionated by Superdex
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N-terminal and internal amino acid sequence analyses of the purified protein indicated that halysase belongs to class P-III snake venom metalloprotease containing a protease domain, a disintegrin-like domain, and a cysteine-rich domain (data not shown). Based on the information of its polypeptide sequence, full-length cDNA encoding halysase was cloned from cDNA library which was constructed with G. halys venom gland as described in our previous report [10] (GenBank Accession No. AY149647). Degradation of fibrinogen Fig. 1. Purification of halysase from G. halys venom. Metalloprotease fractions in the purification process were analyzed with 4–20% gradient SDS–PAGE under reducing condition. Lane 1, crude venom; lane 2, fractionated with 50 mM NaCl in Q-Sepharose; lane 3, Superdex 75 gel filtration; lane 4, final preparation obtained from Mono-Q column.
75 gel filtration (Fig. 1, lane 3) and Mono-Q anion-exchange chromatography (Fig. 1, lane 4), the homogeneous enzyme preparation was revealed to be a single-chain polypeptide with an apparent molecular weight of 66 kDa on the SDS–PAGE analysis under reducing condition. The
Snake venom metalloproteases, either hemorrhagic or non-hemorrhagic, generally have fibrinogenolytic activities [3]. The proteolytic activity of halysase was investigated by reacting with human fibrinogen. When the reaction products were analyzed, it was evident that halysase is able to completely degrade the fibrinogen Aa chain in 2 h without significant enzymatic cleavage of b and c chains (Fig. 2A). Among the various types of protease inhibitors, metal-chelating agents such as EDTA and EGTA completely inhibited the enzymatic activity of halysase to degrade the a chain of human fibrinogen (Fig. 2B). To examine whether
Fig. 2. Fibrinogenolytic activity of halysase. (A) Human fibrinogen was reacted with halysase in 20 mM Tris–HCl (pH 8.0) containing 150 mM NaCl and 2.5 mM CaCl2 at 37 C for 120 min, and was also reacted with apohalysase in the above reaction buffer containing additional 1 mM ZnCl2 at 37 C for 120 min. Apohalysase was prepared as described in Materials and methods. (B) Fibrinogenolytic activity was measured under the above reaction condition in the presence of several protease inhibitors such as EDTA, EGTA, iodoacetamide, pepstatin, antipain or PMSF at 37 C for 60 min. The reaction product was analyzed on SDS–PAGE under reducing condition. (C) CD spectra of halysase and apohalysase. CD spectra were obtained with 0.2 mg/ml of each sample protein at room temperature in the wavelength range 190–250 nm.
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apohalysase recovers its proteolytic activity by reconstituting it with metal ions, the apoenzyme was incubated with fibrinogen in the presence of CaCl2 and ZnCl2. There is a report demonstrating that Zn2+ and Ca2+ ions are required for proteolytic and hemorrhagic activities of snake venom metalloproteases [15]. However, apohalysase failed to hydrolyze human fibrinogen Aa chain in the presence of the metal ions (Fig. 2A). As shown in Fig. 2C, it is interesting to note that there is no considerable difference between the CD spectrum of native halysase and that of apohalysase. This CD spectroscopic evidence suggests that apohalysase undergoes no significant conformational change by removing metal ions from the enzyme molecule (Fig. 2C). Proteolysis of oxidized insulin B-chain by halysase To characterize the enzymatic cleavage site by halysase, oxidized insulin B-chain was reacted as a model substrate with the enzyme. The proteolytic fragmentation of insulin B-chain was confirmed by reverse-phase HPLC, and then the cleaved peptide fractions were analyzed by MALDIMS spectroscopy. Based on the mass of the hydrolyzed peptide, the susceptible peptide bonds of the model substrate were determined. Experimental data indicated that halysase preferentially cleaves His10-Leu11, Glu13-Ala14, Ala14-Leu15, Leu15-Tyr16, Tyr16-Leu17, Gly23-Phe24, and Phe24-Phe25 peptide bonds in the oxidized insulin B-chain (Fig. 3). Platelet aggregation Halysase strongly inhibited the collagen-induced platelet aggregation of human whole blood in a dose-dependent manner (Fig. 4). The IC50 of native halysase was determined to be 87 nM, and that of apohalysase was 166 nM in the collagen-induced human platelet aggregation system. Apohalysase, which is devoid of its proteolytic activity, was less potent than native enzyme to inhibit the platelet aggregation even though their protein conformations appear to be similar to each other in terms of CD spectra (Fig. 4). These results clearly demonstrated that enzymatically inactive halysase also exhibits significant degree of the inhibitory function on human platelet aggregation. We have shown in a previous report that the yeast-expressed recombinant disintegrin-like protein, one of the multifunctional domains in halysase, is capable of inhibiting the collagen-induced platelet aggregation. The
Fig. 4. Inhibition of platelet aggregation by halysase. Platelet aggregation was measured in human whole blood system as described in Materials and methods. The IC50 value of native halysase was determined to be 87 nM and that of apohalysase to be 166 nM, respectively. The concentration of halysase or apohalysase was plotted in a log scale.
inhibitory activity of the recombinant disintegrin-like protein was comparable to that of apohalysase [13]. Taking together these experimental data, it is possible to suggest that not only the disintegrin-like domain but also the metalloprotease domain of halysase is responsible for inhibiting the human platelet aggregation. Integrin binding It is well known that the RGD-containing disintegrins inhibit platelet aggregation by binding to the GP IIb–IIIa on the platelet surface [6,16,17]. Since halysase has a disintegrin-like domain containing a DECD sequence, we investigated by the solid phase inhibition assay whether the enzyme interferes with the fibrinogen binding to GP IIb– IIIa. Salmosin, a typical disintegrin having an RGD sequence, remarkably inhibits the binding of GP IIb–IIIa to immobilized fibrinogen (Fig. 5) [18]. Even though the inhibitory function of halysase was remarkably weaker than that of salmosin, halysase was also able to interrupt the binding of GP IIb–IIIa to fibrinogen in a dose-dependent manner (Fig. 5). On the other hand, the interaction between GP IIb–IIIa and fibrinogen could not be interfered by apohalysase. Similar results were obtained in a previous work with the recombinant disintegrin-like domain of halysase, named as halydin [13]. Based on the experimental evidence, it appears that the interrupted GP IIb–IIIa binding to fibrinogen in the presence of halysase is not associated with the antagonism of GP IIb–IIIa which is mediated by the disintegrin-like domain but with the metalloprotease activity of the enzyme that is capable of degrading fibrinogen. Integrin-mediated platelet adhesion
Fig. 3. Proteolytic fragmentation of oxidized insulin B-chain by halysase. The arrowheads indicate the cleavage sites of oxidized insulin B-chain. The cleavage sites were determined by MALDI-MS spectroscopy.
To further clarify the platelet surface integrin that specifically interacts with halysase, human platelet adhesion to fibrinogen and to type I collagen was examined. As
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Discussion
Fig. 5. Inhibition of integrin binding to immobilized fibrinogen. The binding assay of GP IIb–IIIa to fibrinogen was performed by solid-phase ELISA as described in Materials and methods. The IC50 values of salmosin and halysase to inhibit GP IIb–IIIa binding to fibrinogen were determined to be 2.6 and 22 nM, respectively. The concentration of proteins was plotted in a log scale.
shown in Fig. 6, halysase and apohalysase were much more powerful to inhibit the platelet adhesion to type I collagen than the adhesion to fibrinogen. Halydin also exhibits similar inhibitory patterns in the above platelet adhesion systems [13]. Conversely, salmosin showed more potent activity to suppress the platelet adhesion to fibrinogen than to type I collagen. Anti-integrin b1 monoclonal antibody was also capable of preferentially inhibiting the platelet adhesion to type I collagen comparing with the cell adhesion to fibrinogen (Fig. 6). Since integrin a2b1 is the major receptor of type I collagen in the platelet adhesion, it is postulated that halysase containing a DECD motif in its disintegrin-like domain interacts with integrin a2b1, which results in interruption of platelet adhesion to collagen.
Fig. 6. Inhibition of platelet adhesion to immobilized collagen and fibrinogen. Platelet adhesion was measured by the acid phosphatase activity in the adherent platelets as described in Materials and methods. Platelets were pre-incubated with 50 nM each protein sample or 10 lg/ml anti-integrin b1 monoclonal antibody at 37 C for 10 min. Results were expressed as percent of control adhesion in which platelets were preincubated without inhibitor.
Snake venom is a rich source of proteolytic enzymes that induce diverse toxic effects on platelet aggregation and blood coagulation [3]. Generally, hemorrhage is due to snake venom metalloproteases, Zn2+-dependent enzymes that hydrolyze ECM proteins resulting in bleeding [1,7]. However, the defined mechanism of venom-induced hemorrhage is not fully understood. Recently, our group reported the work on the identification and molecular cloning of a novel hemorrhagic metalloprotease from G. halys snake venom, named as halysase, which is capable of inducing endothelial cell apoptosis [10]. The deduced amino acid sequence of halysase revealed that it belongs to the class P-III snake venom metalloprotease containing a protease domain, a disintegrin-like domain, and a cysteine-rich domain. However, the disintegrin-like domain of halysase has a DECD sequence instead of the typical RGD/KGD motif. In this study, we have demonstrated that halysase is able to strongly inhibit the collagen-induced human platelet aggregation. Since both native halysase and enzymatically inactive apohalysase exhibited distinct potency to inhibit human platelet aggregation (Fig. 4), it was possible to suggest that the metalloprotease activity of halysase plays an independent role in anti-platelet aggregation function. Leonardi et al. [19] also demonstrated that the inactivation of proteolytic activity significantly abolished the hemorrhagic function of Vipera ammodytes hemorrhagin. We have shown in a previous report [13] that the recombinant disintegrin-like domain of halysase, named halydin, is also able to inhibit the platelet aggregation to a certain extent which is comparable to that of apohalysase as demonstrated in this work. Therefore, it is reasonable to speculate that the observed residual activity of apohalysase to inhibit human platelet aggregation might be associated with the other functional domain(s) in the protein molecule. Disintegrins containing the typical RGD or KGD sequence are known to be responsible elements of snake venom-induced hemorrhage [6,16,18,20]. Platelet aggregation plays a key role in hemostasis, and occurs by binding of fibrinogen to GP IIb–IIIa receptor complex, a glycoprotein on the platelet surface membrane, where the RGD sequence of fibrinogen is essentially engaged. As demonstrated in Fig. 5, salmosin strongly inhibits the platelet aggregation by blocking the binding of GP IIb–IIIa and fibrinogen [18]. It is interesting, however, that the disintegrin-like domain of halysase or apohalysase exhibited a potent inhibitory activity on the binding of integrin a2b1 to type I collagen which is closely associated with the anti-platelet aggregation (Fig. 6). There are reports indicating that integrin receptors of collagen play important roles in platelet aggregation [21,22]. Taking together the experimental evidence, snake venom-induced hemorrhage by high molecular weight metalloproteases (class P-III) is closely related with the disruption of platelet function. The hemorrhagic mechanism of halysase could be summarized as follows. First, halysase is capable of degrading
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fibrinogen which is one of the key molecules in blood coagulation and platelet aggregation. Second, it has the proteolytic activity on several extracellular matrix proteins which are critical components in the structure and function of the vascular basement. Finally, the disintegrin-like domain of halysase might be interacting with platelet surface integrins such as a2b1 resulting in the inhibition of platelet adhesion and aggregation. It is possible to hypothesize that the two distinct domains, the metalloprotease and the disintegrin domains, of the high molecular weight metalloprotease may cooperatively contribute to the hemorrhagic function of the enzyme. Such a hypothesis can be strongly supported by the reports that the class P-III metalloproteases usually exhibit more hemorrhagic activity than the class P-I metalloprotease containing only a protease domain [3,8]. Halysase preferentially cleaves X-Leu, X-Ala, X-Tyr, and X-Phe peptide bonds in oxidized insulin B-chain, which are generally hydrophobic amino acid residues including aliphatic and aromatic hydrocarbons (Fig. 3). Crystal structures of several snake venom metalloproteases revealed that the active site of venom metalloprotease is a large hydrophobic pocket that is suitable for accommodating bulky and hydrophobic amino acid side chains [23–25]. However, the structural motifs responsible for hemorrhagic activity are not fully elucidated yet. The hemorrhagic mechanism that is associated with in vivo substrates of the metalloprotease also remains to be understood [26,27]. Further investigation on the substrate specificity of the metalloprotease would be valuable to understand the structure–function relationship of hemorrhagic metalloproteases. Several lines of experimental results obtained in this work will provide useful information in understanding the physiological mechanism of hemorrhage that is induced by metalloprotease having multifunctional domains. Further investigation will be helpful to develop novel antithrombotic agents.
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgment [18]
This work was supported by Research Funds of the National Research Laboratory Program (Project No. M1-0400-00-0043) from the Ministry of Science and Technology in Korea.
[19]
References [20] [1] J.B. Bjarnason, J.W. Fox, Snake venom metalloendopepidase: reprolysins, Methods Enzymol. 248 (1995) 345–368. [2] L.A. Hite, L.G. Jia, J.B. Bjarnason, J.W. Fox, cDNA sequences for four snake venom metalloproteinases: structure, classification, and their relationship to mammalian reproductive proteins, Arch. Biochem. Biophys. 308 (1994) 182–191. [3] T. Matsui, Y. Fujimura, K. Titani, Snake venom proteases affecting hemostasis and thrombosis, Biochim. Biophys. Acta 1477 (2000) 146– 156. [4] H. Takeya, S. Nishida, N. Nishino, Y. Makinose, T. Omori-Satoh, H. Nikai, H. Sugihara, S. Iwanaga, Primary structure of platelet aggregation inhibitors (disintegrins) autoproteolytically released from
[21] [22]
[23]
969
snake venom hemorrhgic metalloproteinase and new fluorogenic peptide substrates for these enzymes, J. Biochem. 113 (1993) 473– 483. N. Marsh, Inventory of haemorrhagic factors from snake venoms. Registry of Exogenous Hemostatic Factors. Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis, Thromb. Haemost. 71 (1994) 793–797. S. Niewiarowski, M.A. McLane, M. Kloczewiak, G.J. Stewart, Disintegrins and other naturally occurring antagonists of platelet fibrinogen receptors, Semin. Hematol. 31 (1994) 289–300. E.N. Baravoma, J.D. Shannon, J.B. Bjarnason, J.W. Fox, Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases, Arch. Biochem. Biophys. 275 (1989) 63–71. G.D. Laing, A.M. Moura-da-Silva, Jararhagin and its multiple effects on hemostasis, Toxicon 34 (2005) 987–996. J.M. Gutierrez, A. Rucavado, T. Escalante, C. Diaz, Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage, Toxicon 45 (2005) 997–1011. W.K. You, H.J. Seo, K.H. Chung, D.S. Kim, A novel metalloprotease from Gloydius halys venom induces endothelial cell apoptosis by its protease and disintegrin-like domains, J. Biochem. 134 (2003) 739– 749. O.H. Jeon, D.S. Kim, Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease, Biochem. Mol. Biol. Int. 47 (1999) 417–425. O.H. Jeon, D.S. Kim, Molecular cloning and functional characterization of a snake venom metalloprotease, Eur. J. Biochem. 263 (1999) 526–533. W.K. You, Y.J. Jang, K.H. Chung, D.S. Kim, A novel disintegrinlike domain of a high molecular weight metalloprotease inhibits platelet aggregation, Biochem. Biophys. Res. Commun. 309 (2003) 637–642. P. Bellavite, G. Andrioli, P. Guzzo, P. Arigliano, S. Chirumbolo, F. Manzato, C. Santonastaso, A colorimetric method for the measurement of platelet adhesion in microtiter plates, Anal. Biochem. 216 (1994) 444–450. J. Zhang, Z. Chen, Y. He, X. Xu, Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom, Toxicon 22 (1984) 931–935. T.F. Huang, J.R. Sheu, C.M. Teng, S.W. Chen, C.S. Liu, Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex, J. Biochem. 109 (1991) 328–334. M.A. McLane, E.E. Sanchez, A. Wong, C. Paquette-Straub, J.C. Perez, Disintegrins, Curr. Drug Targets Cardiovasc. Haematol. Disord. 4 (2004) 327–355. I.C. Kang, K.H. Chung, S.J. Lee, Y.D. Yun, H.M. Moon, D.S. Kim, Purification and molecular cloning of a platelet aggregation inhibitor from the snake (Agkistrodon halys brevicaudus) venom, Thromb. Res. 91 (1998) 65–73. A. Leonardi, F. Gubensek, I. Krizaj, Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes, Toxicon 40 (2002) 55–62. R.M. Scarborough, J.W. Rose, M.A. Hsu, D.R. Phillips, V.A. Fried, A.M. Campbell, L. Nannizzi, I.F. Charo, Barbourin. A GPIIb-IIIaspecific integrin antagonist from the venom of Sistrurus m. barbouri, J. Biol. Chem. 266 (1991) 9359–9362. K.J. Clemetson, J.M. Clemetson, Platelet collagen receptors, Thromb. Haemost. 86 (2001) 189–197. H. Ni, J. Freedman, Platelets in hemostasis and thrombosis: role of integrins and their ligands, Transfus. Apheresis Sci. 28 (2003) 257– 264. S. Bilgrami, S. Yadav, P. Kaur, S. Sharma, M. Perbandt, C. Betzel, T.P. Singh, Crystal structure of the disintegrin heterodimer from sawscaled viper (Echis carinatus) at 1.9 A resolution, Biochemistry 44 (2005) 11058–11066.
970
W.-K. You et al. / Biochemical and Biophysical Research Communications 339 (2006) 964–970
[24] W. Gong, X. Zhu, S. Liu, M. Teng, L. Niu, Crystal structures of acutolysin A, a three-disulfide hemorrhagic zinc metalloproteinase from the snake venom of Agkistrodon acutus, J. Mol. Biol. 283 (1998) 657–668. [25] D.H. Souza, H.S. Selistre-de-Araujo, A.M. Moura-da-Silva, M.S. Della-Casa, G. Oliva, R.C. Garratt, Crystallization and preliminary X-ray analysis of jararhagin, a metalloproteinase/disintegrin from Bothrops jararaca snake venom, Acta Crystallogr. D Biol. Crystallogr. 57 (2001) 1135–1137.
[26] M.B. Bolger, S. Swenson, F.S. Markland Jr., Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the X-ray structure of adamalysin II and atrolysin C, AAPS Pharm. Sci. 3 (2001) E16. [27] F.X. Gomis-Ru¨th, L.F. Kress, J. Kellermann, I. Mayr, X. Lee, R. Huber, ˚ X-ray crystal structure of the snake venom zincW. Bode, Refined 2.0 A endopeptidase adamalysin II. Primary and tertiary structure determination, refinement, molecular structure and comparison with astacin, collagenase and thermolysin, J. Mol. Biol. 239 (1994) 513–544.
Functional roles of the two distinct domains of halysase, a snake venom metalloprotease, to inhibit human platelet aggregation q Weon-Kyoo You a, Yoon-Jung Jang b, Kwang-Hoe Chung c, Ok-Hee Jeon b, Doo-Sik Kim a
b,*
Cardiovascular Research Institute, Comprehensive Cancer Center, and Department of Anatomy, University of California, San Francisco, CA, USA b Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Republic of Korea c Cardiovascular Biology Laboratory, BioBud Research Center, Seoul 120-110, Republic of Korea Received 9 November 2005 Available online 28 November 2005
Abstract Halysase, a hemorrhagic metalloprotease, has an apparent molecular weight of 66 kDa and belongs to the class P-III snake venom metalloprotease. Class P-III snake venom metalloproteases have multifunctional domains including a protease domain and a disintegrinlike domain. Halysase was able to preferentially hydrolyze the a-chain of fibrinogen. Proteolytic activity of the enzyme was completely inhibited by metal chelating agents but not by other typical protease inhibitors. The enzyme principally cleaves X-Leu, X-Tyr, X-Phe, and X-Ala peptide bonds of the oxidized insulin B-chain. Halysase strongly suppresses collagen-induced human platelet aggregation in a dose-dependent manner. Apohalysase that is devoid of its metalloprotease activity was also able to inhibit the platelet aggregation to a certain extent. Experimental evidence clearly indicates that each of the two distinct domains of halysase, the metalloprotease and the disintegrin-like domains, plays its characteristic role to inhibit human platelet aggregation. 2005 Elsevier Inc. All rights reserved. Keywords: Hemorrhagic metalloprotease; Snake venom; Platelet aggregation; Disintegrin-like domain; Fibrinogen; Collagen
Snake venoms contain various metalloproteases that are highly toxic, resulting in a severe bleeding by interfering with the blood coagulation and by degrading the basement membrane or extracellular matrix (ECM) components [1]. Most of the venom metalloproteases are classified into four major groups by their domain structures of proteins or q Abbreviations: BSA, bovine serum albumin; CD, Circular dichroism; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethylether)tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; GP IIb–IIIa, glycoprotein IIb–IIIa; HPLC, high-performance liquid chromatography; MALDI-MS, matrixassisted laser desorption and ionization mass spectrometry; MDC, metalloprotease/disintegrin/cysteine-rich; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; RGD, Arg-Gly-Asp; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SECD, Ser-Glu-Cys-Asp; TACTS, 20 mM Tris–HCl (pH 7.5), 0.02% sodium azide, 2 mM calcium chloride, 0.05% Tween 20, 150 mM sodium chloride. * Corresponding author. Fax: +82 2 312 6027. E-mail address: [email protected] (D.-S. Kim).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.083
their cDNAs [2,3]. Low molecular weight hemorrhagic metalloproteases (class P-I) consist of only the protease domain. Class P-II metalloproteases have a protease and a disintegrin domains. And it has been proposed that disintegrins are generated from class P-II metalloprotease by proteolytic processing [4]. High molecular weight hemorrhagic metalloproteases (class P-III) contain a disintegrinlike and a cysteine-rich domain following the protease domain. The disintegrin-like domain contains a disulfidebonded XXCD sequence in place of RGD or KGD sequence that is the characteristic integrin-binding motif of disintegrins. Class P-IV venom metalloproteases have an additional disulfide-linked C-type lectin domain. The most intensively studied snake venom hemorrhagic proteins are hemorrhagic metalloprotease and disintegrin families. These metalloproteases contain a conserved characteristic Zn2+-binding sequence, HEXXHXXGXXH. It has been suggested that hemorrhagic metalloproteases interact in a specific way with platelet surface proteins
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resulting in an alteration of platelet function [5]. Disintegrins are another class of venom proteins that also obstruct blood homeostasis as inhibitors of platelet aggregation by binding to the platelet glycoprotein IIb–IIIa (GP IIb–IIIa) complex [6]. However, the exact mechanism of venom-induced hemorrhage is not fully understood yet. Snake venom-induced hemorrhage is closely related with degradation of ECM proteins by metalloproteases [7,8]. Proteolytic degradation of these proteins may lead to breakdown of the structural integrity of the ECM resulting in functional disruption. The proteolytic attack of these enzymes on ECM proteins is very slow, while the in vivo hemorrhagic effect of the venom occurs within minutes [8,9]. This indicates that the mechanism of metalloprotease-induced hemorrhage may be more complex. In this study, we report the functional characteristics of halysase on platelet aggregation and adhesion. In our previous report, we have found that halysase, a high molecular weight hemorrhagic metalloprotease from Gloydius halys venom, induces apoptosis of vascular endothelial cells [10]. Experimental results in this work demonstrate that the metalloprotease domain and the disintegrin-like-domain of halysase cooperatively contribute to inhibit collagen-induced human platelet aggregation. Materials and methods Materials. Fresh venom of G. halys was obtained from the local snake farm in Korea. Human fibrinogen, trypsin, oxidized insulin B-chain, PMSF, pepstatin, antipain, and iodoacetamide were purchased from Sigma Chemical (St. Louis, MO). SDS–PAGE gel and molecular weight markers were from Invitrogen (Carlsbad, CA). Collagen was obtained from Chron-log (Havertown, PA). GP IIb–IIIa was from Calbiochem (La Jolla, CA). Q-Sepharose (fast flow), Mono-Q HR 5/5 column, and Superdex 75 H/R 10/30 column were products of Amersham Biosciences (Uppsala, Sweden). Purification of halysase. Halysase was purified from G. halys venom as described in our previous report [10]. Briefly, 1 g of crude venom diluted with 50 ml of 20 mM Tris–HCl (pH 8.0) was loaded onto a Q-Sepharose column pre-equilibrated in the same buffer, sufficiently washed, and then eluted with the buffer containing 50 mM NaCl. Metalloprotease activity was assayed by analyzing the degradation pattern of fibrinogen on SDS– PAGE. Active fractions were pooled and concentrated, followed by separating in Superdex 75 HR 10/30 gel filtration column equilibrated with PBS. Fractions that hydrolyze fibrinogen were collected and dialyzed against 20 mM Tris–HCl (pH 8.0). Dialyzed sample was loaded onto a Mono-Q HR 5/5 column and eluted with a linear gradient of 0–1 M NaCl. Purified halysase was analyzed on SDS–PAGE. CD spectra analysis. CD spectra of halysase and apohalysase were measured at room temperature on J-720 spectrophotometer (Jasco International, Tokyo, Japan) in the wavelength range from 190 to 250 nm. The protein sample concentration was 0.2 mg/ml in PBS. The spectra were obtained by average of 10 scans with a scan speed of 10 nm/min. Degradation of fibrinogen by halysase. Fibrinogenolytic activity was measured according to the method as described in the previous report [11]. Human fibrinogen (10 lg) was reacted with 15 nM halysase in 20 mM Tris–HCl (pH 8.0) containing 150 mM NaCl and 2.5 mM CaCl2. The enzyme reaction was carried out at 37 C for 2 h. Inhibition of its proteolytic activity was examined with various protease inhibitors such as EDTA, EGTA, iodoacetamide, PMSF, antipain, and pepstatin. The reaction was performed at 37 C for 1 h in the above reaction buffer containing each inhibitor (5 mM EDTA, EGTA, iodoacetamide, PMSF, and 10 lM of antipain, pepstatin). Apohalysase was prepared by pre-in-
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cubating halysase with 10 mM EDTA in 20 mM Tris–HCl (pH 7.5) at 4 C for 24 h. Then, the buffer was changed three times with 20 mM Tris–HCl (pH 7.5) using Microcon-10 (Millipore, Bedford, MA). The proteolytic activity of apohalysase was also measured by using fibrinogen as a substrate in the above reaction condition in the presence or absence of metal ions containing 2.5 mM CaCl2 and 1 mM ZnCl2. The reaction products were analyzed by SDS–PAGE. Proteolysis of oxidized insulin B-chain by halysase. Oxidized insulin Bchain (250 lM) was incubated with 0.2 lM halysase at 37 C for 48 h in 20 mM ammonium bicarbonate buffer (pH 8.0) containing 5 mM CaCl2. The proteolytic degradation of oxidized B-chain was confirmed with reverse phase HPLC using Delta Pak C18 column (Waters, Milford, USA). The reaction mixture was lyophilized and then analyzed with MALDI-MS spectroscopy. Platelet aggregation assay. Platelet aggregation assay was performed with human whole blood as described in previous report [12]. Fresh whole human blood was diluted with an equal volume of PBS and then pre-incubated with either purified halysase or PBS alone in a Chrono-log Aggregometer (Chrono-log Co.) at 37 C. Platelet aggregation was initiated by the addition of collagen (2 lg/ml). Light transmittance was recorded and the inhibition of platelet aggregation was measured at the maximum aggregation response. Integrin-binding assay. Fibrinogen/GP IIb–IIIa ELISA was performed with the modified method of the previous report [13]. Microtiter plate (96well) was coated with 0.2 ml of human fibrinogen (20 lg/well) in 0.1 M sodium bicarbonate (pH 7.4) buffer at 4 C for 16 h. After blocking with 1% (w/v) bovine serum albumin (BSA) in TACTS for 1 h, the plate was washed sufficiently with TACTS followed by addition of each protein sample with GP IIb–IIIa (40 lg/ml) in TACTS containing 0.5% BSA. After 2 h incubation, mouse anti-GP IIb monoclonal antibody was added. Following additional 1 h incubation, goat anti-mouse Ig-G conjugated to horseradish peroxidase (Bio-Rad) was added. A final wash was performed, and developing solution, 1-Step ABTS (Pierce, Rockford, IL), was added. Then the plate was incubated until color developed. The reaction was stopped with 1% (w/v) SDS solution followed by absorbance measurement at 405 nm. Platelet adhesion assay. Platelet adhesion assay was carried out according to the previous report [14]. Microtiter plate (96-well) was coated with 0.2 ml of human fibrinogen (40 lg/well) or type I collagen (20 lg/ well) for 1 h at 37 C. Platelets were pre-incubated in TyrodeÕs buffer (1 · 108 platelets/ml) for 10 min with 50 nM each protein sample or 10 lg/ ml anti-integrin b1 monoclonal antibody (Chemicon). TyrodeÕs buffer contains 12 mM NaHCO3, 0.3 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 5.5 mM glucose, 2 mM KCl, and 1 mM MgCl2. After washing with PBS, platelets (0.1 ml) were then added to the coated wells followed by incubation of 90 min at 37 C. After washing the wells with PBS, the adherent platelets were reacted for 60 min at 37 C with 0.1 ml lysis buffer containing 0.1 M sodium citrate, 0.1% Triton X-100, and 5 mM p-nitrophenyl phosphate (Sigma), a chromogenic substrate for platelet acid phosphatase. After the reaction was stopped by the addition of 2 N NaOH (0.1 ml), the adherent platelets were estimated by measuring the absorbance at 405 nm.
Results Purification of halysase A hemorrhagic metalloprotease, designated as halysase, was isolated from the G. halys snake venom as described in our previous report [10]. The crude venom was diluted and initially fractionated by anion-exchange chromatography in a column of Q-Sepharose (Fig. 1, lanes 1 and 2). The proteolytic activity in each fraction was assayed by using fibrinogen as a substrate in the process of purification. When the partially purified protein sample containing the proteolytic activity was further fractionated by Superdex
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N-terminal and internal amino acid sequence analyses of the purified protein indicated that halysase belongs to class P-III snake venom metalloprotease containing a protease domain, a disintegrin-like domain, and a cysteine-rich domain (data not shown). Based on the information of its polypeptide sequence, full-length cDNA encoding halysase was cloned from cDNA library which was constructed with G. halys venom gland as described in our previous report [10] (GenBank Accession No. AY149647). Degradation of fibrinogen Fig. 1. Purification of halysase from G. halys venom. Metalloprotease fractions in the purification process were analyzed with 4–20% gradient SDS–PAGE under reducing condition. Lane 1, crude venom; lane 2, fractionated with 50 mM NaCl in Q-Sepharose; lane 3, Superdex 75 gel filtration; lane 4, final preparation obtained from Mono-Q column.
75 gel filtration (Fig. 1, lane 3) and Mono-Q anion-exchange chromatography (Fig. 1, lane 4), the homogeneous enzyme preparation was revealed to be a single-chain polypeptide with an apparent molecular weight of 66 kDa on the SDS–PAGE analysis under reducing condition. The
Snake venom metalloproteases, either hemorrhagic or non-hemorrhagic, generally have fibrinogenolytic activities [3]. The proteolytic activity of halysase was investigated by reacting with human fibrinogen. When the reaction products were analyzed, it was evident that halysase is able to completely degrade the fibrinogen Aa chain in 2 h without significant enzymatic cleavage of b and c chains (Fig. 2A). Among the various types of protease inhibitors, metal-chelating agents such as EDTA and EGTA completely inhibited the enzymatic activity of halysase to degrade the a chain of human fibrinogen (Fig. 2B). To examine whether
Fig. 2. Fibrinogenolytic activity of halysase. (A) Human fibrinogen was reacted with halysase in 20 mM Tris–HCl (pH 8.0) containing 150 mM NaCl and 2.5 mM CaCl2 at 37 C for 120 min, and was also reacted with apohalysase in the above reaction buffer containing additional 1 mM ZnCl2 at 37 C for 120 min. Apohalysase was prepared as described in Materials and methods. (B) Fibrinogenolytic activity was measured under the above reaction condition in the presence of several protease inhibitors such as EDTA, EGTA, iodoacetamide, pepstatin, antipain or PMSF at 37 C for 60 min. The reaction product was analyzed on SDS–PAGE under reducing condition. (C) CD spectra of halysase and apohalysase. CD spectra were obtained with 0.2 mg/ml of each sample protein at room temperature in the wavelength range 190–250 nm.
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apohalysase recovers its proteolytic activity by reconstituting it with metal ions, the apoenzyme was incubated with fibrinogen in the presence of CaCl2 and ZnCl2. There is a report demonstrating that Zn2+ and Ca2+ ions are required for proteolytic and hemorrhagic activities of snake venom metalloproteases [15]. However, apohalysase failed to hydrolyze human fibrinogen Aa chain in the presence of the metal ions (Fig. 2A). As shown in Fig. 2C, it is interesting to note that there is no considerable difference between the CD spectrum of native halysase and that of apohalysase. This CD spectroscopic evidence suggests that apohalysase undergoes no significant conformational change by removing metal ions from the enzyme molecule (Fig. 2C). Proteolysis of oxidized insulin B-chain by halysase To characterize the enzymatic cleavage site by halysase, oxidized insulin B-chain was reacted as a model substrate with the enzyme. The proteolytic fragmentation of insulin B-chain was confirmed by reverse-phase HPLC, and then the cleaved peptide fractions were analyzed by MALDIMS spectroscopy. Based on the mass of the hydrolyzed peptide, the susceptible peptide bonds of the model substrate were determined. Experimental data indicated that halysase preferentially cleaves His10-Leu11, Glu13-Ala14, Ala14-Leu15, Leu15-Tyr16, Tyr16-Leu17, Gly23-Phe24, and Phe24-Phe25 peptide bonds in the oxidized insulin B-chain (Fig. 3). Platelet aggregation Halysase strongly inhibited the collagen-induced platelet aggregation of human whole blood in a dose-dependent manner (Fig. 4). The IC50 of native halysase was determined to be 87 nM, and that of apohalysase was 166 nM in the collagen-induced human platelet aggregation system. Apohalysase, which is devoid of its proteolytic activity, was less potent than native enzyme to inhibit the platelet aggregation even though their protein conformations appear to be similar to each other in terms of CD spectra (Fig. 4). These results clearly demonstrated that enzymatically inactive halysase also exhibits significant degree of the inhibitory function on human platelet aggregation. We have shown in a previous report that the yeast-expressed recombinant disintegrin-like protein, one of the multifunctional domains in halysase, is capable of inhibiting the collagen-induced platelet aggregation. The
Fig. 4. Inhibition of platelet aggregation by halysase. Platelet aggregation was measured in human whole blood system as described in Materials and methods. The IC50 value of native halysase was determined to be 87 nM and that of apohalysase to be 166 nM, respectively. The concentration of halysase or apohalysase was plotted in a log scale.
inhibitory activity of the recombinant disintegrin-like protein was comparable to that of apohalysase [13]. Taking together these experimental data, it is possible to suggest that not only the disintegrin-like domain but also the metalloprotease domain of halysase is responsible for inhibiting the human platelet aggregation. Integrin binding It is well known that the RGD-containing disintegrins inhibit platelet aggregation by binding to the GP IIb–IIIa on the platelet surface [6,16,17]. Since halysase has a disintegrin-like domain containing a DECD sequence, we investigated by the solid phase inhibition assay whether the enzyme interferes with the fibrinogen binding to GP IIb– IIIa. Salmosin, a typical disintegrin having an RGD sequence, remarkably inhibits the binding of GP IIb–IIIa to immobilized fibrinogen (Fig. 5) [18]. Even though the inhibitory function of halysase was remarkably weaker than that of salmosin, halysase was also able to interrupt the binding of GP IIb–IIIa to fibrinogen in a dose-dependent manner (Fig. 5). On the other hand, the interaction between GP IIb–IIIa and fibrinogen could not be interfered by apohalysase. Similar results were obtained in a previous work with the recombinant disintegrin-like domain of halysase, named as halydin [13]. Based on the experimental evidence, it appears that the interrupted GP IIb–IIIa binding to fibrinogen in the presence of halysase is not associated with the antagonism of GP IIb–IIIa which is mediated by the disintegrin-like domain but with the metalloprotease activity of the enzyme that is capable of degrading fibrinogen. Integrin-mediated platelet adhesion
Fig. 3. Proteolytic fragmentation of oxidized insulin B-chain by halysase. The arrowheads indicate the cleavage sites of oxidized insulin B-chain. The cleavage sites were determined by MALDI-MS spectroscopy.
To further clarify the platelet surface integrin that specifically interacts with halysase, human platelet adhesion to fibrinogen and to type I collagen was examined. As
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Discussion
Fig. 5. Inhibition of integrin binding to immobilized fibrinogen. The binding assay of GP IIb–IIIa to fibrinogen was performed by solid-phase ELISA as described in Materials and methods. The IC50 values of salmosin and halysase to inhibit GP IIb–IIIa binding to fibrinogen were determined to be 2.6 and 22 nM, respectively. The concentration of proteins was plotted in a log scale.
shown in Fig. 6, halysase and apohalysase were much more powerful to inhibit the platelet adhesion to type I collagen than the adhesion to fibrinogen. Halydin also exhibits similar inhibitory patterns in the above platelet adhesion systems [13]. Conversely, salmosin showed more potent activity to suppress the platelet adhesion to fibrinogen than to type I collagen. Anti-integrin b1 monoclonal antibody was also capable of preferentially inhibiting the platelet adhesion to type I collagen comparing with the cell adhesion to fibrinogen (Fig. 6). Since integrin a2b1 is the major receptor of type I collagen in the platelet adhesion, it is postulated that halysase containing a DECD motif in its disintegrin-like domain interacts with integrin a2b1, which results in interruption of platelet adhesion to collagen.
Fig. 6. Inhibition of platelet adhesion to immobilized collagen and fibrinogen. Platelet adhesion was measured by the acid phosphatase activity in the adherent platelets as described in Materials and methods. Platelets were pre-incubated with 50 nM each protein sample or 10 lg/ml anti-integrin b1 monoclonal antibody at 37 C for 10 min. Results were expressed as percent of control adhesion in which platelets were preincubated without inhibitor.
Snake venom is a rich source of proteolytic enzymes that induce diverse toxic effects on platelet aggregation and blood coagulation [3]. Generally, hemorrhage is due to snake venom metalloproteases, Zn2+-dependent enzymes that hydrolyze ECM proteins resulting in bleeding [1,7]. However, the defined mechanism of venom-induced hemorrhage is not fully understood. Recently, our group reported the work on the identification and molecular cloning of a novel hemorrhagic metalloprotease from G. halys snake venom, named as halysase, which is capable of inducing endothelial cell apoptosis [10]. The deduced amino acid sequence of halysase revealed that it belongs to the class P-III snake venom metalloprotease containing a protease domain, a disintegrin-like domain, and a cysteine-rich domain. However, the disintegrin-like domain of halysase has a DECD sequence instead of the typical RGD/KGD motif. In this study, we have demonstrated that halysase is able to strongly inhibit the collagen-induced human platelet aggregation. Since both native halysase and enzymatically inactive apohalysase exhibited distinct potency to inhibit human platelet aggregation (Fig. 4), it was possible to suggest that the metalloprotease activity of halysase plays an independent role in anti-platelet aggregation function. Leonardi et al. [19] also demonstrated that the inactivation of proteolytic activity significantly abolished the hemorrhagic function of Vipera ammodytes hemorrhagin. We have shown in a previous report [13] that the recombinant disintegrin-like domain of halysase, named halydin, is also able to inhibit the platelet aggregation to a certain extent which is comparable to that of apohalysase as demonstrated in this work. Therefore, it is reasonable to speculate that the observed residual activity of apohalysase to inhibit human platelet aggregation might be associated with the other functional domain(s) in the protein molecule. Disintegrins containing the typical RGD or KGD sequence are known to be responsible elements of snake venom-induced hemorrhage [6,16,18,20]. Platelet aggregation plays a key role in hemostasis, and occurs by binding of fibrinogen to GP IIb–IIIa receptor complex, a glycoprotein on the platelet surface membrane, where the RGD sequence of fibrinogen is essentially engaged. As demonstrated in Fig. 5, salmosin strongly inhibits the platelet aggregation by blocking the binding of GP IIb–IIIa and fibrinogen [18]. It is interesting, however, that the disintegrin-like domain of halysase or apohalysase exhibited a potent inhibitory activity on the binding of integrin a2b1 to type I collagen which is closely associated with the anti-platelet aggregation (Fig. 6). There are reports indicating that integrin receptors of collagen play important roles in platelet aggregation [21,22]. Taking together the experimental evidence, snake venom-induced hemorrhage by high molecular weight metalloproteases (class P-III) is closely related with the disruption of platelet function. The hemorrhagic mechanism of halysase could be summarized as follows. First, halysase is capable of degrading
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fibrinogen which is one of the key molecules in blood coagulation and platelet aggregation. Second, it has the proteolytic activity on several extracellular matrix proteins which are critical components in the structure and function of the vascular basement. Finally, the disintegrin-like domain of halysase might be interacting with platelet surface integrins such as a2b1 resulting in the inhibition of platelet adhesion and aggregation. It is possible to hypothesize that the two distinct domains, the metalloprotease and the disintegrin domains, of the high molecular weight metalloprotease may cooperatively contribute to the hemorrhagic function of the enzyme. Such a hypothesis can be strongly supported by the reports that the class P-III metalloproteases usually exhibit more hemorrhagic activity than the class P-I metalloprotease containing only a protease domain [3,8]. Halysase preferentially cleaves X-Leu, X-Ala, X-Tyr, and X-Phe peptide bonds in oxidized insulin B-chain, which are generally hydrophobic amino acid residues including aliphatic and aromatic hydrocarbons (Fig. 3). Crystal structures of several snake venom metalloproteases revealed that the active site of venom metalloprotease is a large hydrophobic pocket that is suitable for accommodating bulky and hydrophobic amino acid side chains [23–25]. However, the structural motifs responsible for hemorrhagic activity are not fully elucidated yet. The hemorrhagic mechanism that is associated with in vivo substrates of the metalloprotease also remains to be understood [26,27]. Further investigation on the substrate specificity of the metalloprotease would be valuable to understand the structure–function relationship of hemorrhagic metalloproteases. Several lines of experimental results obtained in this work will provide useful information in understanding the physiological mechanism of hemorrhage that is induced by metalloprotease having multifunctional domains. Further investigation will be helpful to develop novel antithrombotic agents.
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgment [18]
This work was supported by Research Funds of the National Research Laboratory Program (Project No. M1-0400-00-0043) from the Ministry of Science and Technology in Korea.
[19]
References [20] [1] J.B. Bjarnason, J.W. Fox, Snake venom metalloendopepidase: reprolysins, Methods Enzymol. 248 (1995) 345–368. [2] L.A. Hite, L.G. Jia, J.B. Bjarnason, J.W. Fox, cDNA sequences for four snake venom metalloproteinases: structure, classification, and their relationship to mammalian reproductive proteins, Arch. Biochem. Biophys. 308 (1994) 182–191. [3] T. Matsui, Y. Fujimura, K. Titani, Snake venom proteases affecting hemostasis and thrombosis, Biochim. Biophys. Acta 1477 (2000) 146– 156. [4] H. Takeya, S. Nishida, N. Nishino, Y. Makinose, T. Omori-Satoh, H. Nikai, H. Sugihara, S. Iwanaga, Primary structure of platelet aggregation inhibitors (disintegrins) autoproteolytically released from
[21] [22]
[23]
969
snake venom hemorrhgic metalloproteinase and new fluorogenic peptide substrates for these enzymes, J. Biochem. 113 (1993) 473– 483. N. Marsh, Inventory of haemorrhagic factors from snake venoms. Registry of Exogenous Hemostatic Factors. Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis, Thromb. Haemost. 71 (1994) 793–797. S. Niewiarowski, M.A. McLane, M. Kloczewiak, G.J. Stewart, Disintegrins and other naturally occurring antagonists of platelet fibrinogen receptors, Semin. Hematol. 31 (1994) 289–300. E.N. Baravoma, J.D. Shannon, J.B. Bjarnason, J.W. Fox, Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases, Arch. Biochem. Biophys. 275 (1989) 63–71. G.D. Laing, A.M. Moura-da-Silva, Jararhagin and its multiple effects on hemostasis, Toxicon 34 (2005) 987–996. J.M. Gutierrez, A. Rucavado, T. Escalante, C. Diaz, Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage, Toxicon 45 (2005) 997–1011. W.K. You, H.J. Seo, K.H. Chung, D.S. Kim, A novel metalloprotease from Gloydius halys venom induces endothelial cell apoptosis by its protease and disintegrin-like domains, J. Biochem. 134 (2003) 739– 749. O.H. Jeon, D.S. Kim, Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease, Biochem. Mol. Biol. Int. 47 (1999) 417–425. O.H. Jeon, D.S. Kim, Molecular cloning and functional characterization of a snake venom metalloprotease, Eur. J. Biochem. 263 (1999) 526–533. W.K. You, Y.J. Jang, K.H. Chung, D.S. Kim, A novel disintegrinlike domain of a high molecular weight metalloprotease inhibits platelet aggregation, Biochem. Biophys. Res. Commun. 309 (2003) 637–642. P. Bellavite, G. Andrioli, P. Guzzo, P. Arigliano, S. Chirumbolo, F. Manzato, C. Santonastaso, A colorimetric method for the measurement of platelet adhesion in microtiter plates, Anal. Biochem. 216 (1994) 444–450. J. Zhang, Z. Chen, Y. He, X. Xu, Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom, Toxicon 22 (1984) 931–935. T.F. Huang, J.R. Sheu, C.M. Teng, S.W. Chen, C.S. Liu, Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex, J. Biochem. 109 (1991) 328–334. M.A. McLane, E.E. Sanchez, A. Wong, C. Paquette-Straub, J.C. Perez, Disintegrins, Curr. Drug Targets Cardiovasc. Haematol. Disord. 4 (2004) 327–355. I.C. Kang, K.H. Chung, S.J. Lee, Y.D. Yun, H.M. Moon, D.S. Kim, Purification and molecular cloning of a platelet aggregation inhibitor from the snake (Agkistrodon halys brevicaudus) venom, Thromb. Res. 91 (1998) 65–73. A. Leonardi, F. Gubensek, I. Krizaj, Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes, Toxicon 40 (2002) 55–62. R.M. Scarborough, J.W. Rose, M.A. Hsu, D.R. Phillips, V.A. Fried, A.M. Campbell, L. Nannizzi, I.F. Charo, Barbourin. A GPIIb-IIIaspecific integrin antagonist from the venom of Sistrurus m. barbouri, J. Biol. Chem. 266 (1991) 9359–9362. K.J. Clemetson, J.M. Clemetson, Platelet collagen receptors, Thromb. Haemost. 86 (2001) 189–197. H. Ni, J. Freedman, Platelets in hemostasis and thrombosis: role of integrins and their ligands, Transfus. Apheresis Sci. 28 (2003) 257– 264. S. Bilgrami, S. Yadav, P. Kaur, S. Sharma, M. Perbandt, C. Betzel, T.P. Singh, Crystal structure of the disintegrin heterodimer from sawscaled viper (Echis carinatus) at 1.9 A resolution, Biochemistry 44 (2005) 11058–11066.
970
W.-K. You et al. / Biochemical and Biophysical Research Communications 339 (2006) 964–970
[24] W. Gong, X. Zhu, S. Liu, M. Teng, L. Niu, Crystal structures of acutolysin A, a three-disulfide hemorrhagic zinc metalloproteinase from the snake venom of Agkistrodon acutus, J. Mol. Biol. 283 (1998) 657–668. [25] D.H. Souza, H.S. Selistre-de-Araujo, A.M. Moura-da-Silva, M.S. Della-Casa, G. Oliva, R.C. Garratt, Crystallization and preliminary X-ray analysis of jararhagin, a metalloproteinase/disintegrin from Bothrops jararaca snake venom, Acta Crystallogr. D Biol. Crystallogr. 57 (2001) 1135–1137.
[26] M.B. Bolger, S. Swenson, F.S. Markland Jr., Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the X-ray structure of adamalysin II and atrolysin C, AAPS Pharm. Sci. 3 (2001) E16. [27] F.X. Gomis-Ru¨th, L.F. Kress, J. Kellermann, I. Mayr, X. Lee, R. Huber, ˚ X-ray crystal structure of the snake venom zincW. Bode, Refined 2.0 A endopeptidase adamalysin II. Primary and tertiary structure determination, refinement, molecular structure and comparison with astacin, collagenase and thermolysin, J. Mol. Biol. 239 (1994) 513–544.