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A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma
Journal of Pharmacological and Toxicological Methods 61 (2010) 27–31
Contents lists available at ScienceDirect
Journal of Pharmacological and Toxicological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j p h a r m t ox
Original article
A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma Margaret A. O'Leary a, Geoffrey K. Isbister a,b,⁎ a b
Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle Hospital, Newcastle, New South Wales, Australia Tropical Toxinology Unit, Menzies School of Health Research, Charles Darwin University, Darwin, Australia
a r t i c l e
i n f o
Article history: Received 17 May 2009 Accepted 16 June 2009 Keywords: Snake venoms Procoagulant Turbidimetric assay Clotting studies Coagulant toxins Assay methods
a b s t r a c t Introduction: Assessment of the procoagulant effect of snake venoms is important for understanding their effects. The aim of this study was to develop a simple automated method to measure clotting times to assess procoagulant venoms. Methods: A turbidimetric assay was developed which monitors changes in optical density when plasma and venom are mixed. Plasma was added simultaneously to venom solutions in a 96 well microtitre plate. After mixing, the optical density at 340 nm was monitored in a microplate reader every 30 s over 30 min. The clotting time was defined as the lag time until the absorbance sharply increased. The turbidimetric method was compared to manual measurement of the clotting time defined as the time when a strand of fibrin can be drawn out of the mixture. The two methods were done simultaneously, with the same venom and plasma, and compared by plotting the manual versus turbidimetric clotting times. Within-day and between-day runs were done and the coefficient of variation (CV) was calculated. Results: Plots comparing manual clotting times to the lag time in the turbidimetric assay showed good correlation between the two methods for brown snake (Pseudonaja textilis) venom, including 24 determinations in triplicate over six days for seven different venom concentrations. Good correlation was also found for four other venoms: tiger snake (Notechis scutatus), Carpet viper (Echis carinatus), Russell's viper (Daboia russelii) and Malaysian pit piper (Calloselasma rhodostoma). Between-day CV was in the range 10–20% for both methods, while within-day CV b 10%. Discussion: The turbidimetric assay appears to be a simple and convenient automated method for the measurement of clotting times to assess the effects of procoagulant venoms. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
1. Introduction Many snake venoms are procoagulant and cause whole blood or plasma to clot when the venom is added to plasma in vitro (Isbister, 2009). Bites by snakes with procoagulant venoms cause consumption of clotting factors leading to a coagulopathy, rather than simple clot formation (Isbister, 2009; Hutton & Warrell, 1993; Isbister, O'Leary, Schneider, Brown & Currie, 2007). In addition to the importance of procoagulant venoms in human envenoming, they have been used as diagnostic and laboratory agents and have been investigated as therapeutic agents (Isbister, 2009). An understanding of their action on the clotting pathways is therefore important for a number of reasons. For procoagulant venoms and toxins, a fundamental measure of venom potency is the time it takes for the venom to cause plasma to clot.
⁎ Corresponding author. Calvary Mater Newcastle Hospital, Edith St, Waratah NSW 2298, Australia. Tel.: +612 4921 1211; fax: +612 4921 1870. E-mail address: [email protected] (G.K. Isbister).
The simplest method for measuring this is combining venom (or a procoagulant component or toxin) and plasma, then observing the time until a thin strand of fibrin appears and can be drawn out of the mixture. A series of venom concentrations may be assessed in this way (Isbister, O'Leary, Schneider, Brown & Currie, 2007; Marshall & Herrmann, 1983; Williams, White & Mirtschin,1994). This simple method has a number of disadvantages such as requiring continuous attention, having an endpoint that is dependent on the observer, not always reproducible on different days and being difficult to do at 37 °C. Instrumental methods aim to improve the accuracy and reproducibility of the clotting time end-point and they may be either mechanical methods, such as the fibrometer (Barnett & Pinto, 1966), or methods that depend on changes in the optical properties of the clotting plasma. The fibrometer is used widely for standard clotting studies such as the prothrombin time and the activated partial thromboplastin time, and has been used for measuring the effects of venom in plasma (Sprivulis, Jelinek & Marshall, 1996). Another important mechanical method is the thromboelastograph which measures a number of dynamic parameters such as clot strength, in addition to the time until clot formation (Salooja & Perry, 2001). Thromboelastography has been used for the assessment of venom effects in plasma (Filippovich et al., 2005) but requires a
1056-8719/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2009.06.004
28
M.A. O'Leary, G.K. Isbister / Journal of Pharmacological and Toxicological Methods 61 (2010) 27–31
dedicated instrument that may not be available in many venom research laboratories. Most optical methods are aimed at detecting particular enzyme functions of the clotting pathway using either chromogenic substrates or fluorogenic substrates (Triplett, 1981). These have been used extensively in venom research, including the substrates for prothrombin, thrombin and Factor X because of the importance of these enzymes in snake venom coagulopathy (Isbister, 2009). However, these assays only provide insight into a particular part of the clotting pathway and are less useful for understanding the effects of venoms on clotting in vivo. More complex systems that measure thrombin generation have been used to measure the downstream effects of procoagulant venoms and may provide a useful approach, but again require specialised equipment (Isbister, O'Leary, Schneider, Brown & Currie, 2007; Lincz et al., 2006; Hemker et al., 2002). Despite the existence of a number of instrumental techniques for the investigation of the clotting effects of venom, most studies have used either manual clotting times or chromogenic assays. We now describe a turbidimetric method for the measurement of clotting times aimed mainly for use with assessing the procoagulant features of snake venoms and toxins. The method is simple and convenient and can be carried out with microtitre plates in a kinetic plate reader, an instrument that is readily available in many laboratories. 2. Materials and methods Fresh frozen plasma was obtained from the Australian Red Cross and aliquots of 10 mL were thawed at 37 °C then spun at 2500 rpm for 10 min. Calcium (60 µL 0.2 M CaCl2/mL) was added immediately before the assay if it was used. Tris-buffered saline (TBS) was used in preference to phosphate buffered saline (PBS) because a precipitate of calcium phosphate may occur with PBS and re-calcified plasma. Eastern or common brown snake (Pseudonaja textilis) venom, Tiger snake (Notechis scutatus) venom, Eastern Diamondback Rattlesnake (Crotalus adamanteus) venom and Malaysian Pit Viper (Calloselasma rhodostoma) venom were purchased from Venom Supplies, South Australia. Russell's viper (Daboia russelii) was obtained from the University of Colombo, Sri Lanka and Carpet Viper (Echis carinatus) was purchased from Sigma. 100 µL aliquots (1.0 mg/mL) of venom were prepared in 0.5% BSA/TBS and stored at − 20 °C. Dilutions were prepared in 0.5% BSA/TBS immediately before use. 2.1. Turbidimetric assay Venom solutions (100 µL) were placed in the wells of a 96 well microtitre plate at room temperature or at 37 °C in a BioTek ELx808 plate reader. Plasma (100 µL) was then added simultaneously to each well at the same temperature, using a multichannel pipette. After a 5-second shake step for mixing, the optical density at 340 nm was monitored every 30 s over 30 to 60 min. This produces a plot of optical density versus time (see Fig. 1). Gen5 software (supplied with the Biotek ELx808) fits the data to an exponential association equation after plateau. Y = IF½XbXo; Plateau; Plateau + SpanTð1 − expð−kð X − XoÞÞÞ Plateau is the initial absorbance, Span is the change in absorbance, and Xo is the lag time at which absorbance begins to sharply increase. The lag time as calculated by the Gen5 software is user-defined as the time when absorbance becomes 0.02 units greater than the average of the first two absorbance measurements. The lag time was taken to be the clotting time because it represented the sharp rise in optical density consistent with a clot beginning to form. 2.2. Manual clotting time Manual clotting times were carried out as a comparison to the turbidimetric assay. Citrated plasma (100 µL) was added at room
Fig. 1. Plots of absorbance versus time for no venom and increasing concentration of P. textilis venom in recalcified plasma (A) and in non-recalcified plasma (B), showing the sharp increase in absorbance rising to plateau, after the lag time.
temperature to venom solutions (100 µL) in a 1 mL conical polystyrene vial. The clotting time was defined as the time when a strand of fibrin could be drawn out of the mixture. 2.3. Comparison of tests The two tests (turbidimetric and manual) were run in parallel on the same day with the same venom preparation and plasma. The turbidimetric and manual clotting times were then compared by plotting the manual versus the turbidimetric clotting time. Withinday and between-day runs were done for both clotting time tests and a coefficient of variation (CV) was calculated for each test. In addition to the lag time calculated by the Gen5 plate-reader software, the raw data was also imported into Prism 5 and the lag time calculated by fitting the data to an exponential association curve. All statistical analysis was done using either Prism 5 or Microsoft Excel 2007. 3. Results Fig. 1 shows the plots of absorbance or optical density versus time for a range of venom concentration with and without calcium, including a control sample with no venom. Following the addition of plasma to the venom solution there is an initial lag time after which there is a sharp rise in the optical density due to the cloudiness of clot formation. The change in optical density follows a hyperbolic curve to a maximum value. The lag time was taken to be the clotting time. We
M.A. O'Leary, G.K. Isbister / Journal of Pharmacological and Toxicological Methods 61 (2010) 27–31
found that the difference in optical density between clotted and nonclotted plasma increases with decreasing wavelength, thus measurements were optimally carried out at a wavelength of 340 nm. Fig. 1 demonstrates that calcium alone will eventually cause clot formation but in the absence of calcium the plasma did not clot. There was also a marked attenuation of the venom effects in the absence of calcium. Table 1 compares the calculated clotting times obtained by fitting the series of brown snake venom concentrations in Fig. 1 to an exponential association curve with times extracted directly from the experimental data. Fig. 2 shows good correlation (R2 = 0.92) between the new turbidimetric assay and the manual method for common brown snake (P. textilis) venom, including 24 determinations in triplicate for both methods over six days, using seven different venom concentrations and plasma from two different batches. Good correlation (R2 N 0.97) was also found for venoms from tiger snake (N. scutatus), Carpet Viper (E. carinatus), Russell's viper (D. russelii) and the Malaysian Pit Viper (C. rhodostoma) which is shown in Fig. 3. It can be seen from these plots that at high concentrations of venom the turbidimetric method gives a shorter clotting time because the cloudiness sets in before a tangible fibrin strand may be withdrawn. At low concentrations of venom the reverse tends to occur. Runs carried out on three successive days gave CV in the range 10 to 20% for both manual and turbidimetric methods, while within-day determinations in triplicate gave values of b10%. The greatest variability occurred at the lowest concentration of venom tested which gave the longest clotting times. Comparisons were carried out at room temperature, because of the impracticality of conducting manual tests at 37 °C. Fig. 4 compares six representative venoms with plots of the clotting times versus Log venom concentration, using the turbidimetric method at 37 °C. The clotting curves provide additional information about the extent of clotting by the total increase in absorbance. In nonrecalcified plasma (Fig. 1B), not only are the lag times increased but also the final plateau is much lower and more dependent on venom concentration compared to recalcified plasma (Fig. 1A). This difference in clot density cannot be detected by the manual method. 4. Discussion We present a simple automated method for the measurement of clotting times to assess the effects of procoagulant venoms on plasma. The method has similar reproducibility to the manual method but provides some additional information and can be done on multiple specimens simultaneously. The method only requires a microplate reader and can be set up in a general laboratory for venom research. We used the assay on six different types of procoagulant venoms that have toxins with different clotting activation mechanisms — Factor X activation (D. russelii), prothrombin (Factor II) activation (E. carinatus, N. scutatus and P. textilis), and cleavage of fibrinogen (C. rhodostoma and C. adamanteus). In all cases, the method provides consistent results when compared with those obtained by the manual method. This technique of observing fibrin generation by monitoring optical density changes over time has been previously applied to the investigation of coagulopathies and factor deficient plasmas (He, Antovic & Blomback, 2001). In these studies, clotting is initiated by
Table 1 Comparison of clotting times (min) for brown snake (Pseudonaja textilis) venom carried out in the plate reader using the Gen5 software package, calculating the clotting time in Prism 5 using the raw data from the plate reader, and the manual clotting time. Venom concentration (ng/mL)
250
125
62.5
31.2
15.6
7.8
3.9
Plate reader Calculated Manual method
2.7 2.7 3.3
4.5 4.6 5.0
6.8 7.2 7.4
10.1 10.8 10.3
14.1 14.9 13.8
17.8 19.6 17.4
23.2 24.4 24.0
29
Fig. 2. Comparison between clotting times from the automatic turbidimetric test and manual clotting times using P. textilis (Brown snake) venom.
either the addition of thrombin (He, Antovic & Blomback, 2001) or tissue factor, (Goldenberg et al., 2008) and subsequent fibrinolysis is ensured by the addition of tissue-type plasminogen activator. The generated curves provide information about the overall haemostatic potential associated with hyper- and hypo-coagulable states such as pregnancy and haemophilia. We have modified this approach and in the turbidimetric assay clotting is initiated by the addition of venom to a constant plasma substrate. We also do not add a plasminogen activator so fibrinolysis does not occur and the resulting curves are simpler and more amenable to mathematical modelling. The variability in the manual and turbidimetric methods is acceptable for both within and between-day assays. The greater variability for longer clotting times may have been because over a longer time period other factors such as dust particle nucleation and inherent clotting mechanisms become relatively more significant. It is important that venom concentrations are selected so that the assay is used for an appropriate range of clotting times (less than 30 min) to avoid this increase variability and unreliability. Although the comparison of the turbidimetric assay to the manual method was carried out at room temperature, the turbidimetric assay can and should be done at 37 °C because this is more representative of the in vivo situation (Fig. 4). This is a further advantage of the turbidimetric assay which is easily done at 37 °C compared to the manual method. The additional information provided by the turbidimetric method (Fig. 1) requires further investigation and comparison to other assay techniques. The height of the final plateau may reflect the amount of fibrin formed which is less in non-recalcified plasma and with lower venom concentrations. This difference cannot be detected by the manual method, but may be analogous to the clot strength parameter obtained by the thromboelastograph (TEG) technique (Filippovich et al., 2005). The venom concentrations used in this study are lower than many previous studies. However, we have shown in a number of studies that these low concentrations are consistent with those found in human envenoming cases (Isbister, O'Leary, Schneider, Brown & Currie, 2007; O'Leary, Isbister, Schneider, Brown & Currie, 2006; O'Leary et al., 2008) and therefore the appropriate concentrations for investigating the pathophysiology of procoagulant venoms and the efficacy of antivenom. The use of higher venom concentrations can be misleading and one such study of Australian snake venoms (Sprivulis, Jelinek & Marshall, 1996) lead to inappropriate increases in antivenom use, before further studies were undertaken at the appropriate low concentrations (Isbister, O'Leary, Schneider, Brown & Currie, 2007). Some snake venoms contain anticoagulants, but only one group of snakes (Pseudechis spp; Australian black snakes) are reported to cause in vivo anticoagulant effects (Currie, 2004). Procoagulant effects are the more common coagulant effects seen with snake envenoming and appear to occur at the low venom concentrations seen in humans
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M.A. O'Leary, G.K. Isbister / Journal of Pharmacological and Toxicological Methods 61 (2010) 27–31
Fig. 3. Comparison between clotting times from the automatic turbidimetric test and manual clotting times for four different venoms: (A) tiger snake (N. scutatus), (B) Russell's viper (D. russelii), (C) Carpet Viper (E. carinatus), and (D) Malaysian Pit Viper (C. rhodostoma) — including trend line (non-dashed) and line of unity (dashed).
(Isbister, 2009). At very high concentrations (orders of magnitude greater than those seen in envenomed humans), other snake venoms may have anticoagulant in vitro effects, but these are unlikely to be clinically relevant.
Fig. 4 provides a graphical comparison of six representative procoagulant venoms and can be used as a visual comparison of the procoagulant potency. This data can also be used to determine the minimum coagulant dose (MCD) of the venoms, which has previously been defined as the smallest amount of venom that clots plasma in 60 s (Theakston & Reid, 1983). Funding GKI is supported by an NHMRC Clinical Career Development Award ID300785 and the laboratory research is partly funded by an NHMRC Project Grant 490305. References
Fig. 4. Plots of clotting times versus venom concentration for six representative snake venoms, for a range of venom concentrations including venoms from the Carpet Viper (E. carinatus) [Group A prothrombin activator], Eastern brown snake (P. textilis) [Group C prothrombin activator], Common tiger snake (N. scutatus) [Group D prothrombin activator], Eastern Diamondback Rattlesnake (C. adamanteus) [thrombinlike enzyme], Malaysian Pit Viper (Calloselasma rhodostoma) [thrombin-like enzyme] and Russell's viper (Daboia russelii) [Factor X activator].
Barnett, R. N., & Pinto, C. L. (1966). Reproducibility of prothrombin time determinations between technologists. Comparison of the fibrometer and tilt-tube methods. Technical Bulletin of the Registry of Medical Technologists, 36, 146−149. Currie, B. J. (2004). Snakebite in tropical Australia: A prospective study in the “top end” of the Northern Territory. Medical Journal of Australia, 181, 693−697. Filippovich, I., Sorokina, N., St Pierre, L., Flight, S., de Jersey, J., Perry, N., et al. (2005). Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity. British Journal of Haematology, 131, 237−246. Goldenberg, N. A., Bombardier, C., Hathaway, W. E., McFarland, K., Jacobson, L., & MancoJohnson, M. J. (2008). Influence of factor IX on overall plasma coagulability and fibrinolytic potential as measured by global assay: Monitoring in haemophilia B. Haemophilia, 14, 68−77. He, S., Antovic, A., & Blomback, M. (2001). A simple and rapid laboratory method for determination of haemostasis potential in plasma. II. Modifications for use in routine laboratories and research work. Thrombosis Research, 103, 355−361. Hemker, H. C., Giesen, P., AlDieri, R., Regnault, V., de Smed, E., Wagenvoord, R., et al. (2002). The calibrated automated thrombogram (CAT): A universal routine test for hyper- and hypocoagulability. Pathophysiology Haemostasis Thrombosis, 32, 249−253.
M.A. O'Leary, G.K. Isbister / Journal of Pharmacological and Toxicological Methods 61 (2010) 27–31 Hutton, R. A., & Warrell, D. A. (1993). Action of snake venom components on the haemostatic system. Blood Review, 7, 176−189. Isbister, G. K. (2009). Procoagulant snake toxins: Laboratory studies, diagnosis, and understanding snakebite coagulopathy. Seminars in Thrombosis and Hemostasis, 35, 93−103. Isbister, G. K., O'Leary, M. A., Schneider, J. J., Brown, S. G., & Currie, B. J. (2007). Efficacy of antivenom against the procoagulant effect of Australian brown snake (Pseudonaja sp.) venom: In vivo and in vitro studies. Toxicon, 49, 57−67. Lincz, L. F., Lonergan, A., Scorgie, F. E., Rowlings, P., Gibson, R., Lawrie, A., et al. (2006). Endogenous thrombin potential for predicting risk of venous thromboembolism in carriers of factor V Leiden. Pathophysiology Haemostasis Thrombosis, 35, 435−439. Marshall, L. R., & Herrmann, R. P. (1983). Coagulant and anticoagulant actions of Australian snake venoms. Thrombosis and Haemostasis, 50, 707−711. O'Leary, M. A., Brown, S. G. A., Jacoby, T., Gan, M., Schneider, J., & Isbister, G. K. (2008). A sensitive and specific enzyme immunoassay to distinguish between two closely related Australian elapids: Tropidechis carinatus and Notechis scutatus. 8th Asia– Pacific Congress on Animal, Plant and Microbial Toxins Vietnam: International Society of Toxinology Ref Type: Conference Proceeding.
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O'Leary, M. A., Isbister, G. K., Schneider, J. J., Brown, S. G., & Currie, B. J. (2006). Enzyme immunoassays in brown snake (Pseudonaja spp.) envenoming: Detecting venom, antivenom and venom–antivenom complexes. Toxicon, 48, 4−11. Salooja, N., & Perry, D. J. (2001). Thrombelastography. Blood Coagulation & Fibrinolysis, 12, 327−337. Sprivulis, P., Jelinek, G. A., & Marshall, L. (1996). Efficacy and potency of antivenoms in neutralizing the procoagulant effects of Australian snake venoms in dog and human plasma. Anaesth Intensive Care, 24, 379−381. Theakston, R. D., & Reid, H. A. (1983). Development of simple standard assay procedures for the characterization of snake venom. Bulletin of the World Health Organization, 61, 949−956. Triplett, D. A. (1981). New methods in coagulation. Critical Reviews in Clinical Laboratory Sciences, 15, 25−84. Williams, V., White, J., & Mirtschin, P. J. (1994). Comparative study on the procoagulant from the venom of Australian brown snakes (Elapidae; Pseudonaja spp.). Toxicon, 32, 453−459.
Contents lists available at ScienceDirect
Journal of Pharmacological and Toxicological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j p h a r m t ox
Original article
A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma Margaret A. O'Leary a, Geoffrey K. Isbister a,b,⁎ a b
Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle Hospital, Newcastle, New South Wales, Australia Tropical Toxinology Unit, Menzies School of Health Research, Charles Darwin University, Darwin, Australia
a r t i c l e
i n f o
Article history: Received 17 May 2009 Accepted 16 June 2009 Keywords: Snake venoms Procoagulant Turbidimetric assay Clotting studies Coagulant toxins Assay methods
a b s t r a c t Introduction: Assessment of the procoagulant effect of snake venoms is important for understanding their effects. The aim of this study was to develop a simple automated method to measure clotting times to assess procoagulant venoms. Methods: A turbidimetric assay was developed which monitors changes in optical density when plasma and venom are mixed. Plasma was added simultaneously to venom solutions in a 96 well microtitre plate. After mixing, the optical density at 340 nm was monitored in a microplate reader every 30 s over 30 min. The clotting time was defined as the lag time until the absorbance sharply increased. The turbidimetric method was compared to manual measurement of the clotting time defined as the time when a strand of fibrin can be drawn out of the mixture. The two methods were done simultaneously, with the same venom and plasma, and compared by plotting the manual versus turbidimetric clotting times. Within-day and between-day runs were done and the coefficient of variation (CV) was calculated. Results: Plots comparing manual clotting times to the lag time in the turbidimetric assay showed good correlation between the two methods for brown snake (Pseudonaja textilis) venom, including 24 determinations in triplicate over six days for seven different venom concentrations. Good correlation was also found for four other venoms: tiger snake (Notechis scutatus), Carpet viper (Echis carinatus), Russell's viper (Daboia russelii) and Malaysian pit piper (Calloselasma rhodostoma). Between-day CV was in the range 10–20% for both methods, while within-day CV b 10%. Discussion: The turbidimetric assay appears to be a simple and convenient automated method for the measurement of clotting times to assess the effects of procoagulant venoms. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
1. Introduction Many snake venoms are procoagulant and cause whole blood or plasma to clot when the venom is added to plasma in vitro (Isbister, 2009). Bites by snakes with procoagulant venoms cause consumption of clotting factors leading to a coagulopathy, rather than simple clot formation (Isbister, 2009; Hutton & Warrell, 1993; Isbister, O'Leary, Schneider, Brown & Currie, 2007). In addition to the importance of procoagulant venoms in human envenoming, they have been used as diagnostic and laboratory agents and have been investigated as therapeutic agents (Isbister, 2009). An understanding of their action on the clotting pathways is therefore important for a number of reasons. For procoagulant venoms and toxins, a fundamental measure of venom potency is the time it takes for the venom to cause plasma to clot.
⁎ Corresponding author. Calvary Mater Newcastle Hospital, Edith St, Waratah NSW 2298, Australia. Tel.: +612 4921 1211; fax: +612 4921 1870. E-mail address: [email protected] (G.K. Isbister).
The simplest method for measuring this is combining venom (or a procoagulant component or toxin) and plasma, then observing the time until a thin strand of fibrin appears and can be drawn out of the mixture. A series of venom concentrations may be assessed in this way (Isbister, O'Leary, Schneider, Brown & Currie, 2007; Marshall & Herrmann, 1983; Williams, White & Mirtschin,1994). This simple method has a number of disadvantages such as requiring continuous attention, having an endpoint that is dependent on the observer, not always reproducible on different days and being difficult to do at 37 °C. Instrumental methods aim to improve the accuracy and reproducibility of the clotting time end-point and they may be either mechanical methods, such as the fibrometer (Barnett & Pinto, 1966), or methods that depend on changes in the optical properties of the clotting plasma. The fibrometer is used widely for standard clotting studies such as the prothrombin time and the activated partial thromboplastin time, and has been used for measuring the effects of venom in plasma (Sprivulis, Jelinek & Marshall, 1996). Another important mechanical method is the thromboelastograph which measures a number of dynamic parameters such as clot strength, in addition to the time until clot formation (Salooja & Perry, 2001). Thromboelastography has been used for the assessment of venom effects in plasma (Filippovich et al., 2005) but requires a
1056-8719/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2009.06.004
28
M.A. O'Leary, G.K. Isbister / Journal of Pharmacological and Toxicological Methods 61 (2010) 27–31
dedicated instrument that may not be available in many venom research laboratories. Most optical methods are aimed at detecting particular enzyme functions of the clotting pathway using either chromogenic substrates or fluorogenic substrates (Triplett, 1981). These have been used extensively in venom research, including the substrates for prothrombin, thrombin and Factor X because of the importance of these enzymes in snake venom coagulopathy (Isbister, 2009). However, these assays only provide insight into a particular part of the clotting pathway and are less useful for understanding the effects of venoms on clotting in vivo. More complex systems that measure thrombin generation have been used to measure the downstream effects of procoagulant venoms and may provide a useful approach, but again require specialised equipment (Isbister, O'Leary, Schneider, Brown & Currie, 2007; Lincz et al., 2006; Hemker et al., 2002). Despite the existence of a number of instrumental techniques for the investigation of the clotting effects of venom, most studies have used either manual clotting times or chromogenic assays. We now describe a turbidimetric method for the measurement of clotting times aimed mainly for use with assessing the procoagulant features of snake venoms and toxins. The method is simple and convenient and can be carried out with microtitre plates in a kinetic plate reader, an instrument that is readily available in many laboratories. 2. Materials and methods Fresh frozen plasma was obtained from the Australian Red Cross and aliquots of 10 mL were thawed at 37 °C then spun at 2500 rpm for 10 min. Calcium (60 µL 0.2 M CaCl2/mL) was added immediately before the assay if it was used. Tris-buffered saline (TBS) was used in preference to phosphate buffered saline (PBS) because a precipitate of calcium phosphate may occur with PBS and re-calcified plasma. Eastern or common brown snake (Pseudonaja textilis) venom, Tiger snake (Notechis scutatus) venom, Eastern Diamondback Rattlesnake (Crotalus adamanteus) venom and Malaysian Pit Viper (Calloselasma rhodostoma) venom were purchased from Venom Supplies, South Australia. Russell's viper (Daboia russelii) was obtained from the University of Colombo, Sri Lanka and Carpet Viper (Echis carinatus) was purchased from Sigma. 100 µL aliquots (1.0 mg/mL) of venom were prepared in 0.5% BSA/TBS and stored at − 20 °C. Dilutions were prepared in 0.5% BSA/TBS immediately before use. 2.1. Turbidimetric assay Venom solutions (100 µL) were placed in the wells of a 96 well microtitre plate at room temperature or at 37 °C in a BioTek ELx808 plate reader. Plasma (100 µL) was then added simultaneously to each well at the same temperature, using a multichannel pipette. After a 5-second shake step for mixing, the optical density at 340 nm was monitored every 30 s over 30 to 60 min. This produces a plot of optical density versus time (see Fig. 1). Gen5 software (supplied with the Biotek ELx808) fits the data to an exponential association equation after plateau. Y = IF½XbXo; Plateau; Plateau + SpanTð1 − expð−kð X − XoÞÞÞ Plateau is the initial absorbance, Span is the change in absorbance, and Xo is the lag time at which absorbance begins to sharply increase. The lag time as calculated by the Gen5 software is user-defined as the time when absorbance becomes 0.02 units greater than the average of the first two absorbance measurements. The lag time was taken to be the clotting time because it represented the sharp rise in optical density consistent with a clot beginning to form. 2.2. Manual clotting time Manual clotting times were carried out as a comparison to the turbidimetric assay. Citrated plasma (100 µL) was added at room
Fig. 1. Plots of absorbance versus time for no venom and increasing concentration of P. textilis venom in recalcified plasma (A) and in non-recalcified plasma (B), showing the sharp increase in absorbance rising to plateau, after the lag time.
temperature to venom solutions (100 µL) in a 1 mL conical polystyrene vial. The clotting time was defined as the time when a strand of fibrin could be drawn out of the mixture. 2.3. Comparison of tests The two tests (turbidimetric and manual) were run in parallel on the same day with the same venom preparation and plasma. The turbidimetric and manual clotting times were then compared by plotting the manual versus the turbidimetric clotting time. Withinday and between-day runs were done for both clotting time tests and a coefficient of variation (CV) was calculated for each test. In addition to the lag time calculated by the Gen5 plate-reader software, the raw data was also imported into Prism 5 and the lag time calculated by fitting the data to an exponential association curve. All statistical analysis was done using either Prism 5 or Microsoft Excel 2007. 3. Results Fig. 1 shows the plots of absorbance or optical density versus time for a range of venom concentration with and without calcium, including a control sample with no venom. Following the addition of plasma to the venom solution there is an initial lag time after which there is a sharp rise in the optical density due to the cloudiness of clot formation. The change in optical density follows a hyperbolic curve to a maximum value. The lag time was taken to be the clotting time. We
M.A. O'Leary, G.K. Isbister / Journal of Pharmacological and Toxicological Methods 61 (2010) 27–31
found that the difference in optical density between clotted and nonclotted plasma increases with decreasing wavelength, thus measurements were optimally carried out at a wavelength of 340 nm. Fig. 1 demonstrates that calcium alone will eventually cause clot formation but in the absence of calcium the plasma did not clot. There was also a marked attenuation of the venom effects in the absence of calcium. Table 1 compares the calculated clotting times obtained by fitting the series of brown snake venom concentrations in Fig. 1 to an exponential association curve with times extracted directly from the experimental data. Fig. 2 shows good correlation (R2 = 0.92) between the new turbidimetric assay and the manual method for common brown snake (P. textilis) venom, including 24 determinations in triplicate for both methods over six days, using seven different venom concentrations and plasma from two different batches. Good correlation (R2 N 0.97) was also found for venoms from tiger snake (N. scutatus), Carpet Viper (E. carinatus), Russell's viper (D. russelii) and the Malaysian Pit Viper (C. rhodostoma) which is shown in Fig. 3. It can be seen from these plots that at high concentrations of venom the turbidimetric method gives a shorter clotting time because the cloudiness sets in before a tangible fibrin strand may be withdrawn. At low concentrations of venom the reverse tends to occur. Runs carried out on three successive days gave CV in the range 10 to 20% for both manual and turbidimetric methods, while within-day determinations in triplicate gave values of b10%. The greatest variability occurred at the lowest concentration of venom tested which gave the longest clotting times. Comparisons were carried out at room temperature, because of the impracticality of conducting manual tests at 37 °C. Fig. 4 compares six representative venoms with plots of the clotting times versus Log venom concentration, using the turbidimetric method at 37 °C. The clotting curves provide additional information about the extent of clotting by the total increase in absorbance. In nonrecalcified plasma (Fig. 1B), not only are the lag times increased but also the final plateau is much lower and more dependent on venom concentration compared to recalcified plasma (Fig. 1A). This difference in clot density cannot be detected by the manual method. 4. Discussion We present a simple automated method for the measurement of clotting times to assess the effects of procoagulant venoms on plasma. The method has similar reproducibility to the manual method but provides some additional information and can be done on multiple specimens simultaneously. The method only requires a microplate reader and can be set up in a general laboratory for venom research. We used the assay on six different types of procoagulant venoms that have toxins with different clotting activation mechanisms — Factor X activation (D. russelii), prothrombin (Factor II) activation (E. carinatus, N. scutatus and P. textilis), and cleavage of fibrinogen (C. rhodostoma and C. adamanteus). In all cases, the method provides consistent results when compared with those obtained by the manual method. This technique of observing fibrin generation by monitoring optical density changes over time has been previously applied to the investigation of coagulopathies and factor deficient plasmas (He, Antovic & Blomback, 2001). In these studies, clotting is initiated by
Table 1 Comparison of clotting times (min) for brown snake (Pseudonaja textilis) venom carried out in the plate reader using the Gen5 software package, calculating the clotting time in Prism 5 using the raw data from the plate reader, and the manual clotting time. Venom concentration (ng/mL)
250
125
62.5
31.2
15.6
7.8
3.9
Plate reader Calculated Manual method
2.7 2.7 3.3
4.5 4.6 5.0
6.8 7.2 7.4
10.1 10.8 10.3
14.1 14.9 13.8
17.8 19.6 17.4
23.2 24.4 24.0
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Fig. 2. Comparison between clotting times from the automatic turbidimetric test and manual clotting times using P. textilis (Brown snake) venom.
either the addition of thrombin (He, Antovic & Blomback, 2001) or tissue factor, (Goldenberg et al., 2008) and subsequent fibrinolysis is ensured by the addition of tissue-type plasminogen activator. The generated curves provide information about the overall haemostatic potential associated with hyper- and hypo-coagulable states such as pregnancy and haemophilia. We have modified this approach and in the turbidimetric assay clotting is initiated by the addition of venom to a constant plasma substrate. We also do not add a plasminogen activator so fibrinolysis does not occur and the resulting curves are simpler and more amenable to mathematical modelling. The variability in the manual and turbidimetric methods is acceptable for both within and between-day assays. The greater variability for longer clotting times may have been because over a longer time period other factors such as dust particle nucleation and inherent clotting mechanisms become relatively more significant. It is important that venom concentrations are selected so that the assay is used for an appropriate range of clotting times (less than 30 min) to avoid this increase variability and unreliability. Although the comparison of the turbidimetric assay to the manual method was carried out at room temperature, the turbidimetric assay can and should be done at 37 °C because this is more representative of the in vivo situation (Fig. 4). This is a further advantage of the turbidimetric assay which is easily done at 37 °C compared to the manual method. The additional information provided by the turbidimetric method (Fig. 1) requires further investigation and comparison to other assay techniques. The height of the final plateau may reflect the amount of fibrin formed which is less in non-recalcified plasma and with lower venom concentrations. This difference cannot be detected by the manual method, but may be analogous to the clot strength parameter obtained by the thromboelastograph (TEG) technique (Filippovich et al., 2005). The venom concentrations used in this study are lower than many previous studies. However, we have shown in a number of studies that these low concentrations are consistent with those found in human envenoming cases (Isbister, O'Leary, Schneider, Brown & Currie, 2007; O'Leary, Isbister, Schneider, Brown & Currie, 2006; O'Leary et al., 2008) and therefore the appropriate concentrations for investigating the pathophysiology of procoagulant venoms and the efficacy of antivenom. The use of higher venom concentrations can be misleading and one such study of Australian snake venoms (Sprivulis, Jelinek & Marshall, 1996) lead to inappropriate increases in antivenom use, before further studies were undertaken at the appropriate low concentrations (Isbister, O'Leary, Schneider, Brown & Currie, 2007). Some snake venoms contain anticoagulants, but only one group of snakes (Pseudechis spp; Australian black snakes) are reported to cause in vivo anticoagulant effects (Currie, 2004). Procoagulant effects are the more common coagulant effects seen with snake envenoming and appear to occur at the low venom concentrations seen in humans
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M.A. O'Leary, G.K. Isbister / Journal of Pharmacological and Toxicological Methods 61 (2010) 27–31
Fig. 3. Comparison between clotting times from the automatic turbidimetric test and manual clotting times for four different venoms: (A) tiger snake (N. scutatus), (B) Russell's viper (D. russelii), (C) Carpet Viper (E. carinatus), and (D) Malaysian Pit Viper (C. rhodostoma) — including trend line (non-dashed) and line of unity (dashed).
(Isbister, 2009). At very high concentrations (orders of magnitude greater than those seen in envenomed humans), other snake venoms may have anticoagulant in vitro effects, but these are unlikely to be clinically relevant.
Fig. 4 provides a graphical comparison of six representative procoagulant venoms and can be used as a visual comparison of the procoagulant potency. This data can also be used to determine the minimum coagulant dose (MCD) of the venoms, which has previously been defined as the smallest amount of venom that clots plasma in 60 s (Theakston & Reid, 1983). Funding GKI is supported by an NHMRC Clinical Career Development Award ID300785 and the laboratory research is partly funded by an NHMRC Project Grant 490305. References
Fig. 4. Plots of clotting times versus venom concentration for six representative snake venoms, for a range of venom concentrations including venoms from the Carpet Viper (E. carinatus) [Group A prothrombin activator], Eastern brown snake (P. textilis) [Group C prothrombin activator], Common tiger snake (N. scutatus) [Group D prothrombin activator], Eastern Diamondback Rattlesnake (C. adamanteus) [thrombinlike enzyme], Malaysian Pit Viper (Calloselasma rhodostoma) [thrombin-like enzyme] and Russell's viper (Daboia russelii) [Factor X activator].
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