Purification and characterization of platelet aggregation inhibitors from snake venoms

Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Los Angeles 90033.
Thrombosis Research (Impact Factor: 2.45). 02/1994; 73(1):39-52. DOI: 10.1016/0049-3848(94)90052-3
Source: PubMed


Proteins that inhibit glycoprotein (GP) IIb/IIIa mediated platelet aggregation have been purified from the venom of two snake species. A small platelet aggregation inhibitor (p1.AI), multisquamatin (Mr = 5,700), was purified from Echis multisquamatus venom by hydrophobic interaction HPLC and two steps on C18 reverse phase HPLC. A larger p1.AI, contortrostatin (Mr = 15,000), was purified by a similar HPLC procedure from the venom of Agkistrodon contortrix contortrix. Both p1.AIs inhibit ADP-induced human, canine and rabbit platelet aggregation using platelet rich plasma (PRP). Multisquamatin has an IC50 of 97 nM, 281 nM and 333 nM for human, canine and rabbit PRP, respectively. Contortrostatin has an IC50 of 49 nM, 120 nM and 1,150 nM for human, canine and rabbit PRP, respectively. In a competitive binding assay using 125I-7E3 (a monoclonal antibody to GPIIb/IIIa that inhibits platelet aggregation) both contortrostatin and multisquamatin demonstrated GPIIb/IIIa specific binding to human and canine platelets. The IC50 for contortrostatin displacement of 7E3 binding to human and canine GPIIb-/IIIa is 27 nM and 16 nM, respectively and for multisquamatin it is 3 nM and 63 nM, respectively. Our results indicate that both p1.AIs inhibit platelet aggregation by binding with high affinity to GPIIb/IIIa.

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    • "It is currently unknown if dimeric disintegrins will produce the same type of vomeronasal response as the monomeric disintegrins did in this current study. A primary activity of disintegrins is the inhibition of platelet aggregation by selectively binding integrin receptors expressed on cell surfaces [25]. The majority of monomeric disintegrins, including crotatroxins 1 and 2, contain an active Arg-Gly-Asp (RGD) sequence [26], which has been shown to block numerous classes of integrin receptors with a high degree of selectivity. "
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    ABSTRACT: Background Vertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fractious and potentially dangerous prey. A unique array of predatory strategies occur among snakes, ranging from mechanical modes of constriction and jaw-holding in non-venomous snakes, to a chemical means, venom, for quickly dispatching prey. However, even among venomous snakes, different prey handling strategies are utilized, varying from the strike-and-hold behaviors exhibited by highly toxic elapid snakes to the rapid strike-and-release envenomation seen in viperid snakes. For vipers, this mode of envenomation represents a minimal risk predatory strategy by permitting little contact with or retaliation from prey, but it adds the additional task of relocating envenomated prey which has wandered from the attack site. This task is further confounded by trails of other unstruck conspecific or heterospecific prey. Despite decades of behavioral study, researchers still do not know the molecular mechanism which allows for prey relocation. Results During behavioral discrimination trials (vomeronasal responsiveness) to euthanized mice injected with size-fractionated venom, Crotalus atrox responded significantly to only one protein peak. Assays for enzymes common in rattlesnake venoms, such as exonuclease, L-amino acid oxidase, metalloproteinase, thrombin-like and kallikrein-like serine proteases and phospholipase A2, showed that vomeronasal responsiveness was not dependent on enzymatic activity. Using mass spectrometry and N-terminal sequencing, we identified the proteins responsible for envenomated prey discrimination as the non-enzymatic disintegrins crotatroxin 1 and 2. Our results demonstrate a novel and critical biological role for venom disintegrins far beyond their well-established role in disruption of cell-cell and cell-extracellular matrix interactions. Conclusions These findings reveal the evolutionary significance of free disintegrins in venoms as the molecular mechanism in vipers allowing for effective relocation of envenomated prey. The presence of free disintegrins in turn has led to evolution of a major behavioral adaptation (strike-and-release), characteristic of only rattlesnakes and other vipers, which exploits and refines the efficiency of a pre-existing chemical means of predation and a highly sensitive olfaction system. This system of a predator chemically tagging prey represents a novel trend in the coevolution of predator-prey relationships.
    Full-text · Article · Mar 2013 · BMC Biology
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    • "Venom of Agkistrodon contortrix contortrix was purchased from Miami Serpentarium (Punta Gorda, FL) and CN was purified as previously described (Trikha et al., 1994b). "
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    ABSTRACT: Vicrostatin (VCN) is a chimeric recombinant disintegrin generated in Origami B (DE3) Escherichia coli as a genetic fusion between the C-terminal tail of a viperid disintegrin echistatin and crotalid disintegrin contortrostatin (CN). The therapeutic modulation of multiple integrin pathways via soluble disintegrins was previously shown by us and others to elicit potent anti-angiogenic and anti-metastatic effects in several animal cancer models. Despite these favorable attributes, these polypeptides are notoriously difficult to produce recombinantly in significant quantity due to their structure which requires the correct pairing of multiple disulfide bonds for biological activity. In this report, we show that VCN can be reliably produced in large amounts (yields in excess of 200 mg of active purified disintegrin per liter of bacterial culture) in Origami B (DE3), an E. coli expression strain engineered to support the folding of disulfide-rich heterologous proteins directly in its oxidative cytoplasmic compartment. VCN retains the integrin binding specificity of both parental molecules it was derived from, but with a different binding affinity profile. While competing for the same integrin receptors that are preferentially upregulated in the tumor microenvironment, VCN exerts a potent inhibitory effect on endothelial cell (EC) migration and tube formation in a dose-dependent manner, by forcing these cells to undergo significant actin cytoskeleton reorganization when exposed to this agent in vitro. Moreover, VCN has a direct effect on breast cancer cells inhibiting their in vitro motility. In an effort to address our main goal of developing a clinically relevant delivery method for recombinant disintegrins, VCN was efficiently packaged in liposomes (LVCN) and evaluated in vivo in an animal breast cancer model. Our data demonstrate that LVCN is well tolerated, its intravenous administration inducing a significant delay in tumor growth and an increase in animal survival, results that can be partially explained by potent tumor apoptotic effects.
    Full-text · Article · Feb 2011 · Toxicon
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    • "Venom of Agkistrodon contortrix contortrix was purchased from Miami Serpentarium (Punta Gorda, FL). CN was purified in a four-step high-performance liquid chromatography (HPLC) procedure according to an established protocol [26]. "
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    ABSTRACT: Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., alphavbeta3, alphavbeta5, and alpha5beta1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.
    Full-text · Article · Jun 2010 · PLoS ONE
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