BOTROPASE
It is well known that snake venoms, one of the most
concentrated enzyme sources in nature are valuable
expedients in biochemical research. Some of the snake
venom enzymes eg: thrombin like enzymes have certain
applications in the clinical field. The biochemical and
therapeutic uses of snake venom enzymes have led to
intensive attempts to study the occurrence, purification
and characterization of enzymes.
Blood coagulation can be regarded as a cybernetic
system. Some of the controlling components consist of
positive and negative feedback, retarded chain
reactions, multiple enzyme involvement, apparent stoi-
chiometric reactions, and integration with organ
function. The system can be divided into three basic
events or reactions as follows
1. Formation of Auto prothrombin C (Factor Xa)
2. Formation of Thrombin
3. Formation of Fibrin
Snake venoms contain enzymes concerned with all three
basic reactions of blood coagulation. They contain fibri-
nolytic enzymes and inhibitors. Thrombin like enzyme
from Bothrops venoms activates factor XIII. The thrombin
like enzymes are eliminated via urine, the RES and
neutralisation by inhibitors. Their activity is not
reduced by heparin or hirudin.
The clotting of fibrinogen by the venoms was recognised
at the beginning of this century and most likely earlier.
Thrombin like activity was found in most of the Crotalide
group of venoms.
It is mainly due to Brazilian investigators that it has been
possible to isolate the active principles affecting
coagulation, from the snake venoms.
Especially in the last ten years, there has been a veritable
blossoming of research into the relationship between
ophidic poisons and the coagulation of the blood.
BOTROPASE is the first pharmaceutical preparation to be
used therapeutically and is based on the coagulative and
antihaemorrhagic properties of those fractions isolated
from the poison of BOTHROPS JARARACA OR
BOTHROPS ATROX which affect the coagulation of the
blood.
Following the treatment of hundreds of thousands of
subjects over the last 60 years with coagulative
fraction of ophidic poisons and the first therapeutic
experiments carried out with BOTROPASE in Brazil may
rightly be considered an event of almost historical
importance, which has paved the way for all the
subsequent experimental and clinical research.
MECHANISM OF ACTION :
Through out the literature of the past decades, there
appears a desire to find that which "triggers blood
coagulation". No one factor or component is sufficient to
accelerate the system.
The fibrinogen of plasma, with a molecular weight of near
3,40,000 and having three distinct polypeptide chains,
undergoes limited proteolysis by thrombin.
The process removes fibrino peptide A and fibrino peptide
B from each fibrinogen molecule. The fibrinogen without
fibrinopeptides is refered to as a fibrin monomer and by
a process of self assembly, the monomer forms polymers.
The molecules align end-to-end and side-to-side, and the
fibrin has inter connecting branches. Though the rate of
fibrin formation depends upon various factors, thrombin
concentration is the main determinant. Highest
concentration being associated with the most rapid
clotting. Platelets contain a substance known as
platelet factor 2 which has the property of shortening
clotting time of plasma or fibrinogen.
The thrombin like action of the active principle of Bothrops
Jararaca poison of Botropase is characterised primarily
by the fact that blood coagulation takes place even when
all other clotting factors are absent, in particular calcium,
prothrombin, and various other plasmatic and platelet
factors. The action of Botropase in this way is similar to
that of thrombin, which converts fibrinogen into fibrin
without the intervention of any other factors.
There are however, some significant divergences
between the mode of action of Botropase and that of
thrombin, the principal ones being the following.
1. The action of thrombin may be inhibited by the
antithrombin normally existing in the blood, whereas
the action of Botropase continues, even when
antithrombjp is present.
2. By the action of either thrombin or Botropase, the
terminal aminoacids in the fibrin clots consist of tyrosine
and glycine; however, the ratio is of 1:2, in the former
and in the latter it is 1:1.
3. Unlike thrombin, Botropase is not absorbed by the
fibrin clots and is therefore not neutralised by that
mechanism, as occurs with thrombin.
The most important of these differences, from the
practical point of view, is the fact that Botropase remains
in the blood stream even when antithrombin is present and
is not absorbed by the fibrin clots. This enables the action
of Botropase to be much more prolonged than is possible
with thrombin, which is immediately neutralised by the
fibrin clots.
The most recent research has enabled the mode of action
of the Bothrops Jararaca haemocoagulase to be more
exactly defined. In the first place it has been noticed
(Dinelli & Coil) that it is possible by means of
electrophoresis, to establish the direct attack of Botropase
on fibrinogen, on which thrombin acts in a similar manner.
As far as blood platelets are concerned, it has repeatedly
been confirmed that when Botropase comes into contact
with platelets, it releases thromboplastinic factors from
them. This effect may be demonstrated by means of the
thromboplastin generation test.
Finally, it is worth recording the research carried out by
Del Compo and Fazzi vlSlich has shown that the antigenic
nature of Botropase and particularly the possibility that
specific antibodies are formed subsequent to its injection
may definitely be ruled out.
INDICATIONS
POST OPERATIVE HAEMORRHAGE
* Capillary Haemorrhages
* Laparotomy
* Tonsillectomy & Adenotomy
Laryngectomy
* Tympanoplasty
Dental extractions
Prostatectomy
Ophthalmological surgery
Gastroenterology
* Cosmetic Surgeries
PRIMARY AND SECONDARY HAEMORRHAGIC SYNDROMES
Haemophilic syndromes (Haemophilia A,
Haemophilia B)
Haemophilia - like syndromes
(Eg: parahaemophilia, Hypoproconvertinaemia)
Haemorrhagic syndromes due to circulating
anticoagulants.
Thrombocytopenic purpura
Hypoprothrombinaemia.
INTERNAL & EXTERNAL HAEMORRHAGES
Haematoma
Epistaxis
Haemoptysis
Haematemesis
Melena
Haematuria
Meno & Metrorrhagia
PPH (Post Partum Haemorrhage)
CONTRA-INDICATIONS
In view of its strong coagulating action, Botropase is
contraindicated in cases of venous or arterial thrombosis
and as a general rule in diseases with a tendency to
intravascular coagulation (Thrombophilic conditions).
INEFFICACY
In the extremely rare cases of haemorrhage entirely due
to fibrinogen deficiency (hypofibrinogenaemia, afibrino-
genaemia), Botropase alone is not sufficient to achieve
complete haemostasis and it is therefore
necessary to adopt a therapy of replacement by means
of blood or plasma transfusions, or better still by the
injection of purified fibrinogen.
TOLERANCE
Botropase is well tolerated. Only in exceptional cases
there may be symptoms of mild allergy. But these are
quickly obviated by the administration of antihistaminics.
DOSAGE
Prophylaxis For Haemorrhages - One ampoule of 1 cc.lM
2-3 hours prior to intervention and one ampoule of 1 cc.
IV 15-30 minutes prior to surgical intervention. Prophylaxis
of one ampoule daily for 3-4 or more days before the
intervention.
Treatment In Haemorrhages - One ampoule of 1 cc.lM
or IV repeated after a few hours (8 hourly intervals) or if
required at shorter intervals (30-60 minutes) until the
desired effect is obtained. In serious and urgent cases,
the IV Injection may be followed immediately, if neces-
sary, by an IM Injection.
Injections of Botropase by the i.v. route should be effected
rapidly.
Local Haemorrhagic Therapy - In localised haemor-
rhage, for example in epistaxis, dental extractions, cap-
illary haemorrhage due to surgical intervention, the con-
tents of one or more ampoule should be sprayed on to the
haemorrhagic area.
Paediatric Dosage - 0.50 - 0.75 cc according to the age
of the child and seriousness of the haemorrhage.
CLINICAL APPLICATIONS OF BOTROPACE
The clinical applications of Botropase are of great
practical interest. As previously mentioned, the first
clinical experiment was carried out with a preparation
similar to Botropase, by Vaz and Pereira as far back as
1940, in a case of haemophilia with dental haemorrhage
being arrested within 10 minutes of the injection. On
recurrence of the haemorrhage the following day, it was
definitely stopped by two further injections, one by 1.V, the
other by 1.M. route.
From that time onwards, investigations into the clinical
applications of Botropase have greatly increased, and in
practically every sphere of medicine, surgery and kindred
branches the results achieved have proved more than
satisfactory, in the most varied haemorrhagic conditions
as well as in prophylactic measures adopted for the
prevention of haemorrhage, such as are normally used
preoperatively.
We shall now briefly review the greater part of the
observations which have been published upto the present, but
would point out that the data concerned are only those which
have appeared in prinh and that a great deal more unpublished
information has been collected and extensively documented
in reliable case histories, preserved in nursing homes, hospi-
tals and out patient departments.
Botropace in haemophilic syndromes
Haemophilic syndromes constitute one of the most
important applications of Botropase. This designation, as
is well known, really denotes many different conditions
characterised by the diminution of a plasmatic factor of
thromboplastin.
Haemorrhage in these conditions is variable in volume
but nevertheless particularly severe and at times ex-
tremely serious.
It has been demonstrated that Botropase is able to
combat these conditions by means of its positive, con-
stant and innocuous coagulating action. It increases the
coagulability of the blood in haemophilic syndromes, and
at the same time checks the haemorrhage, in the majority
of cases permanently.
It is therefore evident that Botropase brings about the
following results in haemophilic syndromes.
Reduction of clotting time of whole blood.
Reduction of recalcification time.
Reduction of reaction time and clot formation time.
Increased utilisation of prothrombin.
Increased production of thromboplastin.
In haemophilic syndromes, Botropase should be
administered by Injection either IV or IM whenever there
is an internal haemorrhage, and also in cases of external
haemorrhages. In ~e latter, however, and especially in
all forms of dental haemorrhage usually resistant to other
types of treatment it is advisable to apply Botropase
externally by means of tampons impregnated with this
preparation.
Botropase does not constitute a replacement therapy in
haemophilic syndromes and in many cases,
therefore, especially those of a serious nature, it should
be associated with transfusion therapy to enable the
deficient factor to be introduced into the system.
Botropase is also beneficial in maintenance and
prophylactic therapies in haemophilic syndromes,
because of its thromboplastin like effect at the usual
therapeutic dosage. Many haemophiliacs have been
treated for long period with Botropase, and have remained
immune from haemorrhagic complications as a result.
One of the advantages of Botropase in the treatment of
haemophilia is that its clotting action takes place in every
case, independently of the nature of the haemophilic
syndromes concerned. In contrast, certain plasma
derivatives such as purified antihaemophilic globulin,
whose use necessitates careful differentiation of the
various forms of haemophilia, Botropase may be used in
all forms without the need for a complete haematological
examination to determine the nature of the haemophilic
syndromes.
BOTROPACE IN GENERALISED HAEMORRHAGIC SYNDROMES
Besides haemophilic syndromes, which represent the
most typical field of application of Botropase, many other
haemorrhagic conditions benefit by the use of this
preparation. On the basis of the research so far carried
out, Botropase is indicated in any form in which
alterations in the process of blood clotting have occurred,
and which require the administration of a coagulant by
parenteral route. The use of Botropase is therefore also
warranted in the treatment of haemorrhage occurring in
the following conditions:
(a) deficiency of prothrombin, of Factor V (labile
factor) and of Factor VII (stable factor)
(b) presence of anticoagulant in the blood stream.
The first group comprises forms in which there is
congenital deficiency of prothrombin, factor V and factor
VII, which in many cases have similar characteristics as
those of haemophilia and consequently require similar
treatment. As, however, these forms are seldom
encountered, this group does not constitute an extensive
field of application.
Forms with a deficiency of prothrombin and factor VII are,
on the contrary, much more frequently seen during
treatment with anticoagulants such as dicoumarin, cou-
marin or indandione.
Syndromes due to anticoagulants in the blood circulation
form a further group of haemophilic conditions. For
example anticoagulants may appear in the blood stream
either as a complication of haemophilia or independently
of this disease and are of various types, which may bring
about haemorrhagic symptoms. The effect of Botropase
in such conditions is similar to that described in relation
to haemophilic syndromes.
Anticoagulants may, however, also be found in the blood
stream after introduction for therapeutic purpose, and this
is a normal occurrence in the course of treatment with
heparin and similar drugs. Haemorrhage may also ensue
in these conditions and indicates the suspension of the
anticoagulant therapy, followed by the administration of
drugs with a coagulative effect.
Botropase may be used with advantage in other more
generalized haemorrhagic syndromes, even though the
results obtained are less spectacular than in the
conditions to which we have drawn attention.
It is clear that results of any importance cannot be
expected from the use of Botropase in cases where there
is a deficiency of fibrinogen, such as in the extremely rare
cases of congenital afibrinogenaemia. Obviously,
replacement therapy should be resorted to in such cases.
BOTROPACE IN LOCALISED INTERNAL HAEMORRHAGE
As already stated, amongst the indications of Botropase
are also included localised internal and external
haemorrhages which are not attributed to a systemic
haemorrhagic diathesis. They are in the main "lue to local
alterations in the haemostatic factors and in particular to
the blood vessels. This applies, for instance, to many
types of haemorrhages, such as those observed in
subjects affected with tuberculosis and also other
disorders, in which, Botropase may be administered for
haemostatic purposes by which it arrests the
haemorrhage by strengthening the clotting mechanism.
The possibility of utilising Botropase in epistaxis should
also be borne in mind; it may either be applied locally,
which is easily carried out as the haemorrhage is exter-
nal, or systemically. Although epistaxis is often caused
by some local nasal factor, systemic haemostatic therapy
is indicated for the same reason as those put forward with
regard to haemoptysis.
Haemorrhage of the gastroenteric tract, in the form of
haematemesis, melaena as well as bleeding oesopha-
geal varices can also be treated with Botropase, even
when the cause of the haemorrhage does not come within
the framework of a generalised haemorrhagic diathesis
but originates from the lesion of a superficial blood vessel
through erosion of the mucosa or from other harmful
agents. Such considerations are relevant, for eg.
haemorrhage from gastroduodenal ulcers is the most
common form of haemorrhage of the gastroenteric tract
and appears in the form of haematemesis or melaena.
Among the various localised types of internal
haemorrhages, haematuria must not be overlooked
although this comes to a certain extent within the sphere
of affections of a urological nature. In cases where it is
not attributable to a generalised haemorrhagic diathesis,
Botropase may safely be administered, even whilst
waiting for the exact nature of the haematuria itself to be
determined by means of a more thorough examination.
Menorrhagia comes within the haemorrhagic forms of a
gynaecological character and will therefore be dealt with
later.
BOTROPACE IN SURGERY
The application of Botropase in the surgical field are
extremely important. One of the important considerations
of a surgery is the danger of haemorrhage during and
after intervention. This may occur even when there is no
clinical evidence of any alteration in the coagulative and
haemostatic processes. Especially troublesome is the so
called capillary haemorrhage, and here are few really
effective measures against this at the disposal of the
surgeon. This is one of the most brilliant applications of
Botropase, in as much as it provides the surgeon with an
effective, innocuous and ever ready weapon to combat
such forms of haemorrhages.
BOTROPASE IN ORTHOPAEDIC SURGERY
Amongst the various surgical specialities, coagulative
and antihaemorrhagic therapy with Botropase is of par-
ticular interest in the field of orthopaedics, to which infact
the general indication for its administration apply, ln
orthopaedic intervention, therefore the use of Botropase
is indicated as a prophylactic measure, before
intervention and also during the operation in order to
effectively bring about local haemostasis and post
operatively to arrest possible haemorrhage. A frequent
cause of haemorrhage in orthopaedic surgical
interventions is the removal of cartilage from the bone
surfaces and in general the solution of continuity of bones
subsequent to trauma or in relation to the operation itself
In such conditions, Botropase may be used to advantage
either systemically or locally. Indeed, Lalli used Botropase
by means of a tampon applied to the bleeding surfaces
of the bone, and observed a diminution in the haematic
loss in such cases.
BOTROPACE IN UROLOGICAL SURGERY
The advantage of using Botropase in urological
surgery has been more fully appreciated as a result of the
recent research effected by Coppi, who used it for the
treatment of 22 patients who had undergone operative
therapy for diseases of a urological nature principally
prostatic. As is well known, haemorrhagic complications
are especially frequent during prostatectomy and for this
reason anti-haemorrhagic therapy is particularly indi
cated. According to Coppi's observations, with Botropase
post operative haemorrhage was greatly reduced ir
comparison with cases treated with other coagulants
Capillary haemorrhages appeared to be particularly
Response to treatment with botropace.
In only two cases it was found necessary to resort to
plugging. It was also clearly proved that, together with its
haemostatic effect Botropase also brings about a
reduction of coagulation time.
BOTROPACE IN EYE SURGERY
Inconvenience caused by infra-operative haemorrhage is
more noticeable when the operating field is
restricted to a small area, such as, in particular, in
ophthalmological surgery. It is consequently of greater
importance that pre-operative anti-haemorrhagic
prophylaxis should be carried out by means of suitable
coagulative and haemostatic drugs, and more
especially with Botropase which has been widely used in
opthalmological surgery by Calvi-Zampetti and Minini,
either by intravenous and intramuscular route, or by local
injection. Out of 52 cases who underwent prophylactic
treatment, 40 were given 1 cc of Botropase by intrave-
nous Injection and the remainder 1 cc intramuscularly.
Half this dose was administered to children upto 12 years
of age.
The experience of Calvi/Zampetti and Minini was ac-
quired through a great variety of interventions, and in
every case brilliant results were obtained, particularly in
view of the complications which may arise in the course
of some interventions. In the cases treated, no distur-
bances or symptqms of intolerance either immediate or
delayed were observed.
The effect of the haemocoagulase, Botropase , on the
haemostasis of intraoccular bleeding was evaluated in a
rabbit model by Kim et al. The experimental eyes showed
no abnormal findings histotogically. Infusate containing
Botropase appeared to be a useful adjunct for the control
of intraoccular bleeding during vitreous surgery.
BOTROPACE IN EAR,NOSE AND THROAT SURGERY
The use of Botropase in otorhinolaryngological surgery is
justified by the frequency of haemorrhage both during
and after intervention, even following slight operations
(tonsillectomy, turbinotomy, resection of the septum). It
is justified moreover, by the fact that wounds caused by
operation should be left open in natural cavities, as
haemostasis in such areas is somewhat difficult and not
always very effective (Cattaneo). Furthermore, in the
field of ear, nose and throat surgery, capillary haemor-
rhage seriously obstructs good visualisation of the oper-
ating field and satisfactory carrying out of the interven-
tion.
Subsequent to the observations of Vaz and Pereira in
1944, one of the principal contributions to this subject is
that of Plisnier who, in about hundred patients operated
upon for amygdalectomy and adenoidectomy and three
cases of endolaryngeal neoplasia on which laryngofis-
sure was performed, came to the conclusion that, after
the administration of Botropase the loss of blood during
interventioQ was trifling. Moreover, in the majority of
these case~,, the scar formed was clean and there was
no haemorrhage, either immediate or delayed.
BOTROPACE IN OBSTETRICS AND GYNAECOLOGY
The use of Botropase in the field of obstetrics and
gynaecology is justified by a mass of physiopathologic
and clinical evidence and is supported by a great number
of case histories. Indications for the employment of
coagulants such as Botropase in obstetrics and
gynaecology are two principal conditions, i.e. post-partum
haemorrhage not entirely due to uterine insufficiency or
to retention of ovular residue, and gynaecological sur-
gery. In so far as the latter is concerned, endeavours are
directed not alone towards preventing loss of blood, but
also towards obtaining as clear an operating field as
possible, with consequent improved visualisation of the
area itself.
According to Cazzola in plastic interventions - whatever
the technique employed - and in grafting processes of
longstanding, with extensive and tenacious adherences,
capillary haemorrhage is a frequent occurrence and should
be prevented as far as possible.
Recent case histories of Pescetto and Malagamba deal
with numerous clinical cases treated with Botropase,
concerning puerperae with profuse lochia, dyshormonal
metrorrhagia; patients on whom Vaginaplasty has been
performed, followed, by severe postoperative
haemorrhage ; as well as cases of puberal metrorrhagia
and functional metrorrhagia.
BOTROPACE IN PLASTIC SURGERY
Botropase can be effectively made use of to prevent the
capillary bleeding both at the donor site as well as
receptor site By sprinkling Botropase on the area. This
not only reduces capillary bleeding but also studies have
shown that healing is faster. It is postulated that Botropase
acts as a biological glue and it protects the grafts for
faster healing, thereby rejects are minimized. It is sup-
posed to reduce scar formation or cheloid
formation when sprinkled on the incision before suturing.
In Plastic surgery and grafting processes capillary
haemorrhage is a frequent occurrence and should be
prevented as far as possible
Another important application of Botropase is in the field
of dental haemorrhage, subsequent to dental avulsion or
other operation on the dental apparatus. The use of
Botropase in this connection is fully warranted by what
has been said concerning postoperative haemorrhage,
and haemorrhage in the various forms of specialised
surgery.
As a prophylactic measure, Botropase may be used prior
to dental avulsion or any other dental intervention by
means of
(a) An Injection of Botropase by tM route on the day
(days) preceding intervention.
(b) An Injection of Botropase by IV route, 15-20
minutes before intervention.
(c) An Injection of Botropase combined with the
anaesthetic at the time of anaesthesia.
This last type of prophylaxis is easily carried out and is
especially advisable, in as much as the addition of the
anaesthetic prevents possible local reactions of the soft
periodontal tissues.
Wound healing effect of botropace
I
In view of the importance of blood coagulation in wound
healing, Botropase used as systemic haemocoagulant to
arrest bleeding, is studied on different wound models in
albino rats. Physical, biochemical and histological
evaluation revealed that Botropase promotes reparative
process. Pro-coagulation effect and other enzymatic
actions of Botropase may be responsible for augmenta-
tion of healing.
Wound collagen content was increased by Botropase ir
a dose dependent manner. Presurgical use of Botropase
influences coagulation cascade which increasec
significantly the breaking strength and wound collagen
suggesting that Botropase is a promoter of wound heal
ing.
The literature contains much evidence of enthusiasm fo
the use of snake venoms for the purpose of elucidatinc
the nature of blood cogulation mechanisms. Apparently
the main attraction in the field of hemostasis anc
thrombosis has been the utility of venoms and venon-
materials in practical laboratory tests, in clinical uses
and mnro rprc>ntl\/ fnr annii~atinnc in thPnrPtir ctiiHioc
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