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Coagulopathy following lethal and non-lethal
envenoming of humans by the South American rattlesnake
(Crotalus durissus) in Brazil
I.S. SANO-MARTINS, S.C. TOMY, D. CAMPOLINA
1
,M.B.DIAS
1
,
S.C.B. DE CASTRO, M.C.C. DE SOUSA-E-SILVA, C.F.S. AMARAL
2
,
N.A. REZENDE
2
,A.S.KAMIGUTI
3
,D.A.WARRELL
4
and R.D.G. THEAKSTON
5
From the Laborato´rio de Fisiopatologia, Instituto Butantan, Sa˜ o Paulo, Brazil,
1
Hospital Joa˜o XXIII, Minas Gerais, Belo Horizonte, Brazil,
2
Departamento de Medicina
Interna, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil,
3
University
Department of Haematology, Royal Liverpool Hospital, Liverpool, UK,
4
Centre for Tropical
Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, UK and
5
Liverpool School of Tropical Medicine, Liverpool, UK
Received 16 May 2001 and in revised form 3 August 2001
Summary
The South American tropical rattlesnake (Crotalus
durissus subspp) is responsible for ;10%of bites
from venomous snakes in Brazil. We studied
24 victims of bites by this species over 3 years,
in south-eastern Brazil, particularly investigating
haemostatic alterations. Thirteen patients were
defined as moderately envenomed and 11 as
severe. There were two deaths, which were not
attributed to venom-induced haemostatic dis-
turbances. However, envenoming by C. durissus is
frequently associated with haemostatic disorders,
which are probably attributable mainly to the
action of the thrombin-like enzyme, with possible
additional effects secondary to the powerful
myotoxic activity of the venom.
Introduction
The Crotalus durissus complex, comprising the
South American tropical rattlesnakes, is responsible
for approximately 10%of cases of envenoming by
snakes in Brazil; the majority of these are caused
by the cascavel, Crotalus durissus terrificus. The
venom of this species possesses neurotoxic,
1
myotoxic
2
and thrombin-like activities.
3
The principal toxins present in the venom include
crotoxin,
4
crotamine,
5
convulxin,
6
a thrombin-like
enzyme
7
and gyroxin.
8,9
The high toxicity of
the whole venom is attributable to crotoxin, a
phospholipase A
2
complex, which is the principal
component of the venom.
4
Experimentally, crotoxin
in combination with crotamine exerts a neurotoxic
effect on peripheral nerves
10
and a myonecrotic
effect on muscle.
11
The crotamine content of
C. durissus venoms varies between different popu-
lations of snakes in Brazil and Argentina. Crotamine
was absent from populations in north and eastern
Brazil, present in north-western Sa˜o Paulo State
and adjacent areas of Parana´ and Minas Gerais, and
in Ceara´ there were mixed populations, some
secreting crotamine and some not.
12
An intriguing
possible clinical correlation with the presence of
crotamine is the ‘broken neck’ sign resulting from
paralysis of the cervical flexor muscles, possibly
ßAssociation of Physicians 2001
Address correspondence to Dr Ida S. Sano-Martins, Instituto Butantan, Laborato´rio de Fisiopatologia, Av. Vital Brazil,
1500, 05503 900, Sa˜o Paulo (SP), Brazil. e-mail: lusiada@uol.com.br
Q J Med 2001; 94:551–559
through direct action of crotamine. This feature
has been reported from various parts of Latin
America.
13
Convulxin causes convulsions and
respiratory and circulatory disturbances in mice,
dogs, cats and guinea pigs; it also causes in vitro
platelet aggregation in the platelet rich plasma of
many mammalian species.
6
Gyroxin, when injected
intravenously, induces episodes of opisthotonos
and rotation of the animal’s body in the longitudinal
axis.
8,9
These signs are not observed in human
victims. A thrombin-like enzyme, first isolated and
characterized by Raw et al.,
7
is responsible for
the coagulant action of the venom. More recently,
a comparative study of the biological activities of
the venoms of three different Brazilian subspecies
of C. durissus (C. d. terrificus,C. d. collilineatus
and C. d. cascavella) proved that most of these
activities were common to all these subspecies.
14
In most respects, Rosenfeld’s clinical descrip-
tions of envenoming by Crotalus durissus ssp in
Brazil have not been bettered.
15
Local symptoms at
the site of the bite include pain, paraesthesiae
such as formication or anaesthesia, but little or no
swelling and no local necrosis. Rosenfeld denied
erythema, but we have observed this (Figure 1),
for example in patients 3 and 13 (below). There is
a similar lack of effect when the venom is injected
subcutaneously, intramuscularly or intradermally
in experimental animals.
16
This contrasts with bites
by many Crotalus species in North and Central
America, which commonly cause severe local
necrosis.
17,18
Systemic envenoming usually starts with the
development of symmetrical ptosis, external oph-
thalmoplegia and facial weakness, resulting in the
characteristic myopathic/neurotoxic facies. Paresis
of the pupils may impair visual accommodation
(responsible for patients’ complaints of difficulties
with vision), loss of pupillary reflexes and mydriasis.
Rosenfeld regarded mydriasis as a fatal prognostic
sign.
15
Rarely, respiratory muscle involvement may
lead to respiratory failure.
19
The venom induces
generalized rhabdomyolysis, causing myalgias,
a massive rise in serum myoglobin and creatine
kinase (CK) levels, accompanied by myoglobin-
uria.
2,20–23
Pain throughout the whole body,
possibly explained by rhabdomyolysis, was the
main symptom remembered by one of the first
recorded victims of cascavel bite. Father Luis
Rodrigues was bitten near Bahia in north-eastern
Brazil at Christmas-time in 1560. He suffered
terrible symptoms for the next 20 days.
24
In the fatal case described in Londrina, Sa˜o Paulo
State there were clinical, electrocardiographic,
biochemical and histological features suggestive
of venom-induced myocardial damage.
25
Blood coagulation disturbances have been
described in about 50%of patients bitten by
C. durissus subspp.
26
Amaral et al.
27
reported
patients with afibrinogenaemia but without
thrombocytopenia. Spontaneous bleeding has
only been rarely observed in human patients. The
aim of the present study was therefore to examine
in detail the haemostatic disorders in patients
bitten by this species, and to assess the ability
of antivenom to reverse them.
Methods
Patients
Twenty-four patients were admitted to Hospital
Joa˜o XXIII, Belo Horizonte, Minas Gerais State,
Brazil. There were three females and 21 males, ages
ranging from 11 to 74 years. All had been bitten by
C. durissus subspecies (of which C. d. terrificus
is the most prevalent subspecies in Minas Gerais)
between January 1993 and November 1995.
Diagnosis of Crotalus durissus bite was based on
identification of the snake brought with the
patient (four patients, C. d. terrificus in each case);
detection of specific venom antigen in serum
(11 patients) or on clinical features considered
diagnostic at admission (nine patients). All of these
last nine patients showed characteristic signs of
ab
Figure 1. Extensive erythema and some swelling 3 h after a bite by Crotalus durissus terrificus in Sa˜ o Paulo State.
I.S. Sano-Martins et al.552
systemic envenoming by C. durissus (e.g. ptosis,
myalgia, diplopia or paraesthesia). As none of these
signs occur following bites by any other venomous
snake in this region, it is reasonable to include
these cases in the study. The patients were divided
into two entirely arbitrary groups—‘severe’ and
‘moderate’ envenoming—based on their presenting
symptoms and signs. ‘Moderate envenoming’ was
defined as disturbance of visual accommodation,
ptosis or neurotoxic facies,
15
mild myalgia, slight
or absent urinary pigment with normal urine
output, and altered or normal coagulation time.
‘Severe envenoming’ was defined by the pres-
ence of obvious neurotoxic signs, intense myalgia
accompanied by pigmented urine with the presence
or absence of oliguria/anuria and normal or altered
coagulation time.
28
Patients who had been treated
with antivenom before admission to the hospital
were excluded from the study. Informed consent to
participate in the clinical study was obtained from
the patients or, if young children were involved,
from their parents. The study was approved by the
Ethics Committee of Hospital Joa˜o XXIII.
Blood coagulation
Venous blood was sampled on admission, at 6, 12
and 24 h after the end of antivenom therapy,
and then daily until discharge from hospital. For
coagulation and fibrinolysis assays, 9.0 ml blood
was mixed with 0.8 ml 16 mM trisodium citrate
containing 0.2 ml Instituto Butantan Crotalus
monospecific antivenom
29
to neutralize any venom
present in the sample at the time of collection. The
citrated blood was centrifuged at 2000 gfor 15 min
at 4 8C and the platelet-poor plasma was frozen
in 0.5 ml aliquots at 20 8C until tested. Control
samples, collected from people with no history of
snakebite living in the same region as the victims
were treated as described above.
Standardized reagents purchased from Diag-
nostics Stago (France) were used to measure
prothrombin time (PT) and the activated partial
thromboplastin time (APTT). Levels of coagulation
factors (factors II, VII, VIII, IX, X, XI, XII) were
estimated using deficient plasmas, protein C (PC)
(Staclot protein C assay), cross-linked fibrin frag-
ment (D-dimer, DD) using immunoassay by latex
agglutination, a
2
-antiplasmin (a
2
-AP) (Stachrom
Antiplasmin) and serum fibrin(ogen) degradation
products (FnDP/FgDP) using immunoassay (latex
agglutination). Thrombin-antithrombin antigen (TAT)
was determined using an EIA kit system from
Behring (Enzygnost-TAT). Factor V was estimated
using the standardized technique of Denson
30
and fibrinogen levels were measured using the
method of Ratnoff and Menzie.
31
Haematological tests
Blood (2.5 ml) was mixed with 25 mlof10%
potassium EDTA and 50 mlCrotalus antivenom.
Erythrocytes, leukocytes and platelets were counted
using an automated cell counter (Coulter T 890).
Differential leukocyte counts were carried out on
blood films stained with panchromatic stain.
Biochemical tests
Blood was sampled for creatinine, urea and creatine
kinase (CK, normal values: 10–120 U/L) enzyme
activity determinations using Labtest kits. Serum
myoglobin concentration was measured using the
Rapitex-myoglobin kit (Behring).
Venom antigen levels
Enzyme-linked immunosorbent assay (ELISA) was
used for detection and measurement of C. durissus
venom in admission serum samples according
to the technique described by Cha´vez-Olo´ rtegui
et al.
32
A 96-well Microtitre plate (Hemobag
Produtos Ciru´ rgicos) was coated overnight with
100 mlofa5mg/ml solution of anti-C. durissus
IgG in carbonate buffer. After blocking and
washing, 50 ml serum diluted 1 : 2 in dilution
buffer was added (1 h, room temperature). The
plates were washed and incubated with peroxidase
coupled with anti-C. durissus IgG (1 h, room
temperature). The wells were washed, and the
assay was completed and stopped by the addition
of 20 ml of a 1 : 20 dilution of sulphuric acid.
A reference curve was obtained using dilutions
of known concentrations (4–500 ng/ml) of crude
venom from C. durissus terrificus. The baseline for
the assay was established according to the recom-
mendations of Theakston
33
and Ho et al.
34
A cut-off
was established by testing sera from 103 controls of
the same socio-economic group as the patients. The
mean"SD OD at 492 nm was 0.037"0.016 for
the whole venom assay. Using the mean plus 2 SD
as the negative cut-off value for the assays, which
corresponded to a whole venom concentration
of 20 ng/ml, two of the 103 normal sera were
positive for whole venom, resulting in a specificity
of 98.1%.
Results
Clinical observations
The time between bite and admission to hospital in
the 24 envenomed patients ranged from 2 to 60 h.
In 13/24 (54%) a tourniquet had been applied,
7/22 (32%) had paraesthesiae, 18/22 (82%) had
Envenoming by C. durissus 553
ptosis, and 10/19 (53%) suffered from myalgia.
Only 1/24 (4%) had respiratory insufficiency on
admission. The basic information and clinical
symptoms for each patient are shown in Table 1.
Laboratory findings
On admission and before antivenom therapy, 10/22
(45%) patients had a PT-10%and 12/22 (55%)
had a prolonged APTT, with three patients having
totally incoagulable blood (Table 1). Assays of
clotting and fibrinolytic factors showed fibrinogen
and factor V consumption, consumption of a
2
-AP
and consequent increase of FnDP/FgDP and D-DD
and the formation of thrombin-antithrombin com-
plex in some patients (Table 2). Only 3/5 patients
studied had decreased levels of protein C (0%,
26%,49%).
There were no gross alterations in the haem-
atological picture, although a slight polymorpho-
nuclear leukocytosis was recorded in the majority
of cases (Table 2). Interestingly, there was no
alteration in the platelet count in the majority of
patients; thrombocytopenia was observed only in
the two fatal cases.
Twelve hours after antivenom treatment, fibrino-
gen and FnDP/FgDP levels were within the normal
haemostatic range. Fibrinogen levels increased,
and this was paralleled by a decrease in FnDP/
FgDP levels and normalization of DD levels in
the plasma. Simultaneous consumption of a
2
-
antiplasmin indicated secondary activation of the
fibrinolytic system. All these levels had returned
to normal by the time the patient was discharged
from hospital (Figure 2).
As expected, the increased levels of myoglobin
in serum, highest on admission, decreased rapidly
following antivenom therapy (Figure 3a). Serum CK
levels were significantly elevated compared with
the normal range (10–120 U/l) from admission to
24 h after the start of antivenom (Figure 3b).
However, the differences in CK levels on admission
and 12 and 24 h after antivenom were not
significant (p)0.05).
Admission venom levels were measured in 19/24
patients. The mean venom antigen level in the
Table 1 Clinical features and relevant laboratory findings on admission in 24 patients envenomed by Crotalus durissus
Patient Age
(years)
Time
after
bite
(h)
Dose
AV (ml)
Paraesthesiae Ptosis Myalgia Tourniquet Classification PT (%) APTT (s) Venom
antigenaemia
on admission
(ng/ml)
1
S
39 7 100 qModerate 64 36 0
2 26 4 200 qqqSevere -10 ‘750
3*
a,b,c
15 27 200 ND ND ND Severe -10 ND 325
4 11 2 100 qqqModerate 75 36 162
5
S
53 2 150 qqModerate -10 ND 63
6 19 3 100 qqqModerate 62 30 172
7 14 9 200 q Severe -10 ‘97
8 19 6.5 200 qq Severe 45 36 ND
9 27 60 200 qq Severe ND 55 ND
10 11 3 200 qq q Severe 36 34 417
11 13 3 200 qq Severe -10 )60 250
12 74 4 100 qq q Moderate 16.5 35 ND
13*
c
50 18 200 ND ND ND qSevere 55 44 ND
14 24 2 100 qModerate 70 38 0
15 68 7.5 100 qND Moderate 37 )80 0
16 21 5 200 qq Severe -5‘76
17 40 5 200 qqqqModerate -5)120 25
18 47 24 100 qqqqModerate -10 )120 0
19 12 20 100 q Moderate 5 120 0
20 26 7 200 qqND qSevere -10 )120 95
21 56 5.5 200 qqqqSevere ND )180 0
22
S
64 6 100 Moderate 46 39 0
23
S
45 2.5 100 qModerate 95 29 0
24 24 4 150 qND Moderate 85 32 ND
*Fatal case;
a
developed acute renal and respiratory failure;
b
showed gingival bleeding on admission;
c
developed
thrombocytopenia;
S
dead snake brought with patient to the hospital. ND, not done; AV, Instituto Butantan Crotalus
antivenom; PT, prothrombin time; APTT, activated partial thromboplastin time.
I.S. Sano-Martins et al.554
11 moderately envenomed patients was 38.4 ng/ml
"20.0 ng/ml (SEM) and in the eight severely
envenomed patients it was 251.2 ng/ml"86.9
ng/ml (SEM) (Table 1). The difference between
these levels was statistically significant (p¼0.022).
The majority of the severely envenomed patients
(6/9, 67%) had an intense coagulopathy (PT-10%)
on admission, whereas only 4/13 (31%) of the
moderately envenomed patients had evidence of
severe coagulopathy (PT-10%) on admission
(Table 1).
Severe coagulopathy in two fatal cases
Among the 24 patients, there were two deaths
(8.3%). A 15-year-old boy (patient 3) was bitten on
the back of the hand and, 8 h later, was admitted
to a local hospital, prostrated, sweating, hypo-
tensive and in urinary retention. Urethral catheter-
ization yielded 1000 ml of cloudy urine. He was
transferred to Hospital Joa˜o XXIII, arriving 25 h
after the bite. He was comatose and dehydrated,
with gingival bleeding, and oedema and redness
at the site of the bite. He was tachycardic
(120 bpm) and tachypnoeic (28 breaths/min) with
nasal flaring and audible rhonchi. Blood pressure
was 100/60 mmHg. His blood was incoagulable,
with a grossly prolonged prothrombin time, fibrino-
gen 0.023 g/l, platelets 166310
9
/l, leukocytes
12.3310
9
/l (83%polymorphs), CK 96 000 U/l
(3.9%CKMB), haemoglobin 115 g/l, haematocrit
35%. The venom antigen level was 325 ng/ml.
Urine was strongly positive for blood, haemoglobin
or myoglobin. Twenty ampoules of Crotalus anti-
venom (Anticrota´lico, Instituto Butantan) was given
27 h after the bite. Two hours after admission,
the patient’s respiration had become shallow and
he was intubated, transferred to the intensive care
unit and mechanically ventilated, but he may have
aspirated at this time. There was radiographic evid-
ence of right basal consolidation. There was evid-
ence of renal impairment (urea 760 mg/l, creatinine
20 mg/l) and mild metabolic acidosis. Despite
treatment with intravenous fluids, furosamide and
dopamine, urine output was only 500 ml in 24 h,
and the patient became more comatose with neck
rigidity and extensor plantar responses. A brain scan
showed no evidence of intracranial haemorrhage.
At 42 h after the bite, 12 h after antivenom treat-
ment, blood urea had risen to 1450 mg/l, creatinine
to 36 mg/l, potassium to 7 mequiv/l and the
leukocyte count to 16.3310
9
/l (77%polymorphs).
The prothrombin ratio had improved to 42%,
activated partial prothrombin time (APPT) 50/32
seconds. Following initiation of peritoneal dialysis,
the patient’s level of consciousness improved. The
pupils were equal in size and reacted to light. The
platelet count had fallen to 85310
9
/l. Eighty hours
after the bite, the patient remained oliguric on
peritoneal dialysis. Diffuse consolidation developed
in both lungs, with progressive deterioration in
respiratory and haemodynamic function. He died
110 h after the bite. The key features of this
fatal progression were early shock, coagulopathy
with spontaneous bleeding, renal failure, respiratory
failure, rhabdomyolysis and finally, progressive
respiratory failure compounded by aspiration
pneumonia, adult respiratory distress syndrome
and possibly pulmonary haemorrhage.
Patient 13 was a 50-year-old man bitten on the
dorsum of the foot. On admission to Hospital Joa˜o
XXIII 15 h after the bite, there was redness and
swelling at the site of the bite, blurring of vision,
bilateral ptosis, diplopia and passage of red urine.
The blood was incoagulable, with prolonged pro-
thrombin time and APPT and thrombocytopenia
(78310
9
/l). Clotting factors V, VIII, VII and X were
grossly depleted but with normal levels of factors II,
IX, XI and XII, indicating disseminated intravascular
Table 2 Haematological variables in patients on admis-
sion to hospital (before antivenom therapy) following
bites by Crotalus durissus
Parameters Patients Controls
Mean"SD nMean"SD n
Fibrinogen (g/l) 0.60"0.70 17 2.50"0.36 11
FII (%)60"22 16 96"13 8
FV (%)33"38 13 120"37 4
FVII (%)87"33 16 102"21 11
FVIII (%)71"42 17 117"35 15
FIX (%)75"33 16 98"27 14
FX (%)80"39 7 86"21 7
FXI (%)95"55 10 140"39 8
FXII (%)88"28 16 112"913
FnDP/FgDP (mg/ml) 564"501 15 4"011
D-dimer (mg/ml) 37.3"33.5 17 0.5"011
a
2
-antiplasmin (%)36"27 15 100"53
TAT (mg/l) 73.0"25.4 7 5.3"1.3 3
Platelets310
9
/l 278"80 17 237"16 20
Erythrocytes310
12
/l 4.8"0.4 15 5.5"1.0*
Haemoglobin (g/l) 144"15 15 155"25*
Haematocrit (%)44"41547"7*
Leucocytes310
9
/l 12.8"6.1 15 7.5"3.5*
Neutrophils310
9
/l 10.6"6.4 15 2.0–7.5*
Basophils310
9
/l 0 15 0.02–0.1*
Eosinophils310
9
/l 0.16"0.23 15 0.04 –0.40*
Lymphocytes310
9
/l 1.5"0.7 15 1.5–4.0*
Monocytes310
9
/l 0.2"0.2 15 0.2–0.8*
FnDP/FgDP, fibrin(ogen) degradation products; TAT,
thrombin-antithrombin III complex. *Data from Dacie
and Lewis.
35
Envenoming by C. durissus 555
coagulation. CK was 14 400 U/l (5%CKMB),
haemoglobin 147 g/l, haematocrit 46%, leukocytes
14.8310
9
/l (85%polymorphs). The urine was
strongly positive for blood, haemoglobin or myo-
globin, but there was no evidence of renal impair-
ment. He was treated with 20 ampoules of Crotalus
antivenom (Anticrota´lico, Instituto Butantan) 18 h
after the bite and remained haemodynamically
stable, with normal urine output, no biochemical
evidence of renal impairment, falling levels of CK
and an increase in platelet count. His vision and
ptosis improved. On the fourth day of admission
(107 h after the bite), the patient developed visual
and auditory hallucinations with paranoia. There
was no biochemical or haemostatic abnormality.
Ten days after the bite, the patient suffered a fatal
cardiac arrest while taking a bath. Permission for
autopsy was not granted by his family.
Figure 3. Plasma levels of (a) myoglobin and (b) creatine phosphokinase on admission and following antivenom
administration in patients bitten by Crotalus durissus. Bars show the mean"SEM. Numbers over bars ¼number of patients.
Figure 2. Plasma concentration of (a) fibrinogen, (b) fibrin/fibrinogen degradation products (FnDP/FgDP), (c) cross-linked
fragment D (DD), (d)a
2
-antiplasmin in patients on admission and following treatment with antivenom. Bars represent the
mean"SEM. Numbers over bars¼number of patients.
I.S. Sano-Martins et al.556
Discussion
In this study, the severity of envenoming correlated
with venom antigenaemia and haemostatic abnor-
malities on admission to hospital. The four highest
venom antigen levels were found in the severely
envenomed patients. However, in patient 21,
in whom a negative admission venom level was
recorded, it is likely that the blood sample was
taken before release of the tourniquet. It is pos-
sible that only after release of the tourniquet
were the clinical signs observed, probably due to
the release of the venom, previously retained by
the tourniquet, into the circulation.
Both the fatal cases recorded here had profound
haemostatic abnormalities including thrombo-
cytopenia. Among 12 cases of fatal envenoming
by C. durissus in the State of Sa˜o Paulo, 10 (83%)
presented with coagulopathy.
36
However, the likely
cause of death in patient 3 in the present study was
respiratory complications of probable respiratory
muscle weakness that required intubation and
mechanical ventilation. Other life-threatening
effects of envenoming identified in this study were
hypotension and shock, rhabdomyolysis, possible
myocardial damage (patient 13), renal failure and
spontaneous haemorrhage.
Alterations of the whole-blood clotting time
have been observed in 48%(104/216) of patients
bitten by C. durissus, and incoagulability in 38%
(86/216).
26
Some patients present with no detect-
able fibrinogen.
27
In this study, consumption
coagulopathy, characterized principally by the
consumption of fibrinogen, factor V and protein C,
was observed with slight depletion of factors II
and VIII; however, levels of other coagulation
factors were generally within the normal range.
The coagulopathy was accompanied by an increase
in levels of FnDP/FgDP and DD, and a significant
consumption of a
2
-antiplasmin, indicating a sec-
ondary activation of the endogenous fibrinolytic
system; this is consistent with evidence that
the venoms of C. d. terrificus,C. d. collilineatus
and C. d. cascavella do not have fibrinolytic
activity in vitro.
14
An increase in levels of the
thrombin-antithrombin complex was also observed,
indicating the formation of intravascular thrombin.
The decrease in fibrinogen levels soon after
the bite can be explained by the thrombin-like
enzyme in the venom. However, the alterations in
the levels of some coagulation factors (II, V, VIII and
protein C) and the formation of DD and thrombin-
antithrombin complex, characteristic of physio-
logical thrombin action, are difficult to explain as
a direct effect of C. durissus venom, since no activ-
ator of factors II or X is present.
14
It is possible
that another, not yet identified, activator may be
involved. Alternatively, the thrombin-like enzyme
in C. durissus venom may be different from that
present in Bothrops venoms, and act more like
physiological thrombin. The thrombin-like enzyme
of Bothrops jararaca venom is not capable of
clotting rabbit fibrinogen,
37
whereas C. d. terrificus
venom clots both human (Minimum Coagu-
lant Dose, MCD, 21.5 mg/ml) and rabbit (MCD
33.3 mg/ml) fibrinogen.
14
This suggests that the
thrombin-like component of the major Brazilian
subspecies of C. durissus venom has an action
similar to physiological thrombin. In C. durissus
envenoming, all the changes in the coagulation
system, with the exception of fibrinogen levels,
are milder than in Bothrops envenoming, as we
reported previously.
38
The venom of C. durissus includes a powerful
myotoxin which causes rhabdomyolysis in patients
2
and damages the microvasculature of smooth
muscle, especially that of endothelial cells lining
the capillaries and arterioles.
22
The changes in
vascular smooth muscle may be due directly to
the toxic effect of the venom components or
indirectly to the ischaemia
39
described in patients
envenomed by South American rattlesnakes.
26
It
is possible that endothelial cells thus stimulated
may release various constituents that can activ-
ate the coagulation system. The generation of
intravascular thrombin in C. durissus envenoming
could therefore be a consequence of the secondary
activation of the coagulation system as suggested
in this study.
Ontogenic variations in snake venoms may be
important.
40
In the genus Bothrops, the venoms of
the young snakes of the majority of species contain
larger amounts of both factor II and factor X
activators than those of adult snakes.
41
This results
in relatively more severe haemostatic disorders
following envenoming by young snakes.
42
In
young Crotalus atrox, a direct thrombin-like
clotting action on fibrinogen was observed in 2–8-
month-old specimens; from 11 months onwards,
the venoms of the same individual snakes no longer
clotted fibrinogen directly.
43
However, these
age-related differences have not been described
in envenoming by the South American rattle-
snake,
44
and there is no reference in the literature
demonstrating ontogenic differences in coagulant
activity of the venoms of either C. d. terrificus or
C. d. collilineatus.
Venom-induced systemic haemorrhage is rarely
reported following envenoming by C. durissus
subspecies; slight bleeding at the site of the bite is
usual.
45
However, according to Jorge and Ribeiro,
26
about 4.8%(12/249) patients had systemic bleed-
ing; gingival haemorrhage, epistaxis, and vaginal
bleeding. The figures in our study agree, with only
Envenoming by C. durissus 557
one (4.2%) of our 24 patients having signs of
spontaneous systemic haemorrhage, despite the
prevalence of severe coagulopathy. Various
snake venoms possess activators and inhibitors of
platelets.
46
In patients envenomed by Bothrops
species, thrombocytopenia is very common,
and is frequently associated with a bleeding
tendency
47
and platelet dysfunction.
48
However,
in C. durissus envenoming, thrombocytopenia is
rarely recorded;
27
our results support this observa-
tion. Although it is well known that convulxin is a
potent platelet-aggregating agent
6,49
and that cro-
toxin can also cause platelet aggregation,
50
in our
study only two patients developed thrombo-
cytopenia. The polymorphic leukocytosis observed
in C. durissus envenoming is similar to that seen
after bites by many species of snakes, including
Bothrops.
38
Following treatment with specific C. d. terrificus
antivenom,
29
levels of fibrinogen, FnDP/FgDP, DD,
a
2
-antiplasmin and myoglobin rapidly returned to
normal. In fact, by 12 h after the start of antivenom,
almost all laboratory haemostatic variables had
returned to normal; however as would probably
be expected, CK values remained elevated for
over 24 h. We can thus confirm the efficacy
of antivenom treatment, as with envenoming by
B. jararaca.
38
Clinically moderate and severe systemic
envenoming by C. durissus is often associated
with haemostatic disorders attributable to the
action of the thrombin-like enzyme, possibly in
association with the myotoxic component of the
venom. However, the two fatalities in this series
could not be attributed to bleeding or coagulation
disturbances.
Acknowledgements
Financial support was obtained from the Science
and Technology for Development programme of
the European Community (contract no. TS3-CT91-
0024) and FAPEMIG (contract no. CBS 542/92).
CFSA, NAR and ISS-M are recipients of CNPq
fellowships. We wish to thank Neusa Tadeu Penas
Picon for technical assistance.
References
1. Vital Brazil O. Venenos ofidicos neuroto´xicos. Rev Ass Med
Bras 1980; 26:212–18.
2. Azevedo-Marques MM, Cupo P, Coimbra TM, Hering SE,
Rossi MA, Lavre J. Myonecrosis, myoglobinuria and acute
renal failure induced by South American rattlesnake
(Crotalus durissus terrificus) envenomation in Brazil.
Toxicon 1985; 23:631–6.
3. Nahas L, Denson KWE, Macfarlane RG. A study of the
coagulant action of eight snake venom. Thromb Diath
Haemorrh 1964; 2:355–67.
4. Slotta KH, Fraenkel-Conrat H. Estudos quı´micos sobre
os venenos ofı´dicos. 4. Purificac¸a˜ o e cristalizac¸a˜o do
veneno da cobra cascavel. Mem Ins Butantan 1938;
12:505–12.
5. Moura-Gonc¸alves J, Vieira A. Estudo sobre veneno de
serpentes brasileiras. I. Ana´ lise eletrofore´tica. An Acad Bras
Cieˆnc 1950; 22:141–50.
6. Prado-Franceschi J, Vital Brazil O. Convulxin, a new toxin
from the venom of the South American rattlesnake Crotalus
durissus terrificus.Toxicon 1981; 19:875–87.
7. Raw I, Rocha MC, Esteves MI, Kamiguti AS. Isolation and
characterization of a trombin-like enzyme from the venom
of Crotalus durissus terrificus.Braz J Med Biol Res
1986; 19:333–8.
8. Barrio A. Giroxin, a new neurotoxin of Crotalus durissus
terrificus venom. Acta Physiol Latinoamericana 1961;
11:224.
9. Alexander G, Grothusen J, Zepeda H, Schuartzman RJ.
Gyrotoxin, a toxin from the venom of Crotalus durissus
terrificus, is a thrombin-like enzyme. Toxicon
1988; 26:953–60.
10. Vital Brasil O. Neurotoxins from the South American
rattlesnake venom. J Form Med Ass 1972; 71:394–400.
11. Gopalakrishnakone P, Hawgood BJ. Morphological changes
induced by crotoxin in murine nerve and neuromuscular
junction. Toxicon 1984; 22:791–804.
12. Schenberg S. Geographical pattern of crotamine
distribution in the same rattlesnake subspecies. Science
1955; 129:1361–3.
13. Minton SA, Minton MR. Venomous Reptiles. London,
George Allen and Unwin, 1971.
14. Santoro ML, Sousa e Silva MCC, Gonc¸ alves LRC,
Santos SMA, Ferreira ILL, Saiki M, Peres CA, Sano-Martins IS.
Comparison of the biological activities of three subspecies
of the South American rattlesnake (Crotalus durissus
terrificus, Crotalus durissus cascavella, Crotalus durissus
collilineatus). Comp Biochem Physiol 1999; 112C:61–73.
15. Rosenfeld G. Symptomatology, pathology and treatment of
snake bites in South America. In: Bucherl W, Buckley EE,
eds. Venomous animals and their venoms. New York,
Academic Press, 1971:345–84.
16. Amorim MF, Franco de Mello R, Saliba F. Envenenamento
botro´ pico e crota´ lico. Mem Inst Butantan 1951; 23:63–108.
17. Klauber LM. The bite and its effects, Ch.12. In: Rattlesnakes,
Vol. II. University of California Press, Berkeley and
Los Angeles, 1956:797–859.
18. Russell FE, Walter FG, Bey TA, Fernandez MC. Snakes
and snakebite in Central America. Toxicon 1997;
35:1469–522.
19. Amaral CFS, Magalha˜es RA, Rezende NA.
Comprometimento respirato´ rio secunda´ rio a acidente
ofı´dico crota´ lico. (Crotalus durissus). Rev Inst Med Trop Sa˜o
Paulo 1991; 33:251–5.
20. Azevedo-Marques MM, Hering SE, Cupo P. Evidence that
Crotalus durissus terrificus (South American rattlesnake)
envenomation in humans causes myolysis rather than
hemolysis. Toxicon 1987; 25:1163–8.
I.S. Sano-Martins et al.558
21. Cupo P, Azevedo-Marques MM, Hering SE. Clinical and
laboratory features of South American rattlesnake (Crotalus
durissus terrificus) envenomation in children. Trans Roy Soc
Trop Med Hyg 1988; 82:924–9.
22. Rossi MA, Peres LC, Paola F, Cupo P, Hering SE,
Azevedo-Marques MM. Electron-microscopic study of
systemic myonecrosis due to poisoning by tropical
rattlesnake (Crotalus durissus terrificus) in humans.
Arch Pathol Lab Med 1989; 113:169–73.
23. Cupo P, Azevedo-Marques MM, Hering SE. Acute
myocardial infarction-like enzyme profile in human
victimsof Crotalus durissus terrificus envenoming. Trans Roy
Soc Trop Med Hyg 1990; 84:447–51.
24. Leite S. Cartas dos primeiros jesuı´tas do Brasil III. Sa˜ o Paulo,
Comissa˜ o do IV Centena´ rio da cidade de Sa˜o Paulo,
1954:93 and 536.
25. de Siqueira JE, Higuchi M, de L, Nabut N, Lose A, Souza JK,
Nakashima M. Lesa˜o mioca´rdica em acidente ofı´dico pela
espe´ cie Crotalus durissus terrificus (cascavel), relato de caso.
Arq Bras Cardiol 1990; 54:323–5.
26. Jorge MT, Ribeiro LA. Epidemiologia e quadro clı´nico do
acidente por cascavel Sul-Americana (Crotalus durissus).
Rev Inst Med Trop Sa˜ o Paulo 1992; 34:347–54.
27. Amaral CFS, Rezende NA, Pedrosa TMG, da Silva OA,
Pedroso ERP. Afibrinogenemia secunda´ ria a acidente ofı´dico
crota´ lico (Crotalus durissus terrificus). Rev Inst Med Trop Sa˜o
Paulo 1988; 30:288–92.
28. Ministe´rio da Sau´de. Manual de diagno´stico e tratamento de
acidentes por animais pec¸ onhentos. Brasilia, Fundac¸a˜o
Nacional da Sau´ de, 1998:131.
29. Theakston RDG, Warrell DA. Antivenoms: a list of
hyperimmune sera currently available for the treatment of
envenoming by bites and stings. Toxicon 1991; 29:1419–70.
30. Denson, KWE. Appendix 2. In: Biggs R, ed. Human Blood
Coagulation Haemostasis and Thrombosis. Blackwell
Scientific, Oxford, 1976:634–6.
31. Ratnoff OD, Menzie CA. New method for the determination
of fibrinogen in small samples of plasma. J Lab Clin Med
1951; 37:316–20.
32. Cha´vez-Olortegui C, Penaforte CL, Silva RR, Ferreira AP,
Rezende NA, Amaral CFS, Diniz CR. An enzyme-linked
immunosorbent assay (ELISA) that discriminates between the
venoms of Brazilian Bothrops species and Crotalus durissus.
Toxicon 1997; 35:253–60.
33. Theakston RDG. The application of immunoassay
techniques including enzyme-linked immunosorbent
assay (ELISA) to snake venom research. Toxicon 1983;
21:343–52.
34. Ho M, Warrell MJ, Warrell DA, Bidwell D, Voller AA.
Critical reappraisal of the use of enzyme-linked
immunosorbent assays in the study of snake bite.
Toxicon 1986; 24:211–21.
35. Dacie JV, Lewis SM. Reference ranges and normal values.
In: Dacie JV, Lewis SM, eds. Practical Haematology.
Churchill Livingstone, Edinburgh, 1991:9–17.
36. Ribeiro LA, Albuquerque MJ, Pieres de Campos VAF, Katz G,
Takaoka NY, Lebra˜ o ML, Jorge MT. O
´bitos por serpentes
pec¸ onhentas no estado de Sa˜o Paulo: avaliac¸a˜o de 43 casos,
1988/93. Rev Ass Med Brasil 1998; 44:312–18.
37. Santoro ML, Sano-Martins IS. Different clotting mechanisms
of Bothrops jararaca snake venom on human and rabbit
plasmas, Toxicon 1993, 31:733–42.
38. Cardoso JLC, Fan HW, Franc¸a FOS, Jorge MT, Leite RP,
Nishioka SA, Avila A, Sano-Martins IS, Tomy SC, Santoro ML,
Chudzinski AM, Castro SCB, Kamiguti AS, Kelen EMA,
Hirata MH, Mirandola RMS, Theakston RDG, Warrell DA.
Randomized comparative trial of three antivenons in the
treatment of envenoming by lance-headed vipers (Bothrops
jararaca)inSa˜o Paulo, Brazil. Q J Med 1993; 86:315–25.
39. Williams-Kretschmer K, Majno G. Ischemia of the skin:
Electron-microscopic study of vascular injury. Am J Pathol
1969; 54:327–53.
40. Warrell DA. Geographical and intraspecies variation in
envenoming by snakes. In: Thorpe RS, Wuster W,
Malhotra A, eds. Venomous Snakes Exology, Evolution
and Snake Bite. Clarendon Press, Oxford, 1997:189–203.
41. Furtado MFD, Maruyama M, Kamiguti AS, Antonio LC.
Comparative study of nine Bothrops snake venoms from
adult female snakes and their offspring. Toxicon 1991;
29:219–26.
42. Ribeiro LA, Jorge MT. Acidentes por serpentes do geˆnero
Bothrops:se´rie de 3139 casos. Rev Soc Bras Med Trop
1997; 30:475–80.
43. Reid HA, Theakston RDG. Changes in coagulation effects
by venoms of Crotalus atrox as snakes age. Am J Trop
Med Hyg 1978; 27:1053–7.
44. Jorge MT, Ribeiro LA, Nishioka AS. A comparison of clinical
and epidemiological aspects of bites of small and large
South American rattlesnakes. Trop Doctor 1997; 27:106–7.
45. Magalha˜es RA, Ribeiro MMF, Rezende NA, Amaral CFS.
Rabdomio´ lise secunda´ ria a acidente ofı´dico crota´ lico
(Crotalus durissus terrificus). Rev Inst Med Trop Sa˜o Paulo
1986; 28:228–33.
46. Kini RM, Evans HJ. Effects of snakes venom proteins
on blood platelets. Toxicon 1990; 28:1387–422.
47. Kamiguti AS, Rugman FP, Theakston RDG, Franc¸a FOS,
Ishii H, Hay CRM, BIASG. The role of venom haemorrhagin
in spontaneous bleeding in Bothrops jararaca envenoming.
Thromb Haemostas 1992; 67:484–8.
48. Sano-Martins IS, Santoro ML, Castro SCB, Fan HW,
Cardoso JLC, Theakston DG Platelet aggregation in
patients bitten by the Brazilian snake Bothrops jararaca.
Thromb Res 1997; 87:183–95
49. Marlas G, Joseph D, Huet C. Subunit structure of a potent
platelet-aggregating glycoprotein from the venom of
Crotalus durissus cascavella.Biochemie 1983; 65:619–28.
50. Landucci ECT, Condino-Neto A, Perez AC, Hyslop S,
Corrado AP, Novello JC, Marangoni S, Oliveira B, Antunes E,
de Nucci G. Crotoxin induces aggregation of human washed
platelets. Toxicon 1994; 32:217–26.
Envenoming by C. durissus 559
