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The intravascular test for tissue-fibrinogen.—This is constituted by the sum-total of the reactions of the system to an intravascular injection of tissue-fibrinogen. I shall, in a few words, resume the main facts as elicited by Wooldridge's work upon this subject.

In the dog.-An injection of tissue-fibrinogen into the blood-vessels leads to a modification of the blood in two opposite directions: (a) to a modification in the direction of increased coagulability (constituting Wooldridge's positive phase of coagulability); and (b), to a modification in the direction of decreased coagulability (constituting Wooldridge's negative phase of coagulability). Under ordinary circumstances we see both effects from an intravascular injection of tissue-fibrinogen, the positive phase of coagulation being marked by intravascular thrombi in the whole of the portal venous system, and the negative phase being evidenced by a loss of coagulability in the blood of the extra-portal tracts. Wooldridge also pointed out that the condition of affairs was altered to the advantage of the positive phase of coagulation, when the injection was made into an animal during the period of active digestion. In such cases he observed that the coagula were not limited to the portal tract, but occurred also in the right heart and in the pulmonary artery.

In the rabbit.— Wooldridge described the effects of the injection of his coagulating fluid to be in all cases a complete thrombosis of every part of the vascular system to which it obtained access. I believe that a negative phase was not observed by him.

In the cat.I do not find that Wooldridge published any statements as to the effect of tissue-fibrinogen when injected into the vascular system of the cat. The phenomena are in the highest degree inconstant, sometimes only a very slight coagulation being obtained in the portal system ; at other times extensive coagulations are obtained throughout the venous system. The causes of these variations will be dealt with at a later stage of this Paper.

Such are the ordinary reactions obtained on injection of a simple extract of either the thymus or the testicle ; and we may provisionally accept these as constituting the intravascular test for tissue-fibrinogen. When, however, we come to apply these reactions in experiments upon the isolation of the coagulating principle in these extracts, a great defect of sensitiveness becomes apparent in the intravascular as contrasted with the extravascular tests, and in many cases where an apparently negative result on injection of a solution leaves us in doubt as to whether the solution injected does or does not contain tissue. fibrinogen, the extravascular tests afford us invaluable assistance.

Experiments directed towards the Isolation of the active Constituent of the

coagulative Extracts of the Cellular Organs.

We may first, following Wooldridge, proceed to precipitate the extracts of the organs (thymus, testicle) with dilute acetic acid, and having separated off the filtrate from the precipitate we can apply to both the tests for the presence of tissue-fibrinogen.

When we test the filtrate obtained in this way, by injecting large quantities of it intravascularly after previous neutralization, we find that it appears to act as a perfectly inert fluid. Similarly, when we test it with peptone plasma, we do not find that this filtrate has any greater effect in producing coagulation than dilution with distilled water has.

It is therefore evident that the substance which is the cause of coagulation must be looked for in the acetic acid precipitate.

We can easily verify that it can be found there, by adding a small portion of the precipitate to some peptone plasma, when we obtain firm coagulation in the course of a minute or two.

Having thus assured ourselves that the substance which is the cause of coagulation has not been destroyed by the precipitation, and that it is present in the precipitate, the next step to be taken was to endeavour to extract it from the precipitate, and to obtain it in the form of a solution.

In order to accomplish this I subjected the acetic acid precipitate to either prolonged extraction in the warm chamber or to prolonged agitation in the shaking machine, with salt solutions of various strengths and with dilute solutions of both alkaline carbonates and caustic alkalies, and then proceeded to apply the physiological tests for tissue-fibrinogen to the filtrates obtained. It is easy in this way, especially when applying the tests with extravascular plasma, to satisfy oneself that an appreciable solution of tissue-fibrinogen takes place only where an alkaline solution has been applied as a solvent. On the other hand, the results of intravascular injection of even such solutions as give a very distinct positive reaction with peptone plasma present, at first, certain difficulties of interpretation, as the following protocols will show :

Rabbit 53, Feb 14th, 1891.-Injected into the jugular 19 cc. of a filtered 2.5 NaCl extract of bulls' testicles. Death—Universal clotting in the venous side of the vascular system ; slight clot in left heart; no clot in aorta.

Rabbit 55, Feb. 14th, 1891.-Injected 35 cc. of a filtered 1 per cent., solution of the acetic acid precipitate obtained from the above extract. No apparent result.

(Then ran in 10 cc. of the same solution in which a little of the acetic acid precipitate had been suspended. Death-Coagulation universal throughout the vascular system.)

Here we have an instance of a negative result with the filtered alkaline solution, which I found it difficult to account for at the stage of my work which I had then reached. I repeated the experiment over and over again, with solutions which were as concentrated as I could make them, but I did not in any case succeed in obtaining an immediate intravascular coagulation with these solutions, though the coagulability of the blood was always increased by injecting them, and in some cases was increased to the point at which coagulation took place absolutely instantaneously after the blood had left the vessels.

Finally, however, I found that a solution which gave intravascular coagulation could be obtained by reprecipitating the tissue-fibrinogen from the filtered 1 per cent. Na,CO2 solutions, and then redissolving the precipitate (after decanting off the supernatant fluid) by rendering the solution slightly alkaline.

I will quote one out of several essentially similar protocols in support of the above statements.

Feb. 27th, 1891.–Took a large quantity (exact quantity not noted) of the 1 per cent. Na2CO, solution of the acetic acid precipitate from the watery extract of bulls' testicles. Precipitated the tissue-fibrinogen from this solution by acetic acid and NaCl."

Dissolved the precipitate by the addition of a little Na,CO3, and filtered the more or less viscid solution thus obtained through calico before proceeding to the injection.

Rabbit 98.-Injected a few drops of the solution into the jugular vein, which immediately became so firmly blocked with clot that no more of the solution could be run in. Injection continued through the external iliac vein; death occurs immediately, no clot found in the I. V. C. (too little admixture of blood ?). Right and left heart clotted perfectly solid; aorta blocked with clot.

It is thus plain that we have here, in the increase of the positive phase up to the point of intravascular coagulation, obtained also the intravascular test for the presence of tissue-fibrinogen in the 1 per cent. Na,CO, solution of the acetic acid precipitate, and that we have thus

1 The addition of NaCl aids the precipitation both here and also in case of the nucleo-albumen prepared from yeast.

confirmation of the result previously obtained with the more sensitive extravascular plasma (peptone plasma).

It may, however, be objected that there is a defect in conclusiveness in the above experiments in so far as it has not been shown that intravascular coagulation can be obtained with a clear filtered nonviscid solution of the isolated tissue-fibrinogen, and that there is therefore a possibility of the mechanical properties of the solution (the viscidity) having some 'causal relation to the production of the intravascular coagulation.

That this objection, though a perfectly justified one from the è priori standpoint, does not afford the true explanation of the facts will, I think, become apparent upon the consideration of the following propositions :

(1.) The non-viscid filtered watery extract of the glandular organs inaugurates coagulation, both when added to extravascular plasma and also when injected intravascularly (vide, inter alia, protocol rabbit 53, supra).

(2.) The non-viscid filtered 1 per cent. Na2CO, solution of the acetic acid precipitate obtained from (1) coagulates extravascular plasma; and though it does not produce intravascular coagulation, it increases the coagulability of the blood when injected (vide supra).

(3). The viscid solution obtained by adding more of the acetic acid precipitate to (2)(i.e. by adding more of the precipitate to the alkaline solution than that solution is able to carry through the filter) will coagulate both extravascularly and intravascularly (vide protocol rabbit 55, supra).

(4). The viscid solution obtained by reprecipitating the tissuefibrinogen from (2) will coagulate both extravascularly and, when injected without filtering through paper, will coagulate also intravascularly (vide protocol rabbit 68).

(5.) The viscid solution (3), when boiled, will coagulate extrarascularly, but not intravascularly (Wooldridge, repeatedly verified by myself). N.B.—No apparent mechanical change occurs in these soiutions on boiling.

It is plain, therefore, that viscid solutions, even when they possess a certain amount of coagulative power (a sufficient amount of coagulative power to coagulate extravascular plasma), do not by virtue of their mechanical properties produce intravascular coagulation.

Similarly we have evidence, in the case of solution (1), that the

solutions which produce intravascular coagulation need not possess any noticeable degree of viscidity.

We therefore appear to be justified in assuming that the absence of intravascular coagulation, when solutions (2) and (5) are injected, is referable to an insufficient degree of concentration in the tissue-fibrinogen contained in those solutions, such deficiency being brought about in the case of solution (2) by the retention of the tissue-fibrinogen on the filter, and in the case of solution (5) by the alteration or disintegration of the tissue-fibrinogen under the influence of heat. The fact that every one of these five solutions gives the reaction with extravaseular plasma is evidence that they all contain fibrinogen, though in various degrees of admixture. This is also evidenced by the fact that they all produce, when injected, a modification of coagulation in the direction of increased coagulability.

That we should not be able to produce or obtain a sufficiently concentrated filterable (i.e. non-viscid) solution of tissue-fibrinogen will not appear strange on considering (a) the loss of solubility constantly entailed upon albuminous substances by precipitation, and (b) the fact that the filtered original watery extract (i.e. the saturated filtered solution of unprecipitated tissue-fibrinogen) is only just sufficiently concentrated to produce intravascular coagulation. (We require to inject very considerable quantities to obtain this effect-vide e.g. protocol rabbit 53, where circ. 20 cc. had to be injected.) It will thus be seen that (a) we could hardly expect to be able to reconstitute a solution of tissue-fibrinogen of the strength of the original unprecipitated saturated solution, and (b) that intravascular coagulation could hardly be expected with a solution which was at all less concentrated.

In point of fact, this appears to be the condition of things with which we have to deal with here.

Having thus, I believe, entirely confirmed Wooldridge's statement as to the solubilities of tissue-fibrinogen, I proceeded by means of these solubilities to separate out the tissue-fibrinogen from the albuminous substances of other solubilities present in the watery extracts in order to investigate the chemical properties of the thus far-isolated substance. I have discussed these chemical properties elsewhere; but may, perhaps, shortly recapitulate them here.

1 Transactions of the International Congress for Hygiene : London, 1891. Published in an abbreviated form in the British Medical Journal of Sept. 19th, 1891.

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