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only acts, according to Lilienfeld, by reason of the acid qualities of the
nucleic acid it contains. Any other weak acid, e.g. acetic, will answer
equally well. Thus, if a solution of fibrinogen in NaCl (prepared
according to Hammarsten's method) is precipitated by acetic acid, the
precipitate (thrombosin), if dissolved in weak sodium carbonate, instantly
forms a coagulum (fibrin), on the addition of calcium chloride. The
formation of the thrombosin by the action of an acid upon fibrinogen
is, according to Lilienfeld, a precursor to the production of fibrin, and
is analogous to the change in caseinogen by the action of rennin, which
will occur in the absence of lime salts, although the latter are necessary
for the formation of the casein clot (see p. 135).

I have elsewhere shown ^ that this theory is untenable ; for a solution
of fibrinogen in dilute salt solution, prepared by Hammarsten's method,
will, if sufficiently strong, coagulate, on the addition of calcium chloride,
equally well with a solution of the acetic acid precipitate — the so-called
thrombosin — although somewhat less rapidly.^ The difference in rapidity
depends, no doubt, upon the fact that sodium chloride in a certain
amount retards the formation of the clot, or even may prevent it
altogether. This, as Hammarsten has pointed out, is the reason why
Lilienfeld obtained no coagulum on the addition of calcium chloride
to his fibrinogen solution, although he got a coagulum with his
so-called thrombosin solution, for the former was dissolved by aid
of sodium chloride, and the latter by dilute alkali. As already
stated,* Hammarsten holds that in neither case is the coagulum pro-
duced a true fibrin, but in both cases it is a fibrin-like combination
of fibrinogen with lime. The influence of nucleo-proteid is, however,
not eliminated,^ for, as has been already insisted on, fibrinogen pre-
pared by Hammarsten's method always contains some nucleo-proteid.
This is clear both from my own experiments and from the analyses
of Lilienfeld, who indeed — but as it would appear without sufficient
cause — supposes fibrinogen itself to be a nucleo-proteid. The amount
of nuclein or phosphorus which can be obtained from it certainly does
not warrant the assumption ; nevertheless there is always a distinct

^ ZtscJtr. f. physiol. Chem., Strassburg, 1895, Bd. xx.

" Froc. Physiol. Soc, Journ. Physiol., Cambridge and London, 1895, vol. xvii.
p. xviii.

^ Cf. Cramer, Ztschr. f. physiol. Chem., Strassburg, 1897, Bd. xxiii. S. 74, who has
fully confirmed the conclusion that tlie so-called " tlirombosin " is merely fibrinogen.

■* See note 5, p. 165.

•^ Cf. Wistinghausen, Diss., Dorpat, 1894.



THEORIES OF COAGULATION. 173

residue after gastric digestion of its solution, showing that it at least,
as above stated, contains some nucleo-proteid. Probably this is an
accidental contamination.

Production of intravasctdar coagulation of blood and of uncoagulctble
hlood. — It was discovered by Edelberg^ that intravenous injection of
Schmidt's fibrin ferment may produce thrombosis in the venae cavse,
the right side of the heart, and the pulmonary arteries. Foa and
Pellacani^ showed that the same will occur with extracts of various
organs. The same fact was independently noticed by Wooldridge,^
who found that a substance or substances obtainable from saKne extract
of lymphatic glands, thymus, testicle, and other glandular organs, tend to
produce, when injected rapidly in sufficient amount into the veins of
animals, instant coagulation of the blood whilst still within the blood
vessels. On the other hand, if injected more slowly, or in insufficient
amount to produce intravascular coagulation, the coagulability of the
blood in vitro becomes abolished ; this condition was termed by Wool-
dridge the " negative phase." When the negative phase is once obtained,
a very large dose of the material fails to produce intravascular clotting.*
Wooldridge gave the name " tissue fibrinogens " to the substances thus
extracted, and more extended knowledge has led to the general recog-
nition of the fact that they belong to the class of nucleo-albumins or
nucleo-proteids. The coagulation when it occurs is found, first, in the
portal venous system ; then in the general venous system, and pulmonary
arteries and in the right side of the heart ; and finally, when the effect
is most pronounced, in the general arterial system ; but rarely in the
pulmonary veins. Its occurrence is assisted by an excess of CO2 in the
blood.^ Albino rabbits and the Norway hare in its albino condition are
immune to these effects (Pickering).

It has been supposed by Lilienfeld *^ that this action of the nucleo-
proteid in causing coagulation is due to the nuclein or nucleic acid
which it contains, and that, when the negative phase is obtained, this
result is due to the action of the proteid part of the molecule of
nucleo-proteid in preventing coagulation. This hypothesis is rendered
improbable by the observations of Halliburton and Pickering,'^ who
found that intravascular coagulation can be readily obtained in rabbits
by intravenous injection of artificial colloids (containing no nucleic
acid).8 These colloids likewise yield the negative phase (retardation of
coagulation), if injected in quantity insufficient to produce coagulation ;
and, as with solution of nucleo-proteids, they are without action upon
albino rabbits. Nevertheless, like solutions of nucleo-proteid, they
hasten the coagulation of the blood of other animals, if mixed with it
in vit7-o. These observers also found that the retarding influence of

^ Arch. f. exper. Path. u. Pharmahol., Leipzig, 1880, Bd. xii. S. 283.

^ Riv. din. di. Bologna, 1880, p. 241.

^ Proc. Roy. Soc. London, 1886. See also "Die Gerinnung des Blutes," Leipzig, 1891.

''Wooldridge, Arch. f. Physiol., Leipzig, 1888.

^ Wright, Journ. Physiol. , Cambridge and London, vol. xii. ^ Loc cit.

■^ Journ. Physiol., Cambridge and London, 189.5, vol. xviii. pp. 54 and 285 ; Pickering,
Proc. Roy. Soc. London, 1896, vol. Ix. p. 337.

* The artificial colloids investigated were prepared by Grimaux's methods {Compt. rend.
Soc.de Uol., Paris, 1881, tome xciii. p. 771; 1884, xcviii. pp. 105, 1434, and 1578).
Their chemical properties and mode of preparation have already been described by Professor
Halliburton in a previous article (p. 36). It is possible that they may act, not directly,
but by setting free nucleo-proteid from the white corpuscles. Their solutions do not,
however, cause disintegration either of the red or white corpuscles, nor any apparent
change in the epithelium of the vessels.



174 THE BLOOD.

intravascular injections of soap, peptone, and potassium oxalate on the
coagulation of blood is antagonised Idj previous intravascular injection of
the colloid. The action of the colloids in promoting coagulation is
assisted, hke that of nucleo-proteid, by accumulation of CO2 in the
blood. The same effects are produced by snake venom,^ which contains
no nucleo-proteid, and the active principles of which consist of albumoses.
This produces, although in far more minute doses, effects which are in
every way comparable with those produced by Wooldridge's " tissue
fibrinogen." In doses of O'OOOOl to 0*00002 grm. per kilog. body weight,
the venom of the Austrahan black snake {PseudecMs -porpliyriaca) causes
the blood, after a brief mterval of increased coagulability (positive
phase), to lose its tendency to clot (negative phase), and much larger
doses of the poison will now not restore its coagulabiht}'. On the other
hand, moderate and large doses (more than O'OOOl grm. per kilog.)
produce instantaneous clotting within the vessels. But any blood
which has not imdergone the intravascular coagulation is found to be
incoagulable in vitro, and in this point also there is an exact resemblance
to the phenomena produced by nucleo-proteids and by artificial colloids.

Solutions of certain other chemical substances, such as ether, tannic
acid, arsenic,- glycerin, toluylenediamin,^ are also found when injected
into the circulation to produce thrombosis. But, to produce the effect,
these all requke doses large enough to cause disintegration of the
blood corpuscles, thereby setting free the nucleo-proteids which the
corpuscles contain, so that their action is probably a secondary one. It
is possible that snake venom may also operate in this way,* smce it does
produce to a certain extent such dismtegration, but the rapidity of the
production of the intravascular clotting, and the small amount of such
disintegration which normally occurs, render such an explanation
improbable.

Peptone pilasriia. — Researches of Wooldridge. — Other substances, such
as commercial x^eptone, the action of which is due to the albumoses which
it contains, and leech extract, produce a diminution or loss of coagulability
when injected into the blood vessels, without, in any dose, tending to cause
intravascular coagulation. The mcoagulable blood or plasma obtained
by their employment resembles very closely that obtained in the negative
phase, produced by Wooldridge's tissue fibrinogen, by colloids and by
snake venom. Peptone plasma can be made to coagulate by

1. Addition of lymph cells.

2. Addition of nucleo-proteids.

3. Addition of calcium chloride.

4. Dilution with water, or 0"5 per cent, salt solution.

5. A stream of COg.

6. Neutralisation with acetic acid.

But if an excess of the reagents employed to prevent coagulation
(or to produce the negative jjhase), whether peptones or slowly

^ C. J. Martin, Journ. Pliysiol., Cambridge and London, 1893, vol. xv. p. 380 ; and
Journ. and, Proc. Roy. Soc. New South Wales, S)'dney, Julj'^ 3, 1895. These papers contain
full references to the jirevious literature of the subject.

^ The administration of arsenic and y)ho.sphorus by the mouth diminishes the coagulability
of the blood (cf. Gley and Pachon, Arch, de physiol. norm, et path., Paris, 1896, p. 716).

^ Silbermann, Virchoiv's Archiv, 1889, Bd. cxvii. S. 288.

•* 0. J. Martin, op. cit., Journ. and Proc. Poj/. Soc. Ncvj South Wales, Sydney, pp. 4.')-47
of reprint.



THE OKIES OF CO AG ULA TION. 1 7 5

administered nucleo-proteids or snake venom be employed, then these
additions do not produce coagulation.^ In view of the fact that
calcium chloride will not, under these circumstances, produce coagula-
tion, the hypothesis of Freiuid and Pekelharing, that " peptones "
deprive blood of its coaguIal)ility by combining with its calcium salts,
loses probability.

Peptone injected intravenously rapidly disappears from the blood. '^ The
action of peptone differs from that of leech extract, in that a second dose,
given soon after the action of the first dose has passed off, fails to produce an
effect on coagulabihty. Moreover, the blood of a " peptonised " dog confers
immunity from the action of peptone, if injected intravenously into a second
animal.^

The properties of peptone plasma, and the effects of leucocytes and
their saline extracts upon the coagulability of blood, were carefully
studied by Wooldridge. This observer found, as already stated, that if
peptone plasma be kept for a time at 0° a precipitate forms, which takes
the form, under the microscope, of minute discoid particles, almost exactly
similar to blood platelets. The substance thus precipitated was termed
" A-fibrinogen " by Wooldridge, while he named the substance precipit-
able by half -saturation with NaCl " B-fibrinogen " ; this is the same
thing as fibrinogen as ordinarily understood. After the removal of the
A-fibrinogen, the coagulability of peptone plasma by CO2 and other
conditions is greatly diminished or altogether lost, but is restored on
dissolving the A-fibrinogen again with the aid of warmth.

Wooldridge's A-fibrinogen is also obtainable, as Wright has shown, by
cooling oxalate j)lasma, and it is probably composed mainly, if not entirely,
of nucleo-proteid. It has been shown by Hammarsten that if by prolonged
cooling and filtration it is removed as much as possible from oxalated plasma,
the plasma will not coagulate on the addition of sufficient lime salts to more
than balance the excess of oxalic acid, but that, if the precipitate be collected
and treated with lime salts, it furnishes, on subsequently removing the
lime by oxalate, a powerful thromhin or fibrin ferment. Hammarsten
accordingly terms the substance in question, which is precipitated by cold
from plasma, protlwomhin, and considers that it can only be converted into
thrombin by the action upon it of lime salts. Pekelharing regards the
precipitate in question as composed of nucleo-proteid, and considers that the
lime acts by combining with it to form fibrin ferment.

The addition of lymph cells (washed with 0"6 per cent. NaCl solution)
to peptone plasma causes its coagulation outside the body, and also acceler-
ates the coagulation of ordinary blood in vitro, whereas the intravenous
injection of salt solution holding these washed lymph cells in suspension
produces an incoagulable condition (negative phase) of blood, which
does not then coagulate, even on withdrawal. But on addition of some
of this fluid, holding cells in suspension, to such blood after withdrawal,
coagulation is rapidly produced. The loss of coagulability of peptone
plasma is not due, as was supposed by A. Schmidt, to the disappearance
and disintegration of leucocytes, for, as Wooldridge showed, the addition

^ C. J. Martin, op. cit., pp. .35-40. According to Dastre and Floresco, the chief cause of
the lack of coagulation in peptone plasma is its high alkalinity {Arch, de 'physiol. norm, et
path., Paris, 1897, p. 216).

" Schmidt-Mulheim, loc. cit.

^ Contejean, Arch, dephysiol. norm, et piath., Paris, 189,5.



176 THE BLOOD.

of leucocytes to the circulating blood does not increase its coagulability,
but the contrary; and, moreover, peptone plasma contains many
leucocytes.^ These several facts were explained by Wooldridge by
the supposition that coagulation is produced or prevented in the
absence of leucocytes by the action of one substance in the plasma upon
another, or in the presence of leucocytes by the action of a substance
within the plasma upon these cells, or material yielded by them ; the
kind of interaction being different under different circumstances, and
producing, respectively, the phase of incoagulability or coagulation
(negative or positive phase) according to such circumstances. All such
substances, which by their interaction tended to produce fibrin, were
termed by Wooldridge " fibrinogens " ; but the progress of research has
since rendered it i^robable that Wooldridge's " A-fibrinogen " obtained
from plasma, his " serum fibrinogen " obtained from dog's serum, and
the "tissue fibrinogens," which he obtained from various organs, all
owe their action to the nucleo-proteid which they contain. Translating,
then, the phraseology employed by Wooldridge, the alterations in
blood plasma, which come under the various conditions above noticed, are
due to the interaction of nucleo-proteids and fibrinogen. And it would
appear that, when in the interaction the nucleo-proteids are present in
relatively small amount, the negative phase is the result ; when in
large amount, the positive phase. Also that, when added to the
circulating blood, leucocytes yield but little of their nucleo-proteid to
plasma, and hence a negative phase is the result; but, on the other
hand, when added to plasma in vitro, a larger amount is yielded, and
coagulation results. A remarkable observation, and one very difficult to
explain, is the fact that, if the negative phase is once established by the
intravascular injection of a small amount of nucleo-proteid, artificial
colloid, or snake venom, a large excess of the same will then not only
fail to produce the positive phase, but will even strengthen the negative
phase. It is, therefore, only the initial change which is influenced by
the relative amounts of interacting material ; and, when once this change
is established, it does not again become modified.

Wright's exjjeriinents. — Wooldridge further found that under some cir-
cumstances the amount of fibrin produced was dependent upon the amount
of tissue fibrinogen or A-fibrinogen (nucleo-proteid) added to plasma. He
therefore came to the conclusion, since the extent of action was not in all
cases independent of the amount of these substances, that the action could
not be looked upon as that of a ferment, although under some cir-
cumstances the extent of action did appear to be independent of the
amount of these substances. Wooldridge offered no explanation of the
different effects obtained with large and small doses respectively, his
work upon the subject having been cut short by his untimely death.
It has, however, been continued on the same Lines by Wright,^ who,
whilst confirming most of Wooldridge's observations, has added
materially to our knowledge of the conditions under which " tissue
fibrinogen " or nucleo-proteids produce the negative and positive phase
of coagulability. Wright states that the extracts of glands containing

^ Wriglit found, however, that the nmiiber of leucocytes in peptone blood was extremely-
reduced, much more so than is the case in oxalate oi'- magnesium-sulphate blood, and that
it contains a correspondingly larger amount of nuclco-albumins, Froc. Hoy. Soc. London,
10th Feb. 1893.

- Froc. Hoy. Irish Acad., Dublin, 1891, 3rd series, vol. ii. p. 117.



THEORIES OF COAGULATION. 177

these substances readily yield, under the influence of certain reagents
and conditions, a body or loodies giving albumose reactions ; and he finds
that such a body is also present in the blood after their injection, and
rapidly appears in the urine.^ Wright considers it proljable, therefore,
that the contrary effects of large and rapid, or small and gradual,
administration of these extracts is due in the one case to the immediate
action of the nucleo-proteids in effecting the conversion of the fibrinogen
into fibrin before there has been time for the formation of albumose ;
and in the other case, where there has been time for such formation, to
the action of the albumose thus formed in preventing coagulation (as
in the case of directly injecting albumose into the blood vessels). If
any albumose is formed, the action of this would, by delaying coagula-
tion, give time for the formation of more, when a second dose of
nucleo-proteids is injected. Hence, a dose of nucleo-proteid, which would,
if administered rapidly, produce instantaneous coagulation throughout
the vascular system, may, if administered gradually, tend altogether to
prevent coagulation. But, as Halliburton points out, the explanation of
the action of " peptone " in producing a negative form of coagulation
may be that it liberates small quantities of nucleo-proteid, rather than
that it removes calcium ; and if this is so, the explanation offered by
Wright (and Pekelharing) of the action of nucleo-proteids falls to the
ground. Moreover, it cannot be accepted as proven that a " peptone "
moiety is split off from nucleo-proteid. " Peptone " {i.e. " albumose")-
blood is characterised by extreme diminution of the amount of CO2
which it contains,^ and by diminished alkalinity,^ and the reason for
the uncoagulability of such blood is apparently connected with its
deficiency in COo tension,* since it coagulates on passing a stream of
COo throughout it. For the occurrence of intravascular coagulation,
after injection of nucleo-proteid and similarly acting substances, is
largely influenced by the amount of COo in the blood, and it is due to
its richness in CO2 that the blood coagulates under these circumstances,
first in the systematic veins, and of these most readily in the portal
venous system.^

From what has been before said as to the influence of lime, it will be
understood that the lime-salts of the plasma play an essential part in the
interaction between the nucleo-proteid and the fibrinogen. This parti-
cipation of lime in the reaction had not yet been recognised when
Wooldridge's researches were made, but is freely admitted by Wright,
whose views upon the subject of the combined action of nucleo-proteid
and lime in producing coagulation seem to be in close agreement with
those of Pekelharing (see p. 171). It is, however, still by no means
clear why in " peptone " plasma, where all the necessary factors for the
formation of fibrin are present, coagulation, nevertheless, does not occur,

^ This lias been also shown independently by Pekelharing ("Untersueh. u. d. Fibrin-
ferment," Amsterdam, 1892), who offers a similar explanation of the phenomenon of
negative and positive coagulation. Halliburton and Pickering, on the other hand, con-
sider that, in the case of colloids, the negative phase cannot be regarded as a subsidiary
phenomenon, due to disintegration of the material intravenously injected, but is rather a
result characteristic of the action of small doses, and is comparable to the inhibitory action
of small doses of certain drugs, which act contrary to the action of larger doses (such as the
physiological immunity produced by small doses of alexines).

- Lahousse, Arch. f. Physiol., Leipzig, 1889, S. 77.

^ Salvioli, Arch. ital. de hiol., Turin, 1892, vol. xvii. p. 155.

* Wright, loc. cit., a.n&Journ. Path, and Bacterial. , Edin. and London, 1893, vol. i. p. 434.

^ Wright, Journ. Physiol., Cambridge and London, 1891, vol. xii.
VOL. I. — 12



1 78 THE BLOOD.

although it speedily occurs on further addition of lime or on passing
COo. "Wright ^ assumes that the nucleo-proteid acting as a weak acid
has ousted CO., from the bases of the plasma, and that the action of CO^
is to set the nucleo-proteid free again. But this would not account for
the effect of addition of calcium chloride. It may, on the other hand, be
that the Hme wliich is present in the j)lasma is unable to act upon the
nucleo-proteid also present, owing to the former having entered into some
combination from which it is set free by COg. It must be admitted
that the subject is still, in spite of much research, enveloped largely in
obscurity.

Influence of the liver and lungs upon blood coagidabilUy. — It was shown by
Pawlow - that if blood be allowed to circulate through the heart and kmgs
only, and be cut off from the rest of the body, it gradually loses its
coagulability, and the same observation was made independently by K'ewell
Martin. 3 Bohr* obtained a similar result, on preventing the blood from
reaching the portal circulation by occluding the thoracic aorta. The blood
lost its coagulability in a quarter of an hour, nor was it restored for twenty-four
hours after readmission to the abdominal viscera. This was in the dog. In a
rabbit, ligature of the cceliac axis and mesenteric arteries produced a similar
but rather less pronounced effect. Delezenne has shown that artificial
perfusion of " peptone-blood " through the liver restores its coagulability, but
that other organs do not produce the same result.'^

Gley and Pachon*^ find, in confirmation of Contejean,'^ that every cause
which diminishes or suppresses the functional activity of the liver diminishes
or suspends the anti-coagulating action of " peptone." They thus explain the
experiments of Contejean, who noticed that after extirpation of the coeliac
ganglia the action of " peptone " is not obtained.^

Hedon and Delezenne ^ also found that after the establishment of an Eck's
fistula (communication between portal vein and vena cava) in the dog, and the
subsequent removal of the liver, injection of "peptone," although it produces
a great fall of blood pressure, no longer removes the coagulability of the blood.
These experiments apjDcar to show that the liver has a special function in
connection with the maintenance of the coagulability of the blood, and that in
passing through the lungs an effect of an opposite character is produced, but
in what way exactly these organs exert their influence has not as yet been
ascertained.

Blood or plasma can be temporarily made uncoagulable in the living vessels
by removing the fibrin. Dastre found that, if a large quantity of blood be
drawn from an animal, and this be whipped and filtered and returned to the
blood vessels, and the process repeated two or three times, all the fibrin can
be temporarily removed ; and it is only gradually that the blood resumes its
coagulability, which is not comj^letely restored until the lapse of some
hours.

Conclusions regarding the causes of coagulation. — At least three
factors appear necessary to effect the formation of fibrin, namely,



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 25 of 147)