Factor XII-Deficient Chicken Plasma as a Useful Target for Screening of Pro- and Anticoagulant Animal Venom Toxins.

Benedito C Prezoto,Nancy Oguiura

doi:10.3390/toxins12020079

Abstract

The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [≥ 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to ≥ 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution.

Highlights

IntroductionTests to assess the in vivo effects of animal venoms, such as hemorrhage, myonecrosis, defibrination, edema, and venom lethality/antivenom potencies, still largely rely on animal models (usually involving rodents)
Studies of animal venoms and toxins have focused on one or more of the following objectives:(i) To determine the mode and mechanism of action of the toxins; (ii) to find ways and means to neutralize the toxicity and adverse effects of accidents; (iii) to develop specific research tools that are useful in understanding the normal physiological processes at both cellular and molecular levels;(iv) to develop prototypes of pharmaceutical agents based on the structure of toxins [1].Tests to assess the in vivo effects of animal venoms, such as hemorrhage, myonecrosis, defibrination, edema, and venom lethality/antivenom potencies, still largely rely on animal models
The relatively large recalcification time (RT) presented by chicken plasma (CP) allowed for the elaboration of a dose–response curve, and the determination of the relative potencies of both the agonist and antagonist, which caused its sensitivity to be almost 20-fold higher than that of human plasma

Summary

Introduction

Tests to assess the in vivo effects of animal venoms, such as hemorrhage, myonecrosis, defibrination, edema, and venom lethality/antivenom potencies, still largely rely on animal models (usually involving rodents). Toxins 2020, 12, 79 alternative in vitro assays involving non-animal, or nonsentient research methods, as a way to achieve the ‘Three Rs’ goals of animal experimentation, i.e., Reduce, Refine, and Replace animal tests. Current scientific attention is focused on the validation of alternative methods to the screening and characterization of individual venom proteins, which should, in turn, enable novel in vitro assays to be designed, reducing the number of animals required [2]

Objectives

Methods

Results

Discussion

Conclusion