A.—Effects of Venom on the Coagulation of the Blood.
It was observed long ago by Fontana[29] that after viper-bites the blood remains fluid, and Brainard[30] on the contrary, pointed out that, in the case of animals that succumb very rapidly after having been bitten by a Crotalus, the blood was always found coagulated into a mass, while, when a certain interval of time had elapsed since the bite, it remained fluid. Weir Mitchell[31] explained these differences by the hypothesis that, in cases of rapid death, the blood had not had time to become modified by the venom.
Later on it was found by Sir Joseph Fayrer, and subsequently by Halford,[32] in Melbourne, C. J. Martin,[33] in Sydney, G. Lamb,[34] in Bombay, and recently by Noc, in my laboratory, that the venoms of Colubridæ, especially those of Naja tripudians and Australian species of this family, always leave the blood fluid after death, while the venoms of Viperidæ, on the contrary, are usually coagulant.
On the other hand, it was observed by Phisalix,[35] and at an earlier date by Mosso, of Turin, that the venom of Vipera berus causes the blood of the dog to lose its coagulability, while, on the contrary, the same venom is actively coagulant as regards the blood of the rabbit.
How are these differences of action to be explained? It was found by Delezenne,[36] who made an excellent study of the phenomena following the injection of peptone, extracts of organs, and other anticoagulant substances into the organism, that those of these substances that render the blood non-coagulable always dissolve the leucocytes, and thus set at liberty two antagonistic bodies which they contain. One of these substances is coagulant and is found retained by the liver, while the other remains in solution in the plasma, and keeps the blood fluid after issuing from the vessels.
Now, certain extracts of organs, ricin, abrin and certain venoms in weak doses, retard coagulation, while in large doses, on the contrary, they produce partial or general intravascular clotting.
It is believed by Delezenne that the explanation of this phenomenon may be that the doses, which are weak but sufficient to produce the disintegration of the leucocytes, injure the red corpuscle in only a slight degree, while the stronger doses are equally destructive to the two kinds of blood corpuscles.
It follows that we must understand that there are two phases in the action of venoms: one negative, when the dose absorbed does not injure the leucocytes; the other positive, when the leucocytes are destroyed.
If the blood of the dog remains non-coagulable when mixed with doses of venom which, on the contrary, are actively coagulant for the blood of the rabbit, the reason would be that the leucocytes of these animals are not equally resistant to venom.
This conception, however, does not conform to the facts that I have myself observed. I have always found that viper-venom, mixed with citrate- or oxalate-plasma of the dog, rabbit, or horse, coagulates these various plasmas when the venom is in weak doses, while with strong doses coagulation is not produced. To be quite accurate, it should be stated that the quantity of venom necessary to render the plasma of the dog, or of the horse, non-coagulable is less than that which must be employed in the case of the plasma of the rabbit.
I have caused Noc to take up anew the study of this question in my laboratory, with venoms of nine different origins, and I here give a résumé of the results of his researches.[37]
I. Coagulant Venoms.
The venoms of Viperidæ studied range themselves as follows according to their coagulant power:—
| Crotalinæ: | Lachesis lanceolatus (Fer-de-lance, Martinique). |
| Lachesis neuwiedii (Urutù, Brazil). | |
| Lachesis mutus (Bushmaster, or Surucucu, Brazil). | |
| Lachesis flavoviridis (Japan). | |
| Viperinæ: | Vipera russellii (Daboia, India). |
The venoms of Ancistrodon contortrix and A. piscivorus (Crotalinæ) proved entirely inactive.
No Colubrine venom exhibited coagulant power, whatever the dose employed.
There is, therefore, a very decided difference between venoms of divers origins as regards their effects upon the coagulation of the blood.
Noc has determined more especially the coagulant action of the venom of Lachesis lanceolatus (Fer-de-lance of Martinique) upon 1 per cent. citrate-plasmas, 1 per cent. oxalate-plasmas, 4 per cent. chloridate-plasmas, and upon blood rendered non-coagulable by extract of leeches’ heads. He found that, while weak doses of venom (1 milligramme per cubic centimetre of horse- or rabbit-plasma) produce coagulation in a few minutes in the citrate-plasmas, chloridate-plasmas, or those treated with extract of leeches, the doses of the same venom greater than 4 milligrammes on the contrary suppress the coagulability of these plasmas, even when there be added to them doses of chloride of calcium (for the citrate- and oxalate-plasmas), or of distilled water (for the chloridate-plasma), or of fibrin-ferment (for the plasma treated with leech-extract) sufficient to cause rapid coagulation in the control tubes that do not contain venom.
Noc also observed that the venom of the same species of snake (Lachesis lanceolatus), when heated to 75° C., entirely loses its coagulant properties; and that, with a temperature of 58° C., its coagulant power already commences to diminish. When heated for thirty minutes at a temperature of 65° C., a dose of 1 milligramme does not coagulate more than 1 c.c. of citrate-plasma in one hour. G. Lamb has likewise found that the venom of Vipera russellii loses its coagulant power when heated to 75° C.
The coagulant substance in these venoms is precipitable by alcohol at the same time as the neurotoxin and other active substances. The precipitate, when dissolved again in physiological water, preserves all the properties of the original solution.
Antivenomous anticolubrine serum, that is to say, that furnished by horses vaccinated against the venoms of the Cobra and the Krait, does not prevent coagulation by coagulant venoms. This need not surprise us, since the coagulant substances in venoms are destroyed by heating, and the animals vaccinated in order to obtain antitoxic serum are usually inoculated exclusively with heated venoms.
It is easy, however, to obtain active serums specific against the coagulant venoms; it is sufficient to treat these animals by inoculation with progressively increasing doses of the same venoms unheated. I have had no difficulty in achieving this result with small laboratory animals (guinea-pigs and rabbits) and also with the horse, but I have never had at my disposal a sufficient amount of the venoms of Lachesis or Vipera russellii to undertake with them the regular acquisition of large quantities of horse-serum, at once antineurotoxic and anticoagulant. The preparation of such a serum, nevertheless, presents much interest for certain countries, such as Burma, where the Daboia (Vipera russellii) is almost as common as the Cobra, and Brazil, where nearly all the casualties due to venomous snakes are produced by Lachesis.[38]
II.—Anticoagulant Venoms.
Contrary to what is observed with the venoms of Viperidæ in general, all the venoms of Colubridæ and, as exceptions to the rule, the venoms of some North American Crotalinæ (Ancistrodon contortrix and A. piscivorus) suppress the coagulability of the blood in vivo and in vitro. It is, however, important to observe that, in vivo, the blood remains fluid after death only if the dose of venom absorbed has been sufficient. In vitro this phenomenon is easier to study, and has been the subject of several important memoirs.
Halford,[39] Sir Joseph Fayrer,[40] C. J. Martin,[41] Delezenne,[42] Phisalix,[43] and lastly Noc,[44] have shown that the venoms of Colubridæ exert a manifestly anticoagulant action upon citrate-, chloridate-, or oxalate-plasmas, and also upon blood mixed with venom on issuing from the vessels.
On adding 1 milligramme of Cobra-, Bungarus-, Australian Pseudechis-, or Ancistrodon-venom to 1 c.c. of citrate-, oxalate-, or chloridate-plasma, and supplementing the mixture, after varying periods of contact, with a quantity of chloride of calcium (for the citrate- or oxalate-plasmas), or distilled water (for the saline plasma) sufficient to produce coagulation in a few minutes in the control tubes without venom, we find that coagulation no longer takes place after one hour in the tubes containing Cobra- or Bungarus-venom, and after ten minutes in those that contain the venom of Ancistrodon.
In doses less than 1 milligramme for 1 c.c. of plasma, these venoms by themselves never produce coagulation as do those of Lachesis or Vipera russellii. They are thus sharply differentiated in this respect.
If fresh blood issuing from the arteries of an animal be received in a vessel containing a sufficient quantity of Colubrine-venom (that of the Cobra for example), and steps be immediately taken to ensure the perfect mixture of the venom and the blood, we find that the latter has entirely lost its coagulability, just as though it had been mixed with peptone or extract of leeches’ heads.
Again, if a mixture be made in vitro of coagulant venoms, such as that of the Lachesis, with anticoagulant venoms such as that of the Cobra or of Ancistrodon, it is found that these mixtures, when properly effected, become neutral, so that the respective effects of the component venoms are entirely destroyed. Assuming, for instance, that 1 milligramme of Lachesis-venom coagulates in two minutes 1 c.c. of 1 per cent. citrate rabbit-plasma, if we add to the plasma firstly 1 milligramme of Ancistrodon-, or 1 milligramme of Cobra-venom, and then 1 milligramme of Lachesis-venom, the plasma remains non-coagulated, yet coagulates perfectly on the subsequent addition of 1 c.c. of a ½ per cent. solution of chloride of calcium.
There is, therefore, a real antagonism between the actively coagulant substance contained in certain Viperine venoms and the anticoagulant substance comprised in the venoms of certain other Viperidæ (Ancistrodon), belonging to the subfamily Crotalinæ, and in those of all the Colubridæ.
The conclusion to be deduced from the foregoing facts is that the venoms of Colubridæ and those of certain Viperidæ are decidedly anticoagulant, while the majority of the venoms of Viperidæ, on the contrary, possess strong coagulant properties, even when mixed with blood in infinitesimal doses.
The question therefore arises why these coagulant Viperine venoms suppress the coagulability of the blood when mixed with it in vitro in strong doses (for example, in doses beginning from 4 milligrammes of Lachesis-venom, or 7 milligrammes of the venom of Vipera russellii for 1 c.c. of 1 per cent. citrate rabbit-plasma).
The explanation of this apparently contradictory phenomenon is furnished by the intense proteolysis that these Viperine venoms exert upon fibrin, in solution or coagulated. This proteolysis actually manifests itself with weak coagulant doses, for the compact clots formed at the outset soon become soft and then dissolve, like a cube of egg-albumen in an experiment in artificial digestion by trypsin. We shall revert to the subject later on.
III.—Mechanism of the Anticoagulant Action of Venoms on the Blood.
The anticoagulant action of the venoms of Colubridæ and of Ancistrodon upon the blood appears to take effect in the first place upon the fibrin-ferment, and afterwards upon the fibrin by proteolysis. The action on the fibrin-ferment seems manifest when we experiment with anticoagulant venoms which are feebly proteolytic, like the venom of the Cobra.
I have already stated that a mixture of fresh blood with a sufficient dose of Cobra-venom is non-coagulable, as though the blood on issuing from the animal had been mixed with peptone or leech-extract. But, while blood when peptonised or mixed with leech-extract coagulates readily on the subsequent addition of fibrin-ferment, blood mixed with venom remains positively non-coagulable. It is the same with citrate- or oxalate-plasmas, which no longer coagulate when chloride of calcium is added to them, and with 4 per cent. saline plasma on the addition of distilled water.
The anticoagulant substance in the venoms of Colubridæ and Ancistrodon is precipitable by alcohol, like the coagulant substance in the venoms of Viperidæ and like the neurotoxins, from which it is difficult to separate them. The separation can nevertheless be effected by the aid of heat, if we make use of certain venoms that are particularly resistant to high temperatures, such as those of the Cobra or the Krait. These latter venoms, when heated for one hour at 70° C., cease to be anticoagulant, and preserve their toxicity unimpaired. It is, however, impossible to suppress the toxicity without at the same time destroying the anticoagulant substance.
Antivenomous serum completely protects citrate- or chloridate-plasmas against the anticoagulant action of venoms. It is sufficient to mix ½ c.c. of 4 per cent. saline antivenomous serum with 1 c.c. of 4 per cent. saline plasma to ensure that the subsequent addition of 1 milligramme of Cobra-venom to this mixture remains without effect upon the coagulability of the latter. If, after a contact of two hours or more, 2 c.c. of distilled water be added, coagulation is produced just as in saline plasma without venom.