Not only is the cell-globulin or ferment present in the serum, but it also adheres to the fibrin; this is demonstrated by the fact that shreds of Buchanan's washed blood clot cause coagulation in coagulable fluids like pericardial fluid. This has been thoroughly worked out by Gamgee,' who found that the most convenient fluid with which to extract ferment from a washed blood clot' was an 8 per cent. solution of sodium chloride. The extract contained a globulin, on the removal of which, ferment activity was removed also.

Lastly, a globulin with precisely similar characters, and exhibiting powerful ferment properties, can be obtained from the cells of lymphoid structures, cells which subsequently become white blood corpuscles. The method of separation of cell-globulin from other substances will be described in connection with the chemistry of lymphatic glands and white blood corpuscles.

It has been suggested by Sheridan Lea and Green' that cell-globulin and the ferment are not absolutely identical, but that they are closely adherent, and that the various methods adopted for precipitating the globulin cause the ferment to be carried down with it mechanically. But such a theory will not account for the insolubility of the ferment in water, which is one of the most characteristic properties of a globulin. The apparent solubility of the ferment in the water used in Schmidt's method of preparation is really due to the fact that certain salts in the dried alcoholic precipitate enter into solution at the same time. If these be removed by prolonged dialysis, water is then unable to extract the ferment, but a saline solution must be employed.

Certain facts also, of which the two most important are the action of alcohol and the action of heat, go far to prove that the ferment and the proteid are identical.

The action of alcohol.—The ferment is precipitated by alcohol; and it is generally stated that unlike the proteids it is not rendered subsequently insoluble by the prolonged action of alcohol. It is this fact upon which Schmidt bases his method of preparing the ferment, viz. extracting with water the dried alcoholic precipitate of serum. Hammarsten, however, has noticed the loss of activity which the ferment undergoes after exposure to the action of alcohol; and in my own researches it was found that an exposure of the ferment to the action of alcohol for six to seven months renders it absolutely inactive. The ferment is thus, like proteids, rendered ultimately insoluble by alcohol, though more slowly than ordinary albumin is.

The action of heat.-The most striking fact that appears to prove the identity of the ferment and proteid is that the activity of the ferment is abolished at the same temperature as that at which the distinctive characters of the proteid are destroyed (about 75° C.). In the case of sodium chloride solutions of the proteid, heat-coagulation occurs at a lower temperature, viz. 60°-65° C. When dissolved in serum, this lower temperature is also sufficient to destroy its activity.

The cell-globulin, however, acts like other ferments in producing changes without being itself changed. It does not, as Schmidt supposed paraglobulin did,

1 Gamgee, Journ. of Physiol. ii. 145.

? This term is Buchanan's. It means fibrin obtained from blood which has been diluted with 8-10 times its bulk of water immediately it is shed. The same facts, however, hold for fibrin prepared approximately free from corpuscles in the usual way.

5 Halliburton, Proc. Roy. Soc. xliv. 255.

4 Lea and Green, Journ. of Physiol. iv. 380.

R

become a constituent part of the fibrin formed, though much of it remains adherent to the fibrin, as well as being dissolved in the serum.

There are other globulins which have the same effect upon the formation of fibrin that cell-globulin has. For instance, the myosinogen of muscular tissue is one of these (see Muscle).

The addition of living cells, such as yeast cells, or pieces of many fresh tissues, to such liquids as hydrocele fluid or dilute salted plasma causes a rapid formation of a clot. In these cases, if neither cell-globulin nor myosinogen is present, in all probability there are similar unstable globulins in the cell-protoplasm which act in the same way.

Historical account of the theories of Coagulation

Nearly up to the end of the eighteenth century the clot was supposed to consist of merely adherent corpuscles. This view was held in Britain by Keill, Jurin, Thomas Morgan, John Cook, Arbuthnot, Cowper, Langrish, Berdoe, and others; and on the Continent by Leeuwenhoek, Boerhaave, Van Swieten, Haller, and Marherr. Petit, Quesnay, Senac, Borelli, and Davies were the earliest to have an idea of a coagulable substance in addition to the cells, and this was fully recognised and proved by Hewson (1772), and taught by Fordyce and the Hunters.1

The reason that the blood coagulates outside the vessels and not during life was accounted for in different ways; some considered that coagulation was due to the action of the air on the blood (Borelli, Lower); others that the blood was maintained in its liquid condition during life by its continual movement; others, again, that coagulation was due to the cooling of the blood on its removal from the vessels. We however now know, on the contrary, that blood will clot even if collected over mercury without coming in contact with the air at all, that agitation hastens and cooling hinders coagulation. Hunter considered that coagulation was an act of life, and connected with the vitality of the blood- -a vague statement which implies very little; but, as Gulliver 3 pointed out, if it is a vital act, it is equivalent to saying that we are able to pickle the life of the blood for hours or even days, although decomposition may have begun in other parts of the body.

Hewson not only showed that a coagulable substance we now call fibrin separates from the plasma, which he obtained by skimming it off from the surface of blood which coagulated slowly, but he also discovered the fact that cold, contact with living vessels, and admixture with salts are agencies which hinder or prevent coagulation. In connection with the influence of the living vessels on coagulation, the 1 For the references to the writings of these authors see Hewson's Works, edited by Gulliver, Sydenham Soc. p. xxix. et seq.

2 For a résumé of these earlier views, I am indebted to Gamgee, Physiol. Chem. p. 42. 3 Hewson's Works, note 12, p. 21.

further researches of Lister, Fredericq, and Brücke have been already referred to (see p. 224).

Andrew Buchanan was the next who made noteworthy investigations into this subject. He experimented with fluid obtained from the pericardial sac and from the tunica vaginalis in the dropsical condition of that serous membrane called hydrocele. These liquids do not coagulate spontaneously, but Buchanan found that the addition of small shreds of washed blood clot' caused the formation of fibrin in them. This power was exhibited to a greater extent still by the 'buffy coat' of a clot; he therefore concluded that the power resided in the white blood corpuscles which are so abundant in the buffy coat, and their action he compared to the action of rennet in curdling milk. Then came Denis, who saturated blood plasma with sodium chloride, and thus obtained a proteid precipitate. This precipitate was washed with a saturated solution of sodium chloride and redissolved by the addition of water, the adherent salt rendering it soluble. This solution remained liquid for a short time, but on being allowed to stand a clot of fibrin was produced. Denis had thus obtained the precursor of fibrin from the plasma, and to it he gave the name plasmine; the proteids, which were not precipitated by the salt, he called serine, or, as we now call them, serum-albumin. We now know that Denis' plasmine was a mixture of fibrinogen, serum-globulin, and fibrin-ferment.

3

2

Alex. Schmidt recognised these three substances; he, however, supposed that all three were necessary for the formation of a clot. One of the most important experiments on which he based this view was, that if serum which contains serum-globulin (or fibrino-plastic substance, as he termed it) and ferment be added to hydrocele or pericardial fluid, which he supposed contained fibrinogen but no serumglobulin, the result is the formation of fibrin. He also found that the more serum-globulin he added to a coagulable liquid the larger was the yield of fibrin from it.1

1 A. Buchanan, London Med. Gazette, xviii. (1835), p. 50; also vol. i. new series (1845), p. 617. The latter paper was reprinted by Dr. Gamgee in the Journ. of Physiol. vol. ii. (1879). The influence of leucocytes in bringing about coagulation was very strongly insisted on by Mantegazza (Centr. Med. Wiss. 1871, p. 709).

2 Denis, Mémoire sur le sang, 1859, p. 32.

3 A. Schmidt, Arch. f. Anat. u. Physiol. 1861, p. 545; 1862, pp. 428 and 533. Pflüger's Archiv, vi. 445.

4 In connection with the question whether or not the ferment is a globulin, it is interesting to note that the proteid present in Schmidt's ferment solutions, and which some have considered as an impurity, was one which was precipitable by a stream of carbonic acid; he also found that serum minus its globulin has very little ferment activity; that it still possesses any is due to the fact that Schmidt's carbonic acid method does not completely precipitate the globulin.

O. Hammarsten ascertained the characters of fibrinogen, seramglobulin, and fibrin-ferment with greater exactness, and showed that serum-globulin, or paraglobulin, as he terms it, is not necessary for the formation of fibrin, but that fibrinogen is the only precursor in the plasma of the fibrin in the clot. He pointed out that pericardial and hydrocele fluids contain abundance of serum-globulin, as well as fibrinogen, and therefore the addition of serum causes these fluids to coagulate, not in virtue of the serum-globulin, but of the ferment it contains. He pointed out that serum-globulin is very difficult to separate from ferment, a fact which is easy to understand, as we now know that the ferment is probably itself a globulin; the addition of apparently pure serum-globulin, prepared from serum, to hydrocele fluid causes the formation of fibrin because of its admixture with ferment. A pure serum-globulin prepared from pericardial or hydrocele fluid has on the other hand no fibrinoplastic activity. The most striking proof, however, of Hammarsten's theory is this: that a solution of the ferment added to a solution of pure fibrinogen causes the formation of fibrin,

It should be mentioned that Hammarsten does not regard the ferment as a globulin, because he is able to prepare it from horse's serum which has apparently been deprived of all globulin by saturation with magnesium sulphate. Howells 2 and also Hayem have tried this method with the blood of other animals, but unsuccessfully, and in the course of my own work I have done the same, and again with a negative result. In the case of horse's serum, however, I have found that Hammarsten's statement is correct; and the explanation seems to be that for some reason or other it is exceedingly difficult to precipitate all the globulin from horse's blood by the use of this salt; but after repeated saturations one can remove all the globulin, and with it all ferment activity also.

The researches of Gamgee, of Lea and Green, and of myself, into the nature of the fibrin ferment have been already alluded to (see p. 241). To Green also we owe the discovery that the presence of calcium sulphate is necessary for the proper action of the ferment to take place. This again reminds us of Buchanan's old comparison of the clotting of blood to the curdling of milk, where the phosphate of calcium is a sine qua non.

Although Hammarsten did not consider serum-globulin necessary for the formation of fibrin, he admitted that its presence was advantageous; it can, however, be replaced by other proteids like casein, or even by salts like calcium chloride. He considers that serum-globulin possibly acts like calcium chloride in combining with the alkaline

1 O. Hammarsten, Pflüger's Archiv, xiv. 211; xvii. 413; xviii. 38; xix. 563; xxii. 489. 2 Howells, Studies from the Physiol. Lab. Johns Hopkins Univ. Baltimore, vol. ii. 3 Du sang, Paris, 1889.

carbonates present in the blood, the presence of which would otherwise impede the activity of the ferment. The supposed action of calcium chloride in this respect may be represented by the formula

Na2CO3+CaCl2=2NaCl + CaCO3.

Of recent years an entirely new theory was advanced by the late Dr. Wooldridge, which may be stated as follows:1—The coagulation of the blood is a phenomenon essentially similar to crystallisation; in the plasma there are three constituents concerned in coagulation, A, B, and C fibrinogen. A and B fibrinogen are compounds of lecithin and proteid, and fibrin results from the transference of the lecithin from A-fibrinogen to B-fibrinogen. C-fibrinogen is what has hitherto been called fibrinogen; A-fibrinogen is a substance which may be precipitated by cooling peptone plasma,' and on the removal of this substance coagulation occurs with great difficulty. The precipitate produced by cold consists of rounded bodies resembling the blood tablets in appearance. He further found that other compounds of lecithin and proteid, to which he has extended the name of fibrinogen, exist in the testis, thymus, and other organs, in the fluid of lymph glands, in the stromata of red corpuscles, and in the serum of certain animals; these substances may be extracted from the organs by water, and precipitated from the aqueous extract by acetic acid, and on redissolving this in a saline solution, and injecting it into the circulation of a living animal, intravascular clotting occurs, which results in the death of the animal. The form of fibrinogen that acts thus, he looks upon as the precursor of A-fibrinogen. From these points of view the fibrin-ferment and the white corpuscles are looked upon as of secondary import in causing coagulation, though it is admitted that fibrin-ferment converts C-fibrinogen into fibrin.

I have elsewhere given at some length my reasons for not accepting this theory, and this is not the place for debating a controversial subject. I will merely say that I still consider Wooldridge erred, first, in drawing conclusions from observations on peptone plasma without corroborating them by experiments on pure plasma; and, secondly, in attributing to the corpuscular elements a secondary rôle in the causation of clotting.3

4

The latest theory of coagulation is that of Freund; he considers that, when blood is shed, earthy phosphates derived chiefly from the corpuscles unite with fibrinogen and thus form fibrin.

Serum-Albumin

Serum-albumin, or serine, is the proteid which remains in solution after the separation of serum- -globulin from the serum. Now that Hammarsten's method has been adopted for the separation of serum

I Wooldridge, Ludwig's Festschrift, 1886, p. 221.

2 Journ. of Physiol. ix. 270. Wooldridge defended his views in the same Journal,

x. 339.

5 Krüger (Zeit. Biol. xxiv. 189) and Hayem (Du sang, Paris, 1889), who have also recently examined Wooldridge's views, are unanimous in regarding the corpuscles as most important factors in fibrin-formation.

4 Med. Jahrb. 1888, p. 259.