Crotaline Fab Antivenom Reverses Platelet Dysfunction Induced by Crotalus scutulatus Venom: An In Vitro Study

Authors


  • Presented at the North American Congress of Clinical Toxicology, Washington, DC, September 2011.
  • The authors have no relevant financial information or potential conflicts of interest to disclose.
  • The views expressed in this article are those of the authors and do not represent the official position of the U.S. Navy, Department of Defense, or U.S. Government.

Address for correspondence: Shaun D. Carstairs, MD; e-mail: shaunc@yahoo.com. Reprints will not be available.

Abstract

Background

Patients sustaining rattlesnake envenomation often develop thrombocytopenia, the etiology of which is not clear. Laboratory studies have demonstrated that venom from several species, including the Mojave rattlesnake (Crotalus scutulatus scutulatus), can inhibit platelet aggregation. In humans, administration of crotaline Fab antivenom has been shown to result in transient improvement of platelet levels; however, it is not known whether platelet aggregation also improves after antivenom administration.

Objectives

The objective was to determine the effect of C. scutulatus venom on platelet aggregation in vitro in the presence and absence of crotaline Fab antivenom.

Methods

Blood was obtained from four healthy male adult volunteers not currently using aspirin, nonsteroidal anti-inflammatory drugs, or other platelet-inhibiting agents. C. scutulatus venom from a single snake with known type B (hemorrhagic) activity was obtained from the National Natural Toxins Research Center. Measurement of platelet aggregation by an aggregometer was performed using five standard concentrations of epinephrine (a known platelet aggregator) on platelet-rich plasma over time, and a mean area under the curve (AUC) was calculated. Five different sample groups were measured: 1) blood alone, 2) blood + C. scutulatus venom (0.3 mg/mL), 3) blood + crotaline Fab antivenom (100 mg/mL), 4) blood + venom + antivenom (100 mg/mL), and 5) blood + venom + antivenom (4 mg/mL). Standard errors of the mean (SEM) were calculated for each group, and paired t-tests were used to measure differences between groups.

Results

Antivenom administration by itself (group 2) did not significantly affect platelet aggregation compared to baseline (103.8%, SEM ± 3.4%, p = 0.47). Administration of venom (group 3) decreased platelet aggregation (72.0%, SEM ± 8.5%, p < 0.05). Concentrated antivenom administration in the presence of venom (group 4) normalized platelet aggregation (101.4%, SEM ± 6.8%) and in the presence of diluted antivenom (group 5) significantly increased aggregation (133.9%, SEM ± 9.0%; p < 0.05 for both groups when compared to the venom-only group). To further assess the effects of the venom and antivenom, each was run independently in platelet-rich plasma without epinephrine; neither was found to significantly alter platelet aggregation in the absence of epinephrine.

Conclusions

Crotaline Fab antivenom improved platelet aggregation in an in vitro model of platelet dysfunction induced by venom from C. scutulatus. It is unclear at this time whether this improvement in platelet dysfunction translates into improved clinical outcomes in envenomated patients.

Resumen

El Antiofídico Crotaline Fab Revierte la Disfunción Plaquetaria Inducida por el Veneno de C. scutulatus: Un Estudio In vitro

Introducción

Los pacientes que sufren envenenamiento por serpiente de cascabel a menudo desarrollan trombocitopenia, sin clara etiología. Los estudios de laboratorio han demostrado que el veneno de varias especies, incluida la serpiente de cascabel Mojave (Crotalus scutulatus), puede inhibir la agregación plaquetaria. En humanos, la administración de antiofídico Crotaline Fab resulta en una mejora transitoria del recuento de plaquetas; sin embargo, no se conoce si la agregación plaquetaria también mejora tras la administración del antiofídico.

Objetivos

Determinar el efecto del veneno C. scutulatus en agregación plaquetaria in vitro en presencia y ausencia de antiofídico Crotaline Fab.

Metodología

Se obtuvo sangre de cuatro voluntarios adultos varones sanos que no tomaban en ese momento aspirina, AINE ni otros agentes inhibidores de las plaquetas. Se obtuvo el veneno de C. scutulatus de una única serpiente con actividad tipo B (hemorrágica) conocida del Centro Nacional de Investigación de Toxinas Naturales (National Natural Toxins Research Center). Se realizó la medida de la agregación plaquetaria mediante un agregómetro usando cinco concentraciones estándar de adrenalina (un conocido agregante plaquetario) o plasma rico en plaquetas a lo largo del tiempo, y se calculó el área bajo la curva (ABC). Se midieron cinco grupos de muestras distintas: 1) sangre sola; 2) sangre + veneno (0,3 mg/mL); 3) sangre + antiofídico (100 mg/mL); 4) sangre + veneno + antiofídico (100 mg/mL); 5) sangre + veneno + antiofídico (4 mg/mL). Se calculó el error estándar de la media (EEM) para cada grupo y se usó el test de t Student para datos apareados para valorar las diferencias entre los grupos.

Resultados

La administración de antiofídico, por sí misma (grupo 2), no afectó significativamente a la agregación plaquetria comparada con la agregación basal (103,8% ± 3,4%, p = 0,47). La administración de veneno (grupo 3) disminuyó la agregación plaquetaria (72,0% ± 8,5%, p < 0,05). La administración de antiofídico concentrado en presencia de veneno (grupo 4) normalizó la agregación plaquetaria (101,4% ± 6,8%) y en presencia de antiofídico diluido (grupo 5) incremento significativamente la agregación (133,9%, SD ± 9,0%); p < 0,05 para ambos grupos cuando se comparó al grupo de sólo veneno. Para mayor evaluación de los efectos del veneno y el antiofídico, cada uno de ellos se puso independientemente en plasma rico en plaquetas sin adrenalina, y no se encontró en ninguno de ellos una alteración significativa de la agregación plaquetaria en ausencia de adrenalina.

Conclusiones

El antiofídico Crotaline Fab mejoró la agregación plaquetaria en un modelo in vitro de disfunción plaquetaria inducida por veneno de C. scutulatus. No está claro en estos momentos si la mejora en la disfunción plaquetaria se traduce en una mejoría de los resultados clínicos en los pacientes envenenados.

Crotaline snake venom is a complex mixture of proteins, lipids, carbohydrates, and other fractions. The venom components can be variable between individual snakes; variability is dependent on multiple factors, including snake location, size, age, diet, climate, and time of year.[1] This variability can make prediction of the medical course of an envenomated patient difficult.

Two geographically distinct venoms (type A and type B) are produced by the Mojave rattlesnake (Crotalus scutulatus scutulatus). Type A contains Mojave toxin, a presynaptic neurotoxin, whereas type B lacks Mojave toxin but contains multiple components with proteolytic and hemorrhagic activities.[2] Crotaline venom may cause thrombocytopenia and platelet dysfunction; specifically, the venom of the Mojave rattlesnake with type B activity has been shown to inhibit platelet aggregation.[3] The inhibition of platelet aggregation is partially mediated by a class of proteins called disintegrins, which bind to platelet fibrinogen receptors and prevent the binding of fibrinogen to platelets, thereby inhibiting fibrin cross-linking of platelets.[4]

While crotaline Fab antivenom has been shown previously to have a positive effect on thrombocytopenia due to crotaline bites,[5-7] we sought to determine whether antivenom administration would have a similar effect on the platelet dysfunction induced by crotaline venom. The objective of this study was to determine the effect of C. scutulatus venom on platelet aggregation in vitro in the presence and absence of crotaline Fab antivenom.

Methods

Study Design

This was a laboratory study using blood from volunteers to assess clotting after exposure to crotaline venom. Prior to study commencement, approval was obtained from the local institutional review board.

Study Setting and Population

Four healthy volunteers were recruited for the study from residents and attending physicians in the Department of Emergency Medicine. Inclusion criteria were male sex and age 18 to 50 years. Potential subjects were excluded if they had taken any aspirin, nonsteroidal anti-inflammatory drugs, or other platelet-inhibiting agents within 1 week prior to the start of the study; subjects were also excluded if they had any history of congenital bleeding diathesis (such as von Willebrand disease or hemophilia) or known hepatic dysfunction.

Study Protocol

Venous blood in the amount of 50 mL was collected from the right or left antecubital fossa of each study subject via a 21-gauge hypodermic needle into a plastic tube containing 3.8% wt/vol citrate. C. scutulatus (Mojave) venom from a single snake with known type B activity was obtained from the National Natural Toxins Research Center (Kingsville, TX). Crotaline Fab antivenom (CroFab; BTG International Inc., West Conshohocken, PA) was obtained from the hospital pharmacy and diluted to two concentrations: 100 mg/mL (stock concentration) and 4 mg/mL (a concentration that was felt to approximate more closely the in vivo blood concentration achieved with an initial loading dose of antivenom).

Platelet aggregation was measured by a modification of the method of Born and Cross,[8] a standard method of measuring platelet aggregation. Measured platelet activity using an aggregometer (whole blood lumi-aggregometer, Model 560CA with Aggro/Link 810 software, Chrono-Log Corp., Havertown, PA) was recorded for all groups. For the aggregometer, 50 mL of whole blood was mixed with 10% sodium citrate in a syringe. Two milliliters of blood were placed in a glass tube and centrifuged at 15,000 rpm for 2 minutes; the supernatant was then used for a platelet-poor sample. The remaining blood was centrifuged at 1,000 rpm for 8 minutes and the supernatant was stored as platelet-rich plasma (PRP). Platelet aggregability was measured by taking 500-μL aliquots of PRP and adding varying concentrations of epinephrine (0.1, 0.3, 1, 3, and 10 μM) to achieve a threshold concentration that caused platelets to clump.[9] Samples were stirred at 1,100 rpm at 37°C while in the aggregometer. Each sample was run against platelet-poor plasma for 8 minutes and the Aggro/Link software calculated an area under the curve (AUC) representing the percentage of platelet aggregation over time. A mean AUC for the five epinephrine concentrations was then recorded.

Five different sample groups were measured: 1) blood alone (used as a baseline for platelet aggregation), 2) blood + C. scutulatus venom (0.3 mg/mL), 3) blood + crotaline Fab antivenom (100 mg/mL), 4) blood + venom + antivenom (100 mg/mL), and 5) blood + venom + antivenom (4 mg/mL).

Data Analysis

Data were analyzed using Stata 12.1 software (StataCorp LP, College Station, TX). Since this was a pilot study, no a priori group size analysis was performed. Standard errors of the mean (SEM) were calculated for each group, and paired t-tests were used to measure differences between groups for normally distributed continuous variables. A p-value less than 0.05 was considered significant, with no adjustment for multiple comparisons.

Results

Blood was obtained from a total of four male volunteers (mean ± SD age = 43.5 ± 4.8 years, range = 38 to 48 years). When compared to baseline platelet aggregation (group 1), antivenom administration by itself without the addition of venom (group 2) did not significantly affect platelet aggregation compared to baseline (103.8, SEM ± 3.4% of baseline, p = 0.47). Administration of venom (group 3) significantly decreased platelet aggregation (72.0, SEM ± 8.5% of baseline, p < 0.05). Concentrated antivenom administration in the presence of venom (group 4) normalized platelet aggregation (101.4, SEM ± 6.8% of baseline). In the presence of diluted antivenom (group 5), aggregation was significantly increased (133.9, SEM ± 9.0% of baseline; p < 0.05 for both groups when compared to the venom-only group; see Figure 1). To further assess the effects of the venom and antivenom, each was run independently in PRP without epinephrine; neither was found to significantly alter platelet aggregation in the absence of epinephrine.

Figure 1.

Platelet aggregation by group. B = blood; AV = crotaline Fab antivenom; V = venom (0.3 mg/mL); AV100 = crotaline Fab antivenom (100 mg/mL); AV4 = crotaline Fab antivenom (4 mg/mL). Thin black bars represent 95% confidence intervals.

Discussion

The effects of crotaline venom on the human hematologic system are myriad and complex. Depending on the individual snake species, some venom components have procoagulant effects, while others have anticoagulant effects. Platelets may become activated, aggregated, or inhibited from aggregating depending on the components present in the venom. C. scutulatus venom has been well documented to contain constituents that result in inhibition of platelet aggregation.[10-12]

Crotaline Fab antivenom has been shown to counteract some of the hematologic effects of crotaline snake venom, namely, thrombocytopenia and hypofibrinogenemia.[13] However, while an increase in platelet count after administration of antivenom has been demonstrated, it has been unclear what effects, if any, antivenom administration has on the inhibition of platelet aggregation that is known to occur with venom from some crotaline species. We elected to use venom from C. scutulatus because it is one of the crotaline species best documented in the literature to exhibit platelet aggregation–inhibiting properties.

In our in vitro model of platelet aggregation, C. scutulatus venom reliably inhibited platelet aggregation by approximately 28% compared to baseline. Administration of antivenom prevented the inhibition of platelet aggregation: at a dose of 100 mg/mL, platelet aggregation normalized (101.4% of baseline), whereas at a dose of 4 mg/mL, platelet aggregation was actually increased (133.9% of baseline). It is unclear why the lower concentration of antivenom resulted in an increased effect on platelet aggregation; however, our data demonstrated that antivenom itself (in the absence of venom) did not demonstrate any significant effects on platelet aggregation. Moreover, the mechanism by which antivenom prevents inhibition of platelet aggregation is not yet clear. While it is likely that this occurs by binding of antivenom to specific venom components and inhibiting their binding to platelets, other potential mechanisms may also play a role.

Limitations

We enrolled a small number of subjects in our study; however, within our subjects, the results were highly consistent and reproducible. Additionally, this was an in vitro study, and our results may not necessarily extrapolate to an in vivo model. While it is clear that C. scutulatus venom causes platelet dysfunction, it is not obvious whether this dysfunction results in clinically significant bleeding complications in vivo. The venom used in our study was obtained from a single C. scutulatus snake with known hemotoxic venom effects; platelet effects from the venom of other snake species and even other snakes within the same species may differ.

Conclusions

Crotaline Fab antivenom improved platelet aggregation in an in vitro model of platelet dysfunction induced by C. scutulatus venom. It is unclear at this time whether this improvement in platelet aggregation translates into improved clinical outcomes in envenomed patients. Additional studies should be considered to determine whether these antivenom effects on platelets occur in vivo.

Ancillary