DAKS1, a Kunitz Scaffold Peptide from the Venom Gland of Deinagkistrodon acutus Prevents Carotid-Artery and Middle-Cerebral-Artery Thrombosis via Targeting Factor XIa

Scaffold-based peptides (SBPs) are fragments of large proteins that are characterized by potent bioactivity, high thermostability, and low immunogenicity. Some SBPs have been approved by the FDA for human use. In the present study, we developed SBPs from the venom gland of Deinagkistrodon acutus (D. acutus) by combining transcriptome sequencing and Pfam annotation. To that end, 10 Kunitz peptides were discovered from the venom gland of D. acutus, and most of which peptides exhibited Factor XIa (FXIa) inhibitory activity. One of those, DAKS1, exhibiting strongest inhibitory activity against FXIa, was further evaluated for its anticoagulant and antithrombotic activity. DAKS1 prolonged twofold APTT at a concentration of 15 μM in vitro. DAKS1 potently inhibited thrombosis in a ferric chloride-induced carotid-artery injury model in mice at a dose of 1.3 mg/kg. Furthermore, DAKS1 prevented stroke in a transient middle cerebral-artery occlusion (tMCAO) model in mice at a dose of 2.6 mg/kg. Additionally, DAKS1 did not show significant bleeding risk at a dose of 6.5 mg/kg. Together, our results indicated that DAKS1 is a promising candidate for drug development for the treatment of thrombosis and stroke disorders.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Varespladib Inhibits the Phospholipase A2 and Coagulopathic Activities of Venom Components from Hemotoxic Snakes

Phospholipase A2 (PLA2) enzymes are important toxins found in many snake venoms, and they can exhibit a variety of toxic activities including causing hemolysis and/or anticoagulation. In this study, the inhibiting effects of the small molecule PLA2 inhibitor varespladib on snake venom PLA2s was investigated by nanofractionation analytics, which combined chromatography, mass spectrometry (MS), and bioassays. The venoms of the medically important snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus, Echis ocellatus, and Oxyuranus scutellatus were separated by liquid chromatography (LC) followed by nanofractionation and interrogation of the fractions by a coagulation assay and a PLA2 assay. Next, we assessed the ability of varespladib to inhibit the activity of enzymatic PLA2s and the coagulopathic toxicities induced by fractionated snake venom toxins, and identified these bioactive venom toxins and those inhibited by varespladib by using parallel recorded LC-MS data and proteomics analysis. We demonstrated here that varespladib was not only capable of inhibiting the PLA2 activities of hemotoxic snake venoms, but can also effectively neutralize the coagulopathic toxicities (most profoundly anticoagulation) induced by venom toxins. While varespladib effectively inhibited PLA2 toxins responsible for anticoagulant effects, we also found some evidence that this inhibitory molecule can partially abrogate procoagulant venom effects caused by different toxin families. These findings further emphasize the potential clinical utility of varespladib in mitigating the toxic effects of certain snakebites.

Characterization of Venoms of Deinagkistrodon acutus and Bungarus multicinctus Using Proteomics and Peptidomics

Background: Deinagkistrodon acutus (D. acutus) and Bungarus multicinctus (B. multicinctus) as traditional medicines have been used for hundreds of years in China. The venoms of these two species have strong toxicity on the victims. Objective: The objective of this study is to reveal the profile of venom proteins and peptides of D. acutus and B. multicinctus. Method: Ultrafiltration, SDS-PAGE coupled with in-gel tryptic digestion and Liquid Chromatography- Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS) were used to characterize proteins and peptides of venoms of D. acutus and B. multicinctus. Results: In the D. acutus venom, 67 proteins (16 protein families) were identified, and snake venom metalloproteinases (SVMPs, 38.0%) and snake venom C-type lectins (snaclecs, 36.7%) were dominated proteins. In the B. multicinctus venom, 47 proteins (15 protein families) were identified, and three-finger toxins (3FTxs, 36.3%) and Kunitz-type Serine Protease Inhibitors (KSPIs, 32.8%) were major components. In addition, both venoms contained small amounts of other proteins, such as Snake Venom Serine Proteinases (SVSPs), Phospholipases A2 (PLA2s), Cysteine-Rich Secreted Proteins (CRISPs), 5'nucleotidases (5'NUCs), Phospholipases B (PLBs), Phosphodiesterases (PDEs), Phospholipase A2 Inhibitors (PLIs), Dipeptidyl Peptidases IV (DPP IVs), L-amino Acid Oxidases (LAAOs) and Angiotensin-Converting Enzymes (ACEs). Each venom also had its unique proteins, Nerve Growth Factors (NGFs) and Hyaluronidases (HYs) in D. acutus, and Cobra Venom Factors (CVFs) in B. multicinctus. In the peptidomics, 1543 and 250 peptides were identified in the venoms of D. acutus and B. multicinctus, respectively. Some peptides showed high similarity with neuropeptides, ACE inhibitory peptides, Bradykinin- Potentiating Peptides (BPPs), LAAOs and movement related peptides. Conclusion: Characterization of venom proteins and peptides of D. acutus and B. multicinctus will be helpful for the treatment of envenomation and drug discovery.

Antivenom Neutralization of Coagulopathic Snake Venom Toxins Assessed by Bioactivity Profiling Using Nanofractionation Analytics

Venomous snakebite is one of the world’s most lethal neglected tropical diseases. Animal-derived antivenoms are the only standardized specific therapies currently available for treating snakebite envenoming, but due to venom variation, often this treatment is not effective in counteracting all clinical symptoms caused by the multitude of injected toxins. In this study, the coagulopathic toxicities of venoms from the medically relevant snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus and Echis ocellatus were assessed. The venoms were separated by liquid chromatography (LC) followed by nanofractionation and parallel mass spectrometry (MS). A recently developed high-throughput coagulation assay was employed to assess both the pro- and anticoagulant activity of separated venom toxins. The neutralization capacity of antivenoms on separated venom components was assessed and the coagulopathic venom peptides and enzymes that were either neutralized or remained active in the presence of antivenom were identified by correlating bioassay results with the MS data and with off-line generated proteomics data. The results showed that most snake venoms analyzed contained both procoagulants and anticoagulants. Most anticoagulants were identified as phospholipases A2s (PLA2s) and most procoagulants correlated with snake venom metalloproteinases (SVMPs) and serine proteases (SVSPs). This information can be used to better understand antivenom neutralization and can aid in the development of next-generation antivenom treatments.

Protective Effects of Carbon Dots Derived from Phellodendri Chinensis Cortex Carbonisata against Deinagkistrodon acutus Venom-Induced Acute Kidney Injury

Background As an emerging nanomaterial, carbon dots (CDs) have been the focus of tremendous attention for biomedical applications. However, little information is available on their bioactivity of inhibiting acute kidney injury (AKI) induced by snake venom. Methods This study reports the development of a green, one-step pyrolysis process to synthesize CDs using Phellodendri Chinensis Cortex (PCC) as the sole precursor, and their potential application as a protectant against Deinagkistrodon acutus (D. acutus) venom-induced AKI was investigated for the first time. The AKI model was established by injecting D. acutus venom into the abdominal cavity of mice and the potential protective effects of PCC Carbonisata-CDs (PCCC-CDs) on renal abnormalities including dysfunction, inflammatory reactions, tissue damage, and thrombocytopenia at six time points (1, 3, and 12 h, and 1, 2, and 5 days) were investigated. Results These results demonstrated that PCCC-CDs significantly inhibited the kidney dysfunction (reduced serum creatinine (SCR), blood urea nitrogen (BUN), urinary total protein (UTP), and microalbuminuria (MALB) concentrations) and the production of chemoattractant (monocyte chemotactic protein 1 (MCP-1)), proinflammatory cytokines (interleukin (IL)-1β), and anti-inflammatory cytokine (IL-10) in response to intraperitoneal injection of D. acutus venom. The beneficial effect of PCCC-CDs on the envenomed mice was similar to that on the change in renal histology and thrombocytopenia. Conclusions These results demonstrated the remarkable protective effects of PCCC-CDs against AKI induced by D. acutus venom, which would not only broaden the biomedical applications of CDs but also provide a potential target for the development of new therapeutic drugs for AKI induced by D. acutus snakebite envenomation.