Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms

Barbara M. Zietek,Ben Bruyneel,Julien Slagboom,Freek J. Vonk,Morwarid Mayar,Jeroen Kool,Govert W. Somsen,Nicholas R. Casewell

doi:10.1007/s00216-018-1253-x

Barbara M. Zietek, Ben Bruyneel + Show 6 more

Open Access

https://doi.org/10.1007/s00216-018-1253-x

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Abstract

To better understand envenoming and to facilitate the development of new therapies for snakebite victims, rapid, sensitive, and robust methods for assessing the toxicity of individual venom proteins are required. Metalloproteinases comprise a major protein family responsible for many aspects of venom-induced haemotoxicity including coagulopathy, one of the most devastating effects of snake envenomation, and is characterized by fibrinogen depletion. Snake venoms are also known to contain anti-fibrinolytic agents with therapeutic potential, which makes them a good source of new plasmin inhibitors. The protease plasmin degrades fibrin clots, and changes in its activity can lead to life-threatening levels of fibrinolysis. Here, we present a methodology for the screening of plasmin inhibitors in snake venoms and the simultaneous assessment of general venom protease activity. Venom is first chromatographically separated followed by column effluent collection onto a 384-well plate using nanofractionation. Via a post-column split, mass spectrometry (MS) analysis of the effluent is performed in parallel. The nanofractionated venoms are exposed to a plasmin bioassay, and the resulting bioassay activity chromatograms are correlated to the MS data. To study observed proteolytic activity of venoms in more detail, venom fractions were exposed to variants of the plasmin bioassay in which the assay mixture was enriched with zinc or calcium ions, or the chelating agents EDTA or 1,10-phenanthroline were added. The plasmin activity screening system was applied to snake venoms and successfully detected compounds exhibiting antiplasmin (anti-fibrinolytic) activities in the venom of Daboia russelii, and metal-dependent proteases in the venom of Crotalus basiliscus.Graphical abstractᅟ

Highlights

Plasmin is a trypsin-like enzyme, which plays a crucial role in the fibrinolytic pathway where it hydrolyses blood clots and maintains normal hemostasis
Lyophilized snake venoms were from Daboia russelii (Dr) and Crotalus basiliscus (Cb), which both are from the Viperidae family
Screening snake venoms for plasmin inhibitors andproteinases exhibiting activity similar to plasmin was performed with a fluorescence based bioassay based on the protocol reported by Tervo et al [34]

Summary

Introduction

Plasmin is a trypsin-like enzyme, which plays a crucial role in the fibrinolytic pathway where it hydrolyses blood clots and maintains normal hemostasis. Plasmin originates from plasminogen, and is activated by tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA) [1]. Examples of plasmin inhibitors currently used successfully in the clinic are ε-aminocaproic acid (EACA) and tranexamic acid (TXA). Their potency and selectivity are low, and these drugs have to be administered in high doses, which significantly increases the risk of adverse effects [1]. There is a high demand for new, more potent and selective, anti-fibrinolytic agents

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