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1897, Bd. xxiii. S. 74. According to Hammarsten, this coagulum, like that produced in a solu-
tion of the original fibrinogen, is not fibrin, but a fibrin-like combination of lime and fibrin-
ogen. To me, however, it has often appeared difficult to distinguish from fibrin.

"Pekelharing, " Untersuch. ii. d. Fibrin-Ferment," Amsterdam, 1892.

* Journ. Physiol., Cambridge and London, 1894, vol. xv. p. 375.

^ lUd., 1894-5, vol. xvii. p. 159.



1 66 THE BLOOD.

substance which possesses the property of converting fibrinogen into
fibrm, and is, according to Pekelharing, a combination of the nucleo-
proteid with lime, and identical with the fibrin ferment of A. sSchmidt.
The librin ferment is sometimes spoken of as "thrombin," and the
nucleo-proteid material in the plasma from which it is produced is then
termed " prothrombin."

Wooldridge^ found that, on subjecting peptone plasma to cold, he
obtained a finely granular deposit, which had the property of producing
clotting in fibrinogenous iiuids, which are not themselves spontaneously
coagulable, and of accelerating the process of clotting in coagulable fluids.
To the material thus obtained, and which he described as havmg, under
the microscope, an appearance similar to masses of blood platelets, he
gave the name " A-fibrinogen," because he found that on adding it to
peptone plasma it produced fibrin, and that the amount of coagulation
was more or less proportional to the amount of A-fibrinogen added. It
is not fibrinogen as the term is ordinarily used, but is probably either a
nucleo-proteid, or a mixture of nucleo-proteid with globuhn. A similar
deposit occurs, as already stated, in oxalate plasma, on standing in the
cold. A precipitate containing the same substance is also produced by
adding magnesium sulphate solution in considerable amount to blood, and
in both plasma and serum of certain animals on acidulation with acetic
acid, but m both cases it is liable to be mixed with serum globuhn.
It also occasionally occurs in serum, on standing, even without the
application of cold. Halhburton has suggested that the deposit in
peptone plasma may be a part of the proteoses, which were injected into
the blood, for he found that solutions of albumose were liable to give a
similar deposit on coohng by means of ice, but there is not enough proteose
present in peptone plasma to account for such dex^osit, and the fact that it
occurs under other conditions in plasma also negatives this supposition.
These experiments of Wooldridge, and the behaviour of the body termed
by him A-fibrinogen, will be again referred to in a subsequent section.

Fibrin. — Fibrin is the chief substance formed from fibrinogen in the
coagulation of blood plasma, and it is also produced in the coagulation of
lymph and other fibrmogen-containing fluids. It is usually got by whipping
blood as it flows from the blood vessels with a bundle of wires or glass
rods before it has had time to coagulate into a sohd mass. The coagulum
then forms upon the wires or rods, and can be washed free from adherent
red corpuscles by putting it under a stream of water for a few hours.
But to obtain pure fibrin it is necessary first to prepare fibrinogen from
blood plasma by precipitation with NaCl (half -saturated), to purify this
by re-solution and re-precipitation, and finally to cause the coagulation of
the fibrinogen solution by fibrin ferment. The clot thus obtained, which
must be thoroughly washed, is composed of nearly pure fibrin.

When obtained by whipping blood, fibrin is a white stringy substance
when wet, drymg to a glue-like mass. The threads of which it is com-
posed, and which, as may be seen in a microscopic preparation of blood,
interlace with one anotlier and form a network of the finest possible
filaments, entangling the blood corpuscles in its meshes, have a strong-
tendency to retract or shorten when formed ; this is the reason why a
clot shrinks and expresses serum from its interior. Fibrin is slowly
soluble in 5 to 10 per cent, solutions of certam salts, such as sodium
.chloride, sodium sulphate, potassium nitrate, magnesium sulphate, and

^ Wright, Lancet, Londoi), 1892, vol. i. pp. 457, 5] 5.



PR O TEWS OF PL A SMA. 167

ammonium sulphate, and also in iodides and fluorides, and in solutions
of urea.i It is also very slowly dissolved to some extent by normal salt
solution ; the solution is in all cases assisted by moderate warmth. Fibrin
obtained from venous blood is slightly more soluble in salt solutions than
that yielded by arterial blood. The proteid material which is found dis-
solved after solution of fibrin in the above salts is composed of two
globulins,"-^ having heat coagulation temperatures of 55° and 75° respec-
tively. The latter, according to Halhburton, is reduced to 60°-65° in
sodhim chloride solutions, being 73°-75° in magnesium sulphate solutions
only. Albumoses are also present in the fluid (Limbourg, Dastre). This
solution of fibrin in neutral salts occurs in the entire absence of putre-
factive decomposition (Green, Dastre). Fibrin swells in dilute acid (such
as 0-2 per cent. HCl) into a clear jelly, which very slowly undergoes
solution with the formation of acid albumin and proteoses. Stronger acids
and, with the aid of heat, weak acids, effect the conversion more readily.
The addition of pepsin to the acids employed greatly accelerates the con-
version, the fibrin first splitting into two globulins, one coagulating at 56°
and the other at 75°, and then becoming transformed into acid-albumin, pro-
teoses, and peptones.^ Trypsin in alkaline solutions has a similar action.*
Blood yields from -2 to -4 per cent, of its weight of dry fibrin. Ham-
marsten ^ gives the following as the elementary composition of fibrin : —



c


52-68


S


1-10


H


6-83





22-48


N


16-91







It is, however, never free from ash, and the ash invariably contains
lime,'^ but not more than other proteids,'^ nor does it contain more
lime than the fibrinogen from which it is formed. Thus in one
experiment Hammarsten found that a sample of fibrin, obtained by
the action of ferment prepared from oxalated serum, upon fibrinogen
prepared by precipitation from oxalated plasma by acetic acid, yielded
exactly the same amount of lime as a sample of the fibrinogen itself,
namely, 0-055 per cent. This fact completely disposes of the theories of
coagulation which assume that fibrin is merely a combination of
fibrinogen with lime, such as those of Freund, Arthus, Pekelharing, and
Lihenfeld. Fibrin obtained by whipping blood leaves a considerable
phosphorus-containing residue (nuclein) after subjection to peptic
digestion ; this is probably largely derived from the nucleo-proteids of the
entangled leucocytes. But even fibrin obtained from solution of purified
fibrinogen in dilute salt solution yields a certain amount of such residue.^
It is possible that this may be an accidental impurity, but, on the other
hand, it may be an integral constituent of the fibrin.

^ Dastre, Arch, de jjhysiol. norm, et path., Paris, 1895, p. 408 ; Comjjt. rend. Acad. d.
sc, Paris, 1895, tome cxx. p. 589. See also on the solubility of fibrin in neutral salts,
Holzmaun, Arch. f. Physiol., Leipzig, 1884, S. 210; and Arthus, " Coag. des liquides
organiques." Paris, 1894, pp. 105 et seq.

2 Green, Journ. Physiol., Cambridge and London, 1887, vol. viii. p. 372.

^ Hasebrock, Ztsehr. f. lohysiol. Chem., Strassburg, Bd. xi. S. 348.

■* A. Herrmann, ihid., Bd. xi. S. 508. The other literature on this subject will be
found in Halliburton, "Text-Book of Physiol, and Path. Chemistry."

^ Arch. f. d. (jes. Physiol., Bonn, Bd. xxii. S. 484.

" Frederikse, Ztschr. f. i-)hysiol. Chem., Strassburg, 1894, Bd. xix. S. 143.

"^ Hammarsten, ibid., Bd. xxii. S. 392.

^ Sehafer, Proc. Physiol. Sac, Journ. Physiol., Cambridge and London, 1895, vol.
xvii. p. XX.



1 68 THE BLOOD.

Theoeies of Coagulation.

That the coagulation of the blood is due to the formation of an
insoluble substance (fibrin) in the plasma, was proved by Hewson,^ who
showed that a coagulable plasma can be obtained by skimming, after
allowing the corpuscles to subside, in blood the coagulation of which
is delayed in any way, as by cold, by neutral salts, or by its retention
within a living vein. The old theories which ascribed the coagulation to
the cooling of the blood, to its coming to rest, to the running together
of the corpuscles into rouleaux,, were all effectually disproved by the
same careful observer. Hewson also showed that fibrinogen (" coagul-
able lymph ") is precipitable and removable from plasma by a tempera-
ture of a little over 50° C.^ Many of Hewson's observations upon
coagulation were forgotten, and the facts rediscovered by subsequent
observers, but their accuracy was such that until comparatively modern
times no addition of any permanent value to the knowledge of the
subject was made. The most important of such additions (which was
also overlooked for many years) ^ was the observation of Andrew
Buchanan, that a substance could be extracted by water and solutions of
salt from lymphatic glands, from blood clot (especially the buffy coat),
and from various tissues, which had the property of producing the
coagulation of serous fluids, not themselves spontaneously coagulable,
such as hydrocele and pericardial fluid ; such action being comparable
to that of a ferment. But it is only quite recently that the active sub-
stance extracted by Buchanan has been examined, and found to belong
to the class of bodies known as nucleo-proteids.

Schmidt's theory. — A theory of coagulation, which was long accepted,
was that of Alexander Schmidt. Schmidt noticed that fluids which
contained fibrinogen but were not spontaneously coagulable, such
as pericardial or hydrocele fluid, coagulated on the addition of serum.
He ascribed the fibrin formation which resulted to the action (fibrino-
plastic action) of the globulin in the serum upon the fibrinogen of the
pericardial fluid. Since, however, the same globulin is already present
in abundance in pericardial and hydrocele fluid, it became clear that
this explanation of the action of serum was insufficient. It was, how-
ever, shown by Schmidt that a substance is extracted by water from the
alcohol precipitate of blood or serum, which possesses the property of
causing coagulation in these fibrinogenous liquids, or of causing coagula-
tion in plasma, the coagulation of which has been prevented by the
addition of neutral salts. To this substance the name of fibrin ferment
was applied, on account of its action resembling in general that of the
unorganised ferments or enzymes. Thus it was found to have its
activity accelerated by warmth, and destroyed by a high tempera-
ture (65'' C), and also to be capable of producing the coagulation of a
relatively large amount of fibrinogen. It was still held by Schmidt that
the globulin of serum takes an important share in the formation of fibrin.

Hammarsten's earlier researches. — Hammarsten showed that serum

^ Op. cit.

^ For the history of lliis see Scliiifer, Jonni. Physiol., Canihridge and London, 1880,
voL iii. p. 185.

•' Of. Gamgee, "Physiol. Chemistry," 1880, a^oL i., where will also be found an excellent
account of the earlier history of the subject of blood coagulation. See also Arthus, " Coag.
des liquides organiques," Paris, 1894, for a good epitome of the history of the subject up
to til at date.



THEORIES OF COAGULATION. 169

globulin does not take part in forming fibrin. By precipitating fibrin-
ogen by half-saturating plasma with sodium chloride, he obtained it
free from serum globulin, and found that its solution in dilute salt
solution was coagulated by the addition of Schmidt's extract — the so-
called fibrin ferment — alone. Hammarsten proved that coagulation
consists in a conversion of fibrinogen into fibrin; the change being
accompanied by a splitting of the fibrinogen, and not by a combination
of it with the serum globulin, as was supposed by Schmidt.

Infiuence of lime salts. — Theories of Freitncl and of Artlius and Pages.
— The more recent researches since these of Hammarsten have been
in the direction of elucidating the true nature of the substances
contained in Schmidt's extract. Green ^ found the extract to contain
sulphate of lime, and that if lime were removed from plasma by
dialysis its coagulability became lost, but was restored by the addition
of sulphate of lime. Einger and Sainsbury ^ showed that other salts of
lime, such as calcium chloride, might replace the sulphate, and that the
calcium might be replaced by barium and by strontium, although the
salts of these metals are not so efficacious as the corresponding salts
of calcium.

Freund ^ also drew special attention to the important part played by
lime salts in promoting the formation of fibrin. He supposed the
original cause of the deposition of fibrin in fibrinogenous liquids to be
the formation of insoluble tribasic phosphate of lime, by the interaction
of soluble phosphates (which he supposed to be shed out from the
corpuscles whenever they come in contact with and adhere to foreign
surfaces) with soluble lime salts contained in the plasma ; the lime
phosphate combining at the moment of formation with fibrinogen, and
forming fibrin, and no other agency in the shape of a special ferment
being necessary. This inference has not, however, been confirmed by
subsequent observers. Freund supposed neutral salts, peptone, etc., to
act in preventing coagulation, by keeping phosphate of lime in solution,
and the walls of the blood vessels to act in preventing coagulation
because the corpuscles do not adhere to them. Freund based his theory,
partly upon the fact that if blood is drawn from an artery through a
tube smeared with oil or vaseline into a vessel similarly prepared, the
blood remains fluid for a long time, presumably because the adhesion of
the corpuscles to the walls does not occur. Similar experiments with
l^lood kept surrounded by paraffin or oil were performed by Haycraft
with like result.*

Arthus and Pages ^ mixed blood as it flowed from the vessels with a
small quantity of a soluble oxalate ^ (0'07-0"l parts per 100 of blood)
sufficient to precipitate the lime salts dissolved in the plasma. They
found that blood thus treated did not coagulate, however long it might
be kept,'^ but that coagulability of its plasma is immediately restored on
again adding a soluble lime salt, such as calcium chloride. They

^ Journ. Physiol., Cambridge and London, 1887, vol. viii. p. 354.

2 Ibid., 1890, vol. xi. p. 369. » ^/g^_ Jalirb., Wein, 1888, S. 259.

■* Journ. Anat. and Physiol., London, 1888, p. 172 ; Haycraft and Carlier, ibid., p. 582.

® Arch, de physiol. norm, etfath., Paris, 1890, p. 739 ; Arthns, These de Paris, 1892.

^ Solutions ot soap (0'5 parts per 100 of blood) or of soluble fluorides (0"2 parts per 100
of blood) act similarly to those of oxalate.

'' Hammarsten makes a similar statement for horse's blood, but it is certainly not
correct for all kinds of blood. Oxalate plasma, obtained from dog's or sheep's blood, does
undergo coagulation on standing ;. coagulability is therefore not abolished by precipita-
tion of the lime by oxalate, but meiiely deferred. We shall return to this point immediately.



I70 THE BLOOD

inferred that the presence of a sohible salt of hme is necessary to the
formation of fibrin, which, according to them, is produced by a combina-
tion, under the infiuence of fibrin ferment, of a part of the fibrinogen
with lime, the remainder of the fibrinogen — which is assumed to spHt
into two parts — forming a globulin coagulating at 64° C. (Hammarsten's
fibrino-globulin).^

Whilst it would appear from these researches that soluble lime salts are
necessary to the formation of fibrin,^ it has been shown by Home that the
presence of a slight excess of these salts and also those of barium and strontium
will hinder or, in great amount, entirely prevent its formation ; their action
being far more marked in this respect than that of other neutral salts, Avhich
require to be mixed in much greater amount with blood to prevent its co-
agulation. ^ The reason for this is probably to be found in the fact that fibrin
is soluble to some extent in neutral salts of a certain strength (including salts
of calcium, barium, and strontium).

Injiuence of nudco-proteicl. — Theory of Pekclharing. — Halliburton '^ and
Pekelharing ^ both obtained from Schmidt's extract a body giving proteid
reactions, and resembling in many particulars the globulins, to which
class of proteids they at first regarded it as belonging. "^ They showed
that the ferment action of Schmidt's extract is intimately dependent
upon the presence of this substance, which could also, as Halliburton
showed, be obtained from lymphatic glands. Halliburton termed it
cell globulin ] subsequently both observers recognised the fact that the
substance in question was not ' a true globulin but a nucleo-proteid.'^
According to Pekelharing, it possesses the property of combining with
lime, which it does not yield to distilled water by dialysis, nor is the
combination broken up by soluble oxalates, although these, if present
from the first, may prevent the original combination. The albumose in
commercial peptone also prevents such combination, the albumose itself
combining with the lime salts present ; ^ if these are in excess, " peptone "
does not prevent coagulation from taking place. The lime combination
of nucleo-proteid is, according to Pekelharing, the body which has been
known as fibrin ferment (thrombin). It can be formed not only from
the nucleo-proteids contained in plasma or serum, but also from nucleo-
proteids in the cells of the thymus, testicle, and other glands, by

^ Artlms and Pages found that strontium can replace lime in this reaction, but that
barium and magnesium cannot. Ringer and Sainsbury have, however, shown that barium
may take the place of lime in promoting coagulation, although it is less powerful {Joicrn.
Physiol., Cambridge and London, 1890, vol. xi. p. 369). They also found that the salts of
sodium and potassmra act antagonistically to those of lime, barium, and strontium.

-A. Schmidt, even in his last communication upon the subject (" Weitere Beitr. z.
Blutlehre," Wiesbaden, 1895), denied altogether that lime salts have any specific action
or differed from other neutral salts, and considered that the addition of a soluble oxalate to
blood acts either by ])re venting the formation of fibrin ferment or by hindering the action
of ferment, if present, on fibrinogen. Cf., however, Arthus, Arch, dc j^hy^iol, norm, ct
path., Paris, 1896, and Hammarsten, loc. cit.

" Journ. Physiol., Cambridge and London, 1896, vol. xix. p. 356. Wright also noticed
the fact that considerable excess of calcium added to oxalate blood prevents coagulation,
Journ. Path, and Bacterial., Edin. and Loudon, 1893, vol. i. p. 434.

■* Proc. Fi,oy. Soc. London, 1888, vol. xliv. p. 255.

■' Feslschr. Rudolf Virchou; Berlin, 1891, S. 435.

" Lilienfeld has recently i-cpeated this error, Ztschr. f. yhysiol. Chem., Strassburg,
Bd. XX.

''Pekelharing, "Untersuch. li. d. Fibrin-ferment," Amsterdam, 1892; Halliburton,
Journ. Phj/siol., Camljridge and London, 1895, vol. xviii. p. 312.

8 Pekelharing. Cf., however, C. J. Martin, "Venom of Australian Black Snake,"
pp. 36-40, Journ. and Proc. Hoy. Soc. New South Wales, Sydney, July 3, 1895.



THE OKIES OF CO AG ULA TION. 1 7 1

digesting these with calcium chloride, the excess of calcium salt being
afterwards dialysed off. Pekelharing supposes that the ferment action
consists in the transference of lime from its nucleo-proteid combination
to fibrinogen, the lime-compound of this being the insoluble fibrin,^ and
that if there is more lime salt in the solution the nucleo-proteid can
recombine with lime, and thus become reconstituted as an agent for the
conversion of fibrinogen into fibrin. As already pointed out (p. 166),
however, it is not possible to accept this theory in view of the analyses
of fibrin and fibrinogen given by Hammarsten. Pekelharing has himself
shown that even in the entire absence of free lime salts, or in the
presence of soluble oxalates, the transformation of fibrinogen into fibrin
may be produced, provided that the ferment is present.^ This has also
been shown to be the case by A. Schmidt ^ and by myself,* and more
recently in a series of carefully conducted experiments by Hammarsten.
Hammarsten precipitated fibrinogen by oxalated solution of salt, and,
after purifying it by repeated re-solution and re-precipitation, added to
its solution a fibrin ferment obtained from oxalated serum, and obtained
as the result a typical fibrin.^

Exception has been taken to the inference drawn by Pekelharing that
Schmidt's ferment is a compound of nucleo-proteid and lime, on the ground
that the ferment contained in Schmidt's extract differs from nucleo-proteids
in the effect of alcohol upon its solubility in water, and in the fact that
nucleo-proteids cause coagulation in intravascular plasma, which Schmidt's
extract does not, whereas the latter causes coagulation in extravascular (salted)
plasma, and nucleo-proteids do not.*^ The differences may, however, depend,
in part at least, upon the relative amounts of nucleo-proteid and lime. Thus
in Schmidt's extract the amount of nucleo-proteid is small and the amount of
lime large ; in extracts of thymus and the like the amount of nucleo-proteid
is large and the amount of lime small. In part also they depend upon other
circumstances, such as the influence of the magnesium sulphate of the salted
plasma in antagonising the effect of lime.'^

The origin of the nucleo-proteid of plasma and serum is probably the white
corpuscles. It would appear that many of the latter disintegrate after removal
of blood from the body. Glirber ^ found that in coagulated blood the number
of white corpuscles was reduced to one-half, the difference being chiefly in the
number of polynuclear cells. This disappearance has not, however, been
found by all observers, and is not fully admitted. ISTevertheless, Avithout
actually disintegrating, the white corpuscles may shed out or secrete nucleo-
proteid into the plasma. This may occur normally in mere traces, but on with-

1 Centralhl. f. Physiol., Leipzig ii. Wien, 1895, No. 3.

^ It would appear that a soluble oxalate does not throw down all the lime from a proteid-
containing fluid, and that a trace of lime is still held in solution so that an oxalate plasma
is not lime-free, as was supposed by Arthus and Pages. This is well illustrated by an
observation by Einger upon the frog's heart, who finds that a normal saline solution,
to which a little CaClo has been added, will exhibit the physiological effect of lime,
even after the addition to the fluid of a slight excess of a soluble oxalate. It may be
inferred from this that a trace of lime may be held in solution even in a fluid destitute of
proteid.

3 " Weitere Beitr. z. Blutlehre," AViesbaden, 1895.

^ Proc. Physiol. Soc, Journ. Physiol., Cambridge and London, 1895, vol. xvii.
p. xviii.

^' Ztschr. f. physiol. Chevi., Strassburg, Bd. xxii.

•^ Halliburton and Brodie, Journ. Physiol.. Cambridge and London, 1894, vol. xvii. p. 143.

^ Pekelharing, Centralbl. f. Physiol., Leipzig u. Wien, 1895, Bd. ix. S. 102. Halliburton
in a recent paper (./oiM-?i. Physiol., Cambridge and London, 1895, vol. xviii.) comes to a
similar conclusion, namely, that Schmidt's fibrin ferment is a Aveak solution of nucleo-
proteid. It produces Wooldridge's negative phase when intravenously injected.

^ Sitzungsh. d. jihys.-mcd. Gesellsch. zu Wilrzhurg, 1892, No. 6.



172 THE BLOOD.

dravval from the body in larger amoant. It is noteworthy in this connection
that certain forms of lymph, such as the aqueous humour, which contain no
cells, contain also no nucleo-proteid, and are only coagulable on the addition of
nucleo-proteid.

Theory of Lilienfeld. — Lilienfeld,^ like Arthus and Pekelharing, con-
siders that fibrin is formed by a combination of fibrinogen with lime, any
soluble lime salt being equally effectual to produce the combination.
Lilienfeld differs, however, from them in denying the necessity for the
intervention of a ferment in the ordinary sense of the word. He considers
that what the nucleo-proteid effects is not the combination of fibrinogen
with lime, but a transformation or splitting of the fibrinogen into a sub-
stance which he terms " thrombosin," and a globulin ; the thrombosin then
combines with lime, if any be present, to form fibrin. The nucleo-proteid



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 24 of 147)