THE HASTENING OF COAGULATION OF BLOOD BY ADRENIN

The primary value of blood to the body must have been one of the earliest observations of reasoning beings. When we consider the variety of fundamental services which this circulating fluid performs—the conveyance of food and oxygen to all the tissues, the removal of waste, the delivery of the internal secretions, the protection of the body against toxins and bacterial invasion, and the distribution of heat from active to inactive regions—the view of the ancient Hebrews that the “life of the flesh is in the blood” is well justified. It is naturally of the utmost importance that this precious fluid shall be safeguarded against loss. And its property of turning to a jelly soon after escaping from its natural channels assures a closure of the opening through which the escape occurred, and thus protection of the body from further bleeding. The slight evidence that adrenin hastens the clotting process has already been hinted at. When we found that adrenin is set free in pain and intense emotion, it seemed possible that there might exist in the body an arrangement for making doubly sure the assurance against loss of blood, a process that might nicely play its rôle precisely when the greatest need for it would be likely to arise.

It was in 1903, while tracing in dogs the rise and fall of sugar in the blood after administering adrenin, that Vosburgh and Richards[1] first noted that simultaneously with the increase of blood sugar there occurred more rapid coagulation. In some cases the diminution was as much as four-fifths the coagulation time of the control. Since this result was obtained by painting “adrenalin” on the pancreas, as well as by injecting it into the abdominal cavity, they concluded that “the phenomenon appears to be due to the application of adrenalin to the pancreas.” Six years later, during a study of the effect of adrenalin on internal hemorrhage, Wiggers[2] examined incidentally the evidence presented by Vosburgh and Richards, and after many tests on five dogs found “never the slightest indication that adrenalin, either when injected or added to the blood, appreciably hastened the coagulation process.” In 1911 von den Velden[3] reported that adrenin (about 0.007 milligram per kilo of body weight) decreased the coagulation time in man about one-half—an effect appearing 11 minutes after administration by mouth, and 85 minutes after subcutaneous injection. He affirmed also, but without describing the conditions or giving figures, that adrenin decreases coagulation time in vitro. He did not attribute the coagulative effect of adrenin in patients to this direct action on the blood, however, but to vasoconstriction disturbing the normal circulation and thereby the normal equilibrium between blood and tissue. In consequence, the tissue juices with their coagulative properties enter the blood, so he assumed. In support of this theory he offered his observation that coagulation time is decreased after the nasal mucosa has been rendered anemic by adrenin pledgets. Von den Velden’s claim[3] for adrenin given by mouth was subjected to a single test on man by Dale and Laidlaw,[4] but their result was completely negative.

The importance of Vosburgh and Richards’ observation, the thoroughly discordant testimony of later investigators, as well as the meager and incidental nature of all the evidence that has been adduced either for or against the acceleration of clotting by adrenin, made desirable a further study of this matter. Especially was this further study desirable because of the discharge of adrenin into the blood in pain and emotional excitement. Accordingly, in 1914, H. Gray and I[5] undertook an investigation of the question. In doing so we employed cats as subjects. Usually they were quickly decerebrated under ether, and then continuance of the drug became unnecessary. Body temperature was maintained by means of an electric heating pad. Respiration proceeded normally except in a few instances (in which, presumably, there was hemorrhage into the medulla), when artificial respiration had to be given.

The Graphic Method of Measuring the Coagulation Time

In order to avoid, so far as possible, the personal element in determining when the blood was clotted, the blood was made to record its own clotting. The instrument by means of which this was done was the graphic coagulometer devised by W. L. Mendenhall and myself,[6] and illustrated diagrammatically in Fig. 24. It consists essentially of a light aluminum lever with the long arm nearly counterpoised by a weight W. The long arm is prevented from falling by a support S, and is prevented from rising by a horizontal right-angled rod reaching over the lever at R¹ and fixed into the block B which turns on the axis A. Into the same block is fixed the vertical rod R². When this rod is moved from the post P¹, against which it is held by the weight of the horizontal rod R¹, towards the other post P², the check on the long arm of the lever is lifted, and if the short arm is heavier, the long arm will then rise.

The cannula C, into which the blood is received, is two centimeters in total length and slightly more than two millimeters in internal diameter. It is attached by a short piece of rubber tubing to the tapered glass tube T, five centimeters long and five millimeters in internal diameter. The upper end of this tube is surrounded by another piece of rubber which supports the tube when it is slid into the U-shaped support U, fixed directly below the end of the short arm of the lever.

By drawing the cannulas from a single piece of glass tubing and by making the distance from shoulder to upper end about twelve millimeters, receptacles of fairly uniform capacity are assured. All the dimensions, the reach of the rubber connection over the top of the cannula (2–3 millimeters), the distance of the upper rubber ring from the lower end of the glass chamber (4 centimeters), etc., were as nearly standard as possible.

A copper wire D, eight centimeters long and 0.6 millimeters in diameter, bent above into a hook and below into a small ring slightly less than two millimeters in diameter, is hung in a depression at the end of the short arm of the lever. The small ring then rests in the upper part of the cannula (see Fig. 24). The weight of the copper wire makes the short arm of the lever heavier than the long arm by 30 milligrams, when the delicate writing point is moving over a lightly smoked drum. Half a dozen of these standard wires are needed.

For accurate determination of the coagulation time Addis[7] has defined the following conditions as essential:

1. The blood must always be obtained under the same conditions.

2. Estimates must all be made at the same temperature.

3. The blood must always come in contact with the same amount and kind of foreign material.

4. The end point must be clear and definite and must always indicate the same degree of coagulation.

The precautions taken to fulfill these conditions were as follows:

1. Drawing the blood.—The blood was taken from the femoral artery. The artery (usually the right) was laid bare in the groin and freed from surrounding tissue. A narrow artery clip, with each limb enclosed in soft rubber tubing (to prevent injury of the tissues), and with its spring exerting gentle pressure, was placed on the artery immediately below the deep femoral branch, thus allowing no blood to stagnate above the clip. Between the clip and a ligature applied about 1.5 centimeters below, an opening was made. The blood was carefully milked out of the vessels between a blunt dissector moved beneath, and a small forceps, twisted into a pinch of absorbent cotton, moved above.

The cannula, cleaned in water, alcohol, and ether, was set in the rubber connection of the glass tube; the point of the cannula was then lubricated with vaseline and slipped into the artery. The pressure of the clip on the artery was next very slightly released and blood was allowed to flow into the cannula up to the lower border of the rubber connection. Only a good-sized drop of blood was needed. Sometimes the blood ran one or two millimeters above or below, but without appreciably changing the result. Since the clip was situated on the femoral immediately below a branch in which the circulation persisted, the blood received in the cannula was always fresh from the moving stream. As soon as the clip gripped the artery again, the cannula was slipped out. A helper then promptly milked the vessel in the manner described above, and covered it with a pad of absorbent cotton smeared with vaseline to prevent drying. Thereby blood was not permitted to stagnate; and when a new sample was to be taken, the vessel was clean and ready for use.

The tip of the cannula was at once plugged by plunging it into a flat mound of plasticine about three millimeters high. It was drawn off sidewise lest the plasticine plug be pulled out again. One of the copper wires D was now slid into the tube and cannula, the tube slipped into the U-support, and the wire lifted and hung on the lever. This procedure, from the moment blood began to flow until the wire was hung, consumed usually about twenty seconds.

2. Uniform temperature.—Under the U-support was placed a large water bath, in which the cannula and the tapering part of the tube were submerged. A thermometer was fixed to the U-support so that the bulb came near the cannula in the bath. The water was kept within a degree of 25° C. This temperature was chosen for several reasons: (a) The cannula has room temperature and rapidly cools the small volume of blood that enters it. To heat blood and cannula to body temperature would take time. A bath near room temperature, therefore, seems preferable to one near body temperature. (b) The test of clotting was conveniently made at intervals of a half-minute, and if the clotting process were hastened by higher temperatures, this interval would become relatively less exact. (c) A temperature of 25° C. rather than lower was selected because, as Dale and Laidlaw[8] have shown, the coagulation time is much slower for a given change in temperature below 25° than for the same change above. And with slowing of the process the end point, when the determination depends on supporting a weight, is less likely to be sharp. (d) The researches undertaken with use of this coagulometer were concerned with factors hastening the process. For that reason and for reason (b), a long rather than a short coagulation time for normal conditions was desirable.

3. Uniformity in the amount and kind of contact with foreign surface.—The capacity of the cannulas was fairly uniform, as stated above; the amount received in them was fairly constant; and the wire hanging in the blood presented approximately the same surface in different observations.

A further condition for insuring consistent treatment of the blood in different cases was that of making the tests for coagulation always at the same intervals. Below the writing point of the lever was set an electromagnetic signal E, which recorded half-minutes. At the moment a record was made by the signal (see first signal mark, Fig. 25) the clip on the artery was opened, the blood taken, and the process thus begun. In about 20 seconds the cannula was suspended in the water bath and the wire was hanging on the lever. At the next record by the signal and at every subsequent record the vertical rod R² was pushed with the index finger from post P¹ to post P² and allowed to move back. This motion was uniform and lasted about one second. The check R¹ on the long arm of the lever was thus raised, and as the wire sank in the blood the writing point rose, recording that coagulation had not taken place (see Fig. 25).

4. Definite end point.—As soon as the blood clotted, the weight of 30 milligrams was supported, and the failure of the lever to rise to the former height in the regular time allowed, recorded that the change had occurred.

Very rarely the swing of the lever would be checked for a moment and would then begin to move rapidly, indicating that a strand of fibrin had formed but not sufficiently strong to support the weight, and that when the strand broke, the weight quickly sank in the blood. If this occurred, the next record almost always was the short line, which signified that the weight was well supported.

A very slight strand of fibrin was able to prevent the weight from dropping, though at different times the amount of support differed, as shown by the varying length of the final lines (compare first and last series, Fig. 25). These variations are probably a rough indication of the degree of coagulation. In our experiments, however, the length of the final line was disregarded, and merely the fact that the lever failed to swing through its usual distance was taken as evidence of a clot, and the consequent short record was taken as the end point.

As soon as this end point was registered, the tube, wire and cannula were lifted out of the bath; the cannula was then separated from the tube and pulled away from the wire. The clot was thus disclosed, confirming the graphic record.

The method, at least when used at half-minute intervals, did not reveal in all instances the same degree of clotting. Usually, when the process was very rapid, the revealed clot was a thick jelly; whereas, when the process was slow, a strand of fibrin or at most a small amount of jelly was found. This difference in the degree of coagulation introduced, of course, an element of inexactness. In our experiments, however, this inexactness was unfavorable to the result we were seeking for, i. e., the acceleration of the process—because the jelly is a later stage than the fibrin strand; and since we nevertheless obtained good evidence of acceleration, we did not in these experiments attempt to determine more accurately differences in the stage of the clotting process.

5. Cleaning of apparatus.—After the wire was removed from the tube, the clot attached to its ring-tip was carefully brushed away under cool running water. Under the running water, also, a trimmed feather was introduced into the cannula and the tube to push out the plasticine and to wash out the blood. Wire, cannula and tube were then dropped into a beaker receiving running hot water (about 80° C.) and there allowed to remain for about five minutes. On removal from this the parts were shaken free from water, passed through 95 per cent alcohol and again shaken free, passed through ether and let dry.

By having a half-dozen cannulas and wires of standard size, it was possible to save trouble by cleaning a number at one time.

Not infrequently the first few samples of blood taken from an animal showed rapid or somewhat irregular rates of clotting. Some causes for these initial variations will be presented in following pages. The fairly uniform rate of clotting in any individual after the initial stage, varied in twenty-one different animals from an average of 3 to an average of 10.6 minutes, with a combined average of 5.9 minutes. The conditions for these variations among the individuals have not been wholly determined.

The Effects of Subcutaneous Injections of Adrenin

The first observations were of this class.

Oct. 27. A cat weighing about 3 kilos was given 3 cubic centimeters of adrenin 1:1,000, i. e., 1 milligram per kilo, under the skin. The animal, in this instance, was kept in uniform ether anesthesia. Following is a record showing when blood was taken, and the coagulation time in each instance:

In this case the coagulation time remained at its usual level for about 20 minutes after the subcutaneous injection.[*] Thereafter for about an hour the coagulation time averaged 45 per cent of its previous duration. And widely separated tests made during the following hour indicated that approximately the initial rate of clotting had been regained.

The rather long period (nearly 30 minutes), in the case just cited, between the injection and the first appearance of rapid clotting was not the rule. As the following figures show, the coagulation time may become shortened quite promptly after subcutaneous injection.

In this case nine minutes after the injection the change in the rate of clotting had begun, and it continued more rapid for the subsequent half-hour.

We did not attempt to find the minimal subcutaneous dose which would shorten clotting. A dose of 0.01 milligram per kilo, however, has proved effective, as shown by the following figures:

As will be shown later, the dose in this instance was ten times the minimal effective intravenous dose. On the basis of these figures, less than a milligram of adrenin given subcutaneously would be necessary to shorten clotting to a marked degree in a man of average weight (70 kilograms).

Not many observations were made by us on the effects of adrenin administered subcutaneously. The amount reaching the vascular system and the rate of its entrance into the blood could be so much more accurately controlled by intravenous than by subcutaneous introduction that most of our attention was devoted to the latter method.

The Effects of Intravenous Injections

In this procedure a glass cannula was fastened in one of the external jugular veins and filled with the same solution as that to be injected. A short rubber tube was attached and tightly clamped close to the glass. Later, for the injection, the syringe needle was inserted through the rubber and into the fluid in the cannula, the clip on the vein was removed, and the injection made.

The solutions employed intravenously were adrenin 1:10,000, 1:50,000, and 1:100,000, in distilled water.

The smallest amount which produced any change in clotting time was 0.1 cubic centimeter of a dilution of 1:100,000 in a cat weighing two kilos, a dose of 0.0005 milligram per kilo. Four tests previous to the injection averaged 5 minutes, and none was shorter than 4 minutes. Immediately after the injection the time was 2 minutes, but at the next test the effect had disappeared. Doubling the dose in the same cat—i. e., giving 0.2 cubic centimeter (0.001 milligram per kilo)—shortened the coagulation time for about 40 minutes:

From 10.47, immediately after the second injection, till 11.20 the average time for clotting was 2.5 minutes, whereas both before and after this period the time was 4 minutes or longer. At 11.00 o’clock and 11.05, when the end point was reached in 1.5 minutes (a reduction of 63 per cent), a thick jelly was found on examining the cannula. The changes in clotting time in this case are represented graphically in Fig. 26.

In another case a dose of 0.0005 milligram per kilo failed to produce any change, but 0.001 milligram per kilo (0.28 cubic centimeter of adrenin, 1:100,000, given a cat weighing 2.8 kilos) brought a sharp decline in the record, as follows:

In these instances the animals were decerebrated. For decerebrate cats, the least amount of adrenin, intravenously, needed to produce shortening of coagulation time is approximately 0.001 milligram per kilo.

In the above cases rapid clotting was manifest directly after minute doses. Larger doses, however, may produce primarily not faster clotting but slower, and that may be followed in turn by a much shorter coagulation time. The figures below present such an instance:

This unexpected primary increase of coagulation time, lasting at least six minutes, is in striking contrast to the later remarkable shortening of the process from 3 to an average of 1.7 minutes for more than 20 minutes (see Fig. 27, A).

If a strong solution, i. e., 1:10,000, is injected rapidly, the process may be prolonged as above, but not followed as above by shortening, thus:

There was in this case no decrease in coagulation time at any test for a half-hour after the injection, but instead a lengthening (see Fig. 27, B). Howell[9] has reported the interesting observation that repeated massive doses of adrenin given to dogs may so greatly retard coagulation that the animals may be said to be hemophilic. These two instances show that on coagulation large doses have the contrary effect to small, just as Hoskins[10] showed was true for intestinal and Lyman and I[11] showed was true for arterial smooth muscle.

In a few experiments the brain and the cord to midthorax were destroyed through the orbit. Artificial respiration then maintained the animal in uniform condition. Under these circumstances, adrenin intravenously had more lasting effects than when given to the usual decerebrate animals with intact cord. Fig. 28 illustrates such a case. For thirty minutes before injection the clotting time averaged 5.4 minutes. Then, about ten minutes after one cubic centimeter of adrenin, 1:50,000, had been slowly injected, clotting began to quicken; during the next twenty minutes the average was 3.4 minutes, and during the following forty-five minutes the average was 1.9 minutes—only 35 per cent as long as it had been before the injection.

In another case in which the brain and upper cord were similarly destroyed, the clotting time, which for a half-hour had averaged 3.9 minutes, was reduced by one cubic centimeter of adrenin, 1:100,000, to an average for the next hour and forty minutes of 2.3 minutes, with 1.5 and 3 minutes as extremes. During the first forty minutes of this period of one hour and forty minutes of rapid clotting all of eight tests except two showed a coagulation time of 2 minutes or less. The explanation of this persistent rapid clotting in animals with spinal cord pithed is not yet clear.

As indicated in Figs. 26, 27 and 28, the records of coagulation show oscillations. Some of these ups and downs are, of course, within the limits of error of the method, but in our experience they have occurred so characteristically after injection of adrenin, and so often have appeared in a rough rhythm, that they have given the impression of being real accompaniments of faster clotting. It may be that two factors are operating, one tending to hasten, the other to retard the process, and that the equilibrium disturbed by adrenin is recovered only after interaction to and fro between the two factors.

The oscillations in coagulation time after the injections suggest that clotting might vary with changes in blood pressure, for that also commonly oscillates after a dose of adrenin (see, e. g., Fig. 23). Simultaneous recording of blood pressure and determining of coagulation time have revealed that each may vary without corresponding variation in the other. Within ordinary limits, therefore, changes of blood pressure do not change the rate of clotting.

The Hastening of Coagulation by Adrenin Not a Direct Effect on the Blood

As previously stated, von den Velden has contended that shortening of coagulation time by adrenin is due to exudation of tissue juices resulting from vasoconstriction. The amount of adrenin which produces markedly faster clotting in the cat, is approximately 0.001 milligram per kilo. As Lyman and I[12] showed, however, this amount when injected slowly, as in the present experiments, results in brief vasodilation rather than vasoconstriction. Von den Velden’s explanation can therefore not be applied to these experiments.

He has claimed, furthermore, that adrenin added to blood in vitro makes it clot more rapidly, but, as already noted, he gives no account of the conditions of his experiments and no figures. It is impossible, therefore, to criticise them. His claim, however, is contrary to Wiggers’s[13] earlier observations that blood with added adrenin coagulated no more quickly than blood with an equal amount of added physiological salt solution. Also contrary to this claim are the following two experiments: (1) Ligatures were tied around the aorta and inferior vena cava immediately above the diaphragm, and thus the circulation was confined almost completely to the anterior part of the animal. Indeed, since the posterior part ceases to function in the absence of blood supply, the preparation may be called an “anterior animal.” When such a preparation was made and 0.5 cubic centimeter of adrenin, 1:100,000 (half the usual dose, because, roughly, half an animal), was injected slowly into one of the jugulars, coagulation was not shortened. Whereas for a half-hour before the injection the clotting time averaged 4.6 minutes, for an hour thereafter the average was 5.3 minutes—a prolongation which may have been due, not to any influence of adrenin, but to failure of the blood to circulate through the intestines and liver.[14] In another experiment after the gastro-intestinal canal and liver had been removed from the animal, the average time for coagulation during twenty-five minutes before injecting adrenin (0.23 cubic centimeter, 1:100,000, in an animal weighing originally 2.3 kilos) was 5.5 minutes, and during forty minutes after the injection it was 6.8 minutes, with no case shorter than 6 minutes. In the absence of circulation through the abdominal viscera, therefore, adrenin fails to shorten the clotting time. (2) The cannulas were filled with adrenin, 1:1,000, and emptied just before being introduced into the artery. The small amount of adrenin left on the walls was thus automatically mixed with the drawn blood. Alternate observations with these cannulas wet by adrenin and with the usual dry cannulas showed no noteworthy distinction.

The results of these experiments have made it impossible for us to concede either of von den Velden’s claims, i. e., that clotting occurs faster because adrenin is added to the blood, or because adrenin by producing vasoconstriction causes tissues to exude coagulant juices.

Vosburgh and Richards found that coagulation became more rapid as the blood sugar increased. Conceivably faster clotting might result from this higher percentage of blood sugar. Against this assumption, however, is the fact that clotting is greatly accelerated by 0.001 milligram adrenin per kilo of body weight, much less than the dose necessary to increase the sugar content of the blood.[15] And furthermore, when dextrose (3 cubic centimeters of a 10 per cent solution) is added to the blood of an anterior animal, making the blood sugar roughly 0.3 per cent, the coagulation time is not markedly reduced. Adrenin appears to act, therefore, in some other way than by increasing blood sugar.

Since adrenin makes the blood clot much faster than normally in the intact animal, and fails to have this effect when the circulation is confined to the anterior animal, the inference is justified that in the small doses here employed adrenin produces its remarkable effects, not directly on the blood itself, not through change in the extensive neuro-muscular, bony, or surface tissues of the body, but through some organ in the abdomen.

That exclusion of the liver from the bodily economy, by ligature of its vessels or by phosphorus poisoning, will result in great lengthening of the coagulation time has been clearly shown. The liver, therefore, seems to furnish continuously to the blood a factor in the clotting process which is being continuously destroyed in the body. It is not unlikely that adrenin makes the blood clot more rapidly by stimulating the liver to discharge this factor in greater abundance. But proof for this suggestion has not yet been established.

REFERENCES