NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
11
of snake poison will have to be considered separately. I propose, then, to take
my considerations and observations in more or less chronological order ; firstly,
stating briefly the action of cobra and daboia venoms on the coagulation of
the blood as observed in vivo ; and secondly, passing on to consider more in
detail their effect on the coagulability of the whole blood and of the blood plasma
in vitro.
Wall 2 3
s t a t e s i n h i s w o r k o n I n d i a n s n a k e v e n o m s , t h a t t h e b l o o d i n t h e
human subject at post-mortem examination of cases of cobra venom intoxication
is, as a rule, in a fluid condition and clots badly on being withdrawn from the
vessels. In animal experiments, however, this observer found that the blood
on post-mortem examination was always clotted solidly. On the other hand,
Cunningham 24 has shown that, if an animal received a large quantity of cobra
venom and died very shortly after the injection, the blood drawn just before or
immediately after death remained unclotted for a considerable length of time,
and the clot which did ultimately form was semi-fluid, loose and soft ; and no
serum separated out. Neither of these observers makes any record of finding
immediately after death any intravascular thrombosis. This condition, however,
they might easily have overlooked, in the same way as they both failed to ob-
serve the intravascular clotting which results from an intravenous injection of
daboia venom.
In a recent publication along with Dr. Hanna
2 5
I w a s a b l e t o r e c o r d d e f i n -
itely the fact that cobra venom, even when injected in large quantity into a vein,
does not produce any intravascular clotting. An amount of cobra poison, as
great as fifty times the amount of daboia venom which can cause extensive intra-
vascular thrombosis, was found to have no such effect. It is, therefore, certain
that, in whatever other respects these two venoms may differ from one another,
they do differ in this particular physiological action, namely, cobra venom does
not produce intravascular clotting ; while, as we shall see, daboia venom intro -
duced intravenously even in minute doses does possess this remarkable property.
If a non-lethal dose of cobra venom is injected subcutaneously into an
animal, one seldom sees any symptoms which might point, as is the case with
daboia venom, to any diminution of the blood coagulability. I have, however,
observed on several occasions during the process of immunisation of horses and
donkeys which received an injection of this venom over the shoulder, a large
œdema of the parts dependent from the site of injection and reaching down the
leg for some distance. I have never seen any general œdema. I have also
been able to demonstrate in the case of rabbits that a slight diminution of blood
coagulability does take place as a result of the subcutaneous injection of non-
lethal doses of cobra venom. Such a series of observations, made by means
of Wright's coagulometer tubes, are detailed in Table V of the protocols. A glance
at this table will show that in every experiment a slight lengthening of the
normal coagulation time of rabbit's blood followed the venom injection, and that
this diminution in coagulability was more marked the nearer the quantity of
venom injected approached to the minimum lethal dose. These experiments
might also explain Wall's failure to observe any post-mortem fluidity of the
blood in his animal experiments and Cunningham's success in this respect. For
it is apparent that, if a dose of cobra venom which kills in from 6 to 10 hours
be given, the deficiency in coagulability may not have had time to develop or
may be so slight as to escape observation on post-mortem examination, while a
larger quantity, such as Cunningham used, and which killed within a few minutes,
may cause a marked deficiency in the blood coagulability of fairly rapid onset
by reason of " mass action."
To sum up, then, it is certain that cobra venom in vivo does not produce
any increase but rather a deficiency of the blood coagulability, the amount of
this deficiency depending upon the quantity of venom injected, the mode of its
injection and the time allowed for its action to take place.
Before passing on to the consideration of the action of cobra venom on the
whole blood and on the blood plasma in vitro, it would be well, in order to
secure a consecutive story, to draw attention briefly to some observations which
I have, along with Dr. Hanna, already published2 5
o n t h e a c t i o n o f d a b o i a
venom on the blood coagulability in vivo.
Both Wall2 6
a n d C u n n i n g h a m
2 7
a t t r i b u t e d t h e r a p i d d e a t h w h i c h f o l l o w s t h e
intravenous or subcutaneous injection of daboia venom to a direct action of the
poison on the central nervous system. They considered that daboia venom in all
instances caused a fluidity of the blood, sometimes amounting to complete loss of
coagulability. Hanna and I, 2 8
h o w e v e r , w e r e a b l e t o s h o w t h a t t h i s h y p o t h e s i s
was erroneous and that the rapid death which follows an injection of daboia
venom is invariably caused by extensive intravascular thrombosis. Further, we
showed that, if the amount of venom injected is not sufficient to cause this
thrombosis, a negative phase of diminished coagulability results, sometimes
amounting to complete inhibition of clotting, and that this diminished coagula-
bility is probably an important factor in the production of some of the symptoms
which are observed in these cases. We found that, while 0. 1 milligramme of
daboia venom per kilo of weight when injected intravenously into a rabbit causes
extensive intravascular clotting, 0·4 milligramme has the same effect when in
- j e c t e d s u b c u t a n e o u s l y i n t o a p i g e o n . T h i s t h r o m b o s i s m a y b e c o n f i n e d , i f t h e
quantity of venom injected has not been excessive, to the portal veins, the right
heart and the pulmonary arteries : in these cases the blood collected from the
other veins, and from the left heart specially, remains unclotted or clots only
after a long interval of time, when the clot is very loose and gelatinous. We
13
also showed that, when the negative phase of diminished coagulability had once
set in, it was impossible to produce increased coagulability and thrombosis by
the injection of any quantity of venom which under normal circumstances injected
intravenously would have been ample to bring about these phenomena. The
action, then, of daboia venom on the blood coagulability in vivo is very definite
and well marked, and corresponds exactly, as far as can be judged from the
observation of the symptoms and the results of post-mortem examinations, to the
action, as described by Martin, of the venoms of the two Australian colubrine
snakes, namely, Hoplocephalus and Pseudechis. Until, however, researches
have been made with the specific sera prepared with these poisons, it would
be premature even to suggest that daboia venom was of the same nature as Hop-
locephalus and Pseudechis venoms even as regards its blood poison.
The action of cobra and daboia venoms on the coagulability
of the blood in vitro.
We may now pass on to consider if these snake venoms have any definite
action on the coagulation of the blood when mixed with this in vitro, In this
connection I shall have to put on record some experiments which I have made
with cobra and daboia venoms with the whole blood and with the blood plasma
freed from red cells.
With the poison of Pseudechis, Martin 29 found that by running fresh blood
into a solution (·01 per cent.) of this venom, clotting was considerably delayed,
and the clot which did form was soft, and on shrinking failed to entangle the
corpuscles. Working with crotalus poison, Weir Mitchell and Reichert 30w e r e
able to prevent coagulation of the blood altogether in vitro. These observers
drew the blood directly into a vessel containing a strong solution of venom sur-
rounded by a freezing mixture. On allowing the temperature to rise gradually
they found the blood remained permanently liquid.
Further, both Cunningham 31a n d K a n t h a c k
3 2
h a v e s h o w n t h a t w h e n f r e s h
blood was run into a strong solution of cobra venom, either it remained perma-
nently unclotted, or the clot which formed after a considerable delay was soft
and gelatinous and no serum exuded. The former of these observers also
states that in three experiments, in which blood was run into solutions of daboia
venom, although clotting took place with normal rapidity, the clots which formed
were loose, soft, and tremulous.
These observations, therefore, would tend to show that in vitro all snake
venoms have a similar action on the blood coagulation, namely, an inhibitory
action. In view of the facts recorded above as regards the action of cobra and
daboia venoms on the blood coagulability in vivo, it appeared to me necessary
14
to investigate further this action in vitro of these two venoms, the only two
which were available to me at the time.
After many failures to get any definite results by the method followed by
the older investigators, namely, the method of allowing fresh blood to flow
directly into a solution of venom, I was led to use blood which was kept liquid
by the addition of citrate of soda. At a later date I found that similar results were
obtained with citrate and oxalate plasma, which had been completely freed from
the red corpuscles by allowing these latter to sediment, and then syphoning off
the clear supernatant fluid.
The observations, therefore, which I have now to record naturally fall into
two groups, as follows :—
(1) Observations on the action of cobra and daboia venoms on the
coagulability of citrate whole blood.
(2) Observations on the action of cobra and daboia venoms on the
coagulability of oxalate and citrate plasma freed from red cells.
The action of cobra and daboia venoms on the coagulability
of citrate whole blood.
The blood which was used in these experiments was got from rabbits in the
following manner. By means of a cannula in the femoral artery, a measured quan-
tity of blood was run into a measured amount of a 20 per cent. solution of sodium
citrate. These quantities were so arranged that the citrate of soda was in the
pr opor t i on t o t he bl ood of 1 t o 100 or 1 t o 50. I n t he pr ot ocol s of t he exper i ment s
it is invariably recorded in which of these proportions the citrate was present.
The citrate blood was then divided up into a series of small test tubes, 1 c.c.
being accurately measured into each test tube. By direct experiment was
then ascertained the minimum amount of a solution of calcium chloride of
known strength which it was necessary to add to each cubic centimetre of cit-
rate blood so as to cause solid clotting in from 1 to 3 minutes, and likewise the
maximum amount of calcium chloride solution which could be added without
causing any clot in from 4 to 6 hours or longer.
It was found that these quantities of lime solution varied slightly with each
sample of citrate blood used, however carefully the solutions of citrate and of
calcium chloride were prepared, and however accurately the blood was measured
and divided up. It was, therefore, necessary to make careful control experi
- m e n t s , s u c h a s I h a v e i n d i c a t e d , f o r e a c h s e r i e s o f o b s e r v a t i o n s , t h a t i s t o s a y ,
for every different sample of rabbit's blood which was used.
Let us first consider, then, the action of cobra venom on plasma as shown by
its effect on the coagulability of citrate whole blood. In view of the fact that
cobra venom does not, even when introduced in large quantity directly into the
blood stream, produce intravascular clotting, it was not to be expected that this
poison would have the property of clotting citrate blood in vitro. Such a
surmise was found to be correct. For it will be seen on reference to Table VI
of the protocols that when 20 or even 30 milligrammes of cobra venom were
a d d e d t o 1 c . c . o f c i t r a t e b l o o d , n o c l o t f o r m e d a f t e r 2 h o u r s , a n d f u r t h e r , t h a t
on the addition, after this interval of time, of such an amount of soluble lime
salt as clotted the control in 3 minutes, the blood remained permanently liquid.
As we shall see later, this is a very different result to that got in similar
experiments with daboia venom. We can say, then, that a large quantity of cobra
venom does not increase blood coagulability in vitro but completely prevents
clotting taking place.
We have also seen above that cobra venom, injected subcutaneously or in-
travenously even in small sub-lethal doses, has the effect of diminishing the
coagulability of the blood. By means of a technique such as I have described
above, the optimum quantity of lime solution necessary for rapid clotting of the
control being added to each tube two hours after the addition of the cobra
venom, a result comparable to what obtains in vivo can be got in vitro
with citrate whole blood.
Thus, I was able to estimate fairly accurately, as judged from a number of
experiments, the minimum quantity of cobra venom which can keep 1 c.c. of
citrate rabbit blood permanently liquid in vitro, and the minimum quantity
which distinctly inhibits, but does not altogether prevent, coagulation.
The result of such an estimation is detailed in Table VII of the protocols.
From this table it will be seen that milligramme of cobra venom kept 1 c.c.
of blood liquid for at least 20 hours, and that 0·01 milligramme had a distinct
inhibitory action on the coagulability of the same amount of blood, lengthening
the coagulation time, as compared with the control, by about half an hour. It
may be mentioned in passing that the interval of time allowed to elapse
between the addition of the venom and the soluble lime salt in some cases affects
the results of experiments such as I have collated above. Especially is this
the case when the quantity of poison used is not greatly in excess of the mini-
mum amount requisite to keep the blood permanently liquid. A reference to
Table VIII will make the meaning of this statement clear. It requires no further
explanation, except to state that it was, however, found in many experiments
that even when the cobra venom and the lime solution were added together,
the same inhibition of coagulation resulted, as when a certain interval of time
was allowed to elapse. Such a result as I have tabulated in Table VIII is
exceptional and cannot be depended on.
In view of Martin's hypothesis, that the proteids coagulable by heat are
the constituents of snake venoms which affect the blood, it is important to be
C
16
able to record an observation bearing on the effect which heating a solution of
cobra venom, so as to coagulate the coagulable proteids, has on this anti-clot-
ting power of the poison. Such experiments are detailed in Table IX of the
protocols. Inasmuch as the same technique was used in these experiments as
was employed in the similar series of experiments with unheated venom detailed
in Table VII, one is able to compare the results recorded in these two Tables.
From such a comparison it is evident that, while heating a solution of cobra
venom at 75° C. for half an hour, that is to say, so as to coagulate all proteids
coagulable by heat, does not deprive the venom of its anti-clotting action on the
blood, such heating, nevertheless, diminishes this particular physiological action
of the poison to a considerable extent. For a reference to these two Tables
will show that, while 0·4 milligramme of the unheated venom suffices to keep 1 c.c.
of citrate blood permanently liquid after the addition of the lime solution, 0·4
milligramme of the heated venom, from which the coagulable proteids had been
removed, delayed clotting for a considerable interval of time, but did not pre-
vent it from ultimately taking place. Larger quantities of the heated venom
had a still greater inhibitory action on the coagulation of this blood, but even
2 milligrammes did not completely prevent clotting. Such a result would
appear to point to the conclusion, that it is not the coagulable proteids contained in
cobra venom which affect the blood coagulability, but rather some non-coagu-
lable substance which, however, is considerably diminished in activity by heating.
This result, therefore, lends no support to Martin's theory, a conclusion the
same as was arrived at with respect to the hæmolysing constituent of cobra
venom. These two similar results, in fact, are strong proof against the cor-
rectness of this hypothesis. Such, then, is the effect of the action of cobra
venom on the coagulability of citrate whole blood. We may now pass on to
the consideration of some similar experiments which were made with daboia
venom.
We have seen above that daboia venom, injected into animals in sufficient
quantity, so increases the blood coagulability as to lead to extensive and rapid
intravascular clotting, and further that, when the quantity injected is not suffi-
cient to cause thrombosis, a negative phase of marked diminution in the blood
coagulability sets in. As regards the in vitro experiments with citrate rab-
bit blood, now to be put on record, it is unnecessary to detail again the tech-
nique used, which was similar to that employed in the experiments with cobra
venom. But inasmuch as the first series of experiments was made with the
object of ascertaining if daboia venom had any effect on increasing the coagula-
bility of citrate blood, it is apparent that, in order to demonstrate such an
action to the best advantage, it was not feasible to add the lime solution to
each tube in such amount as would clot the control in from 2 to 3 minutes.
17
It was found that a good result was obtained by the addition of a very small
amount of calcium chloride, a quantity, in fact, which did not clot the control
even after six hours.
A reference to Table X will show the results which were obtained by work-
ing in this way. The control tube with a small quantity of lime added remained
unclotted after six hours ; the tubes to which from 1 to 0·0078 milligrammes
of daboia venom were added clotted solidly in from 5 to 10 minutes ; and that
to which 0·0002 milligramme was added clotted solidly after one hour. We
have here, then, a perfect demonstration in vitro of the positive phase of
increased coagulability caused by daboia venom. A further series of experi-
ments was then made with the object of ascertaining whether or not the nega-
tive phase of diminished coagulability could be demonstrated with citrate blood
in vitro. A technique similar to that used in the series of experiments noted
above was employed. But much smaller quantities of venom were used, and
the poison was added to the blood one hour before the addition of the lime
solution. Further, the quantity of lime solution added to each tube was slightly
increased, so that a clot formed in the control tube in about four hours. It
is evident, then, that these conditions of experimentation were in every way
favourable to show any diminution in the coagulability of the blood which the
venom might cause. No such diminution, however, took place. In fact, quite
the opposite result was obtained. For a glance at Table XI of the protocols
will show that very minute quantities of daboia venom had a distinct effect in
increasing the coagulability of citrate blood, even 0·0000009 milligramme
added to 1 c.c. of citrate blood causing a slight shortening of the coagulation time
as compared with the control. A similar series of experiments to that recorded
in Table XI was then made, the only difference being that 0·3 c.c., instead of
0·1 c.c., of lime solution (0·5 per cent.) was added to each tube. This amount of
lime clotted the control tube in about two minutes. As it was found that
the contents of all the tubes to which the venom had been added clotted in
about the same time as the control, some of them in fact even more quickly, this
series of experiments need not be further considered. It confirms the results
already plainly demonstrated in Table XI, namely, that daboia venom, even in
minute quantities, does not cause any diminution in the coagulability of citrate
blood in vitro, similar to the effect which is produced by cobra venom.
This result, therefore, throws no light on how the negative phase of coagulabil-
ity caused by daboia venom in vivo is brought about. It does show, how-
ever, that this action of daboia venom is something quite different from the
action of cobra venom in this respect, and that, in all probability, it is not due
to a direct action of the poison on the blood cells or on the plasma.
In the above experiments with daboia venom a small quantity of soluble
lime salt was added to the citrate blood. I have now to put forward the results
of some experiments in which no lime salt was added, daboia venom alone being
C 2
C2
18
run into each tube of citrate blood. Two series of experiments of this descrip-
tion are tabulated in the protocols (Tables XII and XIII).
Firstly, in the series of observations detailed in Table XII the citrate was
present in the proportion of 1 to 100 to the blood, that is to say, the minimum
amount which can be depended on to keep rabbit's blood permanently liquid. 3 3
A reference to this table will show that 0·5 milligramme of venom clotted 1 c.c.
of the citrate blood in a little over half an hour, and 0·0312 milligramme in
about two hours. Smaller quantities than this caused no clot in two hours.
Secondly, in the series of experiments detailed in Table XIII of the proto-
cols the citrate was present in the proportion of 1 to 50 to the blood, namely,
a quantity double the amount which is necessary to keep rabbit's blood unclotted
in vitro. Under this condition it was found that 2 milligrammes of venom
were required to cause clotting of 1 c.c. of blood in half an hour, that 0·6 milli-
gramme caused clotting only after 1¾ hours, and that 0·2 milligramme caused
only a slight clot after 2 hours. Thus, it is seen that the presence of an excess
of citrate, while not preventing the clotting action of the venom, certainly
lessens this power to some extent. The results, then, of these two series of
experiments, taken with the results of the two series detailed in Tables X and XI,
show that daboia venom can of itself cause clotting of well citrated blood in
vitro, but that the clotting is more rapid and the clot is firmer, the nearer the
quantity of lime salts in solution in the blood approaches to that contained in
normal blood. I do not for a moment urge that daboia venom of itself is able
to cause clotting of blood which contains no lime salt in solution, as the citrate
of lime is not an absolutely insoluble salt. However, the fact that daboia ve-
nom does of itself clot citrate blood in vitro is important and interesting,
as I know of no other substance which has this action without the addition of a
small quantity of soluble lime salt.
In a recent paper by Dr. Hanna and myself, 34w e p o i n t e d o u t t h e f a c t t h a t
heating a weak solution of daboia venom completely destroys the property
which this poison has of causing any increase of the blood coagulability in
vivo. We showed that after heating a 0·1 per cent. solution for half an hour
at 75° C. a large quantity of the filtrate can be introduced into the blood
stream of animals without causing any symptoms.
I have found that a comparable result is obtained with citrate blood in
vitro, that is to say, heating a weak solution of daboia venom at 75° C. for half
an hour completely destroys the clotting action the unheated venom has on cit-
rate blood. Such an experiment is detailed in Table XIV of the protocols. From
this Table it will be seen that even 2 milligrammes of venom (0·2 per cent.
solution), heated for half an hour at 75° C., does not clot 1 c.c. of citrate blood after
3 hours. When we consider that 0·03 milligramme of the unheated venom
clots in 2 hours this amount of blood, citrated to the same extent, we must