Multipurpose HTS Coagulation Analysis: Assay Development and Assessment of Coagulopathic Snake Venoms

Abstract

Coagulation assays currently employed are often low throughput, require specialized equipment and/or require large blood/plasma samples. This study describes the development, optimization and early application of a generic low-volume and high-throughput screening (HTS) assay for coagulation activity. The assay is a time-course spectrophotometric measurement which kinetically measures the clotting profile of bovine or human plasma incubated with Ca2+ and a test compound. The HTS assay can be a valuable new tool for coagulation diagnostics in hospitals, for research in coagulation disorders, for drug discovery and for venom research. A major effect following envenomation by many venomous snakes is perturbation of blood coagulation caused by haemotoxic compounds present in the venom. These compounds, such as anticoagulants, are potential leads in drug discovery for cardiovascular diseases. The assay was implemented in an integrated analytical approach consisting of reversed-phase liquid chromatography (LC) for separation of crude venom components in combination with parallel post-column coagulation screening and mass spectrometry (MS). The approach was applied for the rapid assessment and identification of profiles of haemotoxic compounds in snake venoms. Procoagulant and anticoagulant activities were correlated with accurate masses from the parallel MS measurements, facilitating the detection of peptides showing strong anticoagulant activity.

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

“…Recently we developed a 384 well plate-reader based plasma coagulation assay (19), which gave us the opportunity to perform high-throughput profiling of haemotoxic snake venoms. In addition, we developed an ‘at-line’ nanofractionation setup for high-throughput venom screening of individual components in crude venoms towards selected bioactivities (20, 21).…”

“…pro-coagulation) or negative (i.e. anti-coagulation) maxima for each bioactive compound (19). Because each bioactivity chromatogram has a corresponding MS chromatogram obtained in the parallel measurement, the accurate masses of the bioactive peaks can be determined when correlating the peaks from the bioassay chromatograms with the data obtained from MS.…”

Development of high-throughput screening assays for profiling snake venom Phospholipase A2activity after high-resolution chromatographic fractionation

“…The cleaning procedure of the robotic pipetting machine is as reported previously in Still et al. [28] For typical kinetic curve results obtained when running both PLA 2 assay formats with venoms high in PLA 2 abundance, the reader is referred to supporting information S1. The difference in curve slopes allows for the reconstruction of bioactivity chromatograms (more details in Section 3.3).…”

“…For high-resolution fractionation of snake venoms, the analytical system previously described by Still et al [28] and by Mladic et al [29] was used. Samples were injected with a Shimadzu SIL-30AC autosampler and LC separation was performed with a Shimadzu LC system controlled by Lab Solutions software.…”

“…[36,37] The nanofractionation analytics platform connects chromatographic separations of venoms with UV data collection, optionally mass spectrometry, and high-resolution fractionation on well plates followed by bioassaying. This platform for screening snake venoms has been described earlier by others, including for example Mladic et al [29] and Still et al [28] for other assay formats. In this study, snake venoms were separated using RP-LC with a post column split directing 90% of the effluent to a high-resolution fraction collector (e.g.…”