Functional and Sequence Characterization of Agkicetin, a New Glycoprotein IB Antagonist Isolated from Agkistrodon acutus Venom

doi:10.1006/bbrc.1995.1684

Biochemical and Biophysical Research Communications

Volume 210, Issue 2, 16 May 1995, Pages 472-477

https://doi.org/10.1006/bbrc.1995.1684 Get rights and content

Abstract

A new glycoprotein Ib (GPIb) antagonist, agkicetin, was purified from the venom of Agkistrodon acutus and characterized. It is a disulfide-linked heterodimer consisting subunits of 15 and 14 kDa. The subunits are homologous to each other and to other snake venom proteins of the C-type (Ca²⁺-dependent) lectin superfamily. Agkicetin behaved as a potent antagonist of von Willebrand Factor (vWF)-induced platelet agglutination (IC₅₀=12.5 nM) and bound specifically to GPIb of fixed platelets with high affinity (K_d=38 nM). It did not bind coagulation factor IX and thrombin. Monoclonal antibody against epitope on the N-terminal domain of GPIb competed the binding of agkicetin with platelets. Reduced and alkylated agkicetin lost most of its inhibitory efficacy toward vWF-induced platelet agglutination.

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To date, the venom proteomes of several clinical significant snakes have been studied and reported (Tan et al., 2017a; Tang et al., 2016; Ali et al., 2013; Zainal Abidin et al., 2016; Tan et al., 2015a), including 2 other Viperidae snakes, T. stejnegeri and P. mucrosquamatus, in Taiwan (Villalta et al., 2012). So far, a few of the proteins from D. acutus venom have been isolated and studied (Chen and Tsai, 1995; Wang et al., 2005, 1996a); however, our knowledge of the protein profile of whole D. acutus venom proteome is still incomplete. Thus, in this present study, we use reverse-phase chromatography conjugated with shotgun proteomic technology to investigate the venom proteome of D. acutus, in combination with screening the profile of recognition and cross-recognition by DA-AV and FH-AV.
Deinagkistrodon acutus, also known as the hundred-pace viper or Chinese moccasin, is a clinically significant venomous snake in Taiwan. To address the lack of knowledge on the venom proteome of D. acutus, the venom composition was studied by a bottom-up proteomic approach combining reverse phase high-performance liquid chromatography, SDS-PAGE, and LC–MS/MS analysis. The immunoreactivity and cross-reactivity of Taiwanese freeze-dried D. acutus antivenom (DA-AV) and hemorrhagic antivenom (FH-AV) were investigated, as well. The proteomic analysis revealed the presence of 29 distinct proteins from D. acutus venom belonging to 8 snake venom protein families. Snake venom metalloproteinase (SVMP, 46.86%), C-type lectin (CLEC, 37.59%), phospholipase A₂ (PLA₂, 7.33%) and snake venom serine protease (SVSP, 6.62%) were the most abundant proteins. In addition to DA-AV, FH-AV also showed a profile of broad immunorecognition toward the venom of D. acutus. Remarkably, both antivenoms specifically reacted with the HPLC fractions containing SVMPs, and the titer was 5–10 times higher than fractions of other components. This information helps us to deeply understand the pathophysiology of D. acutus envenomation and guide us to development of more effective antivenom for clinical treatment.
Snake venom components in medicine: From the symbolic rod of Asclepius to tangible medical research and application
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Both mythologically and logically, snakes have always fascinated man. Snakes have attracted both awe and fear not only because of the elegant movement of their limbless bodies, but also because of the potency of their deadly venoms. Practically, in 2017, the world health organization (WHO) listed snake envenomation as a high priority neglected disease, as snakes inflict up to 2.7 million poisonous bites, around 100.000 casualties, and about three times as many invalidities on man. The venoms of poisonous snakes are a cocktail of potent compounds which specifically and avidly target numerous essential molecules with high efficacy. The individual effects of all venom toxins integrate into lethal dysfunctions of almost any organ system. It is this efficacy and specificity of each venom component, which after analysis of its structure and activity may serve as a potential lead structure for chemical imitation. Such toxin mimetics may help in influencing a specific body function pharmaceutically for the sake of man’s health. In this review article, we will give some examples of snake venom components which have spurred the development of novel pharmaceutical compounds. Moreover, we will provide examples where such snake toxin-derived mimetics are in clinical use, trials, or consideration for further pharmaceutical exploitation, especially in the fields of hemostasis, thrombosis, coagulation, and metastasis. Thus, it becomes clear why a snake captured its symbolic place at the Asclepius rod with good reason still nowadays.
Venomics of Calloselasma rhodostoma, the Malayan pit viper: A complex toxin arsenal unraveled
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The second major protein component of C. rhodostoma venom is snake venom C-type lectins, or snaclecs, which consist of two major categories with opposite action on platelet aggregation: the platelet aggregation inhibitor, rhodocetin, and the platelet aggregation inducer, rhodocytin (see below). Interestingly, the correlation of significant snaclecs to anti-platelet activity has also been reported for the venoms of the other basal crotalids: an estimation of 5.5% of snaclecs was noted in the proteome of H. hypnale venom [35], while for D. acutus venom, this could be inferred from its abundant venom-gland transcripts of snaclecs [55] and the presence of agkicetin and aglucetin in the venom [56,57]. Wang et al. [58] showed that rhodocetin has a covalent heterodimeric structure comprising alpha (16 kDa) and beta (15 kDa) subunits, which are dissociated from the complex during reversed-phase HPLC.
The venom of Malayan pit viper (Calloselasma rhodostoma) is highly toxic but also valuable in drug discovery. However, a comprehensive proteome of the venom that details its toxin composition and abundance is lacking. This study aimed to unravel the venom complexity through a multi-step venomic approach. At least 96 distinct proteins (29 basic, 67 acidic) in 11 families were identified from the venom. The venom consists of mainly snake venom metalloproteinases (SVMP, 41.17% of total venom proteins), within which the P-I (kistomin, 20.4%) and P-II (rhodostoxin, 19.8%) classes predominate. This is followed by C-type lectins (snaclec, 26.3%), snake venom serine protease (SVSP, 14.9%), L-amino acid oxidase (7.0%), phospholipase A₂ (4.4%), cysteine-rich secretory protein (2.5%), and five minor toxins (nerve growth factor, neurotrophin, phospholipase B, 5′ nucleotidase and phosphodiesterase, totaling 2.6%) not reported in the proteome hitherto. Importantly, all principal hemotoxins unveiled correlate with the syndrome: SVSP ancrod causes venom-induced consumptive coagulopathy, aggravated by thrombocytopenia caused by snaclec rhodocytin, a platelet aggregation inducer, while P-II rhodostoxin mediates hemorrhage, exacerbated by P-I kistomin and snaclec rhodocetin that inhibit platelet plug formation. These toxins exist in multiple isoforms and/or complex subunits, deserving further characterization for the development of an effective, polyspecific regional antivenom.
Advents in proteomics and bioinformatics have vigorously propelled the scientific discoveries of toxins from various lineages of venomous snakes. The Malayan pit viper, Calloselasma rhodostoma, is a medically important species in Southeast Asia as its bite can cause envenomation, while the venom is also a source of bioactive compounds for drug discovery. Detailed profiling of the venom, however, is inadequate possibly due to the complex nature of the venom and technical limitation in separating the constituents into details. Integrating a multi-step fractionation method, this study successfully revealed a comprehensive and quantitative profile of the composition of the venom of this medically important venomous snake. The relative abundance of the various venom proteins is determined in a global profile, providing useful information for understanding the pathogenic roles of the different toxins in C. rhodostoma envenomation. Notably, the principal hemotoxins were identified in great details, including the variety of toxin subunits and isoforms. The findings indicate that these toxins are the principal targets for effective antivenom neutralization, and should be addressed in the production of a pan-regional polyspecific antivenom. In addition, minor toxin components not reported previously in the venom were also detected in this study, enriching the current toxin database for the venomous snakes.
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Snake venom C-type lectins, which interact with GPIb can either be agonists or antagonists of platelet activation. Jararaca GPIb binding protein (Fujimura et al., 1995), tokaracetin (Kawasaki et al., 1995) or agkicetin (Chen and Tsai, 1995) bind to GPIb without subsequent platelet activation. Moreover they can inhibit platelet activation induced by von Willebrand factor (vWF).
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Since 1995, a number of enzymes with structural and functional diversity have been purified from Agkistrodon acutus snake venom. In 1995, Chen YL et al [7] purified Agkicetin (a heterodimer with 14 and 15 kDa subunits), a GP1b antagonist and platelet inhibitor. In 1998, Chia-Hsin Yeh et al [8] purified Accutin (5.241 kDa), a new member of disintegrin family, which potently inhibits human platelet aggregation.
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Regular ArticleFunctional and Sequence Characterization of Agkicetin, a New Glycoprotein IB Antagonist Isolated from Agkistrodon acutus Venom

Abstract

Regular Article
Functional and Sequence Characterization of Agkicetin, a New Glycoprotein IB Antagonist Isolated from Agkistrodon acutus Venom