STUDIES

I. THE

ON THE CHEMISTRY
MEASUREMENT CLOTTING. CHARGAFF, MARGARET

OF BLOOD

COAGULATION
OF BLOOD

OF THE METHODS

INHIBITION AND UNITS*

BY

ERWIN

FREDERIC W. BANCROFT, STANLEY-BROWN

AND

(From

the Departments of Biological Chemistry and Surgery, College Physicians and Surgeona, Columbia University, New York)

of

Downloaded from www.jbc.org by guest, on January 14, 2010

(Received

for publication,

May 25, 1936)

Progress in the study of heparin and synthetic anticoagulants is conditional upon the availability of a simple method for the estimation of the activity of substances which inhibit blood clotting. Workers in this field have not as yet agreed upon one method, so that many of the results reported in the literature are subject to considerable uncertainty. The method chiefly employed is that of Howell (1). According to this procedure 1 heparin unit is the amount of heparin which in the cold prevents 1 cc. of cat blood from clotting in 24 hours. This method has also been used by Charles and Scott (2) in their important work on the heparin content of various organs and on the purification of heparin. Fischer and Schmitz (3), and Schmitz (4), employing chicken plasma, use a coagulation value which is derived from a logarithmic function of the clotting times at 40 at various dilutions of heparin. Jorpes (5) in his work on heparin makes use of ox blood, the coagulation of which is determined at room temperature. From an inspection of the data on heparin activity before and after purification, given in the papers mentioned above, it will be apparent that the methods described can by no means form the basis of a comparative investigation of various anticoagulants. We have tried to develop a simple method for the determination of inhibiting substances and to define an inhibitor unit which,
* Study of the mechanism of thrombosis the Carnegie Corporation of New York. 149 and embolism supported by

150

Chemistry
limit

of Blood Coagulation.
of error, will be constant

I
for a given sub-

within a certain stance.

EXPERIMENTAL

In the experiments here described chicken plasma was used. This, in contrast to that of almost all other animals, can easily be so secured that it will not clot for a relatively long period of time. On the other hand, by the addition of varying amounts of muscle extract any desired clotting time can be attained. Examination of the conditions under which inhibitor activity could be estimated indicated that success depended on the following conditions. (1) The reaction volume must be kept constant, as the numerous substances contained in plasma respond to dilution in different ways. The addition to a series of plasma samples, for instance, of increasing amounts of an inhibitor solution will lead to discordant results. (2) Extreme care must be taken to disturb the plasma as little as possible. Attempts to increase the precision of the end-point may adversely affect its accuracy Plasma which coagulates in the presence of an inhibitor in general gives rise to clots that are very soft and may easily be broken up beyond recognition, if shaken before their formation is complete (3) The period of time over which the determination of inhibitor activity takes place should not be too extended, as the clotting properties of plasma may change quite considerably with time Method Preparation of Plamna--The blood used in our experiments is obtained in the usual manner from the carotid artery of 2 year-old roosters, about 50 cc. being secured per animal. All glass parts with which the blood comes in contact are parafhned. The blood is collected in four fractions, the first and the last of which may clot faster. The chilled glass cups are immediately centrifuged for 20 minutes. The perin the angle centrifuge at 3000 R.P.M. fectly clear plasma is drawn off into parafhned tubes and kept in the refrigerator. In general, no plasma older than a week, nor any sample in which even a partial clot has formed, should be used Preparation of Activator-The activator is prepared according to the method described by Fischer (6) for his muscle coagulin. From 165 gm. of breast muscle of a bled rooster 5.4 gm. of an

Downloaded from www.jbc.org by guest, on January 14, 2010

Chargaff,

Bancroft,

and Stanley-Brown

151

extremely active, slightly yellow powder were obtained. The addition of as little as 0.1 microgram in 0.03 cc. of physiological saline to 0.1 UC. of plasma effected a drop in clotting time from 120 to 90 minutes at 30. DeJinition of Unit-We define the inhibitor unit as the smallest amount of inhibitor which will raise the clotting time of 0.1 cc. of plasma to 4 times its normal value under the experimental conditions described in the next paragraph.
TABLE I Estimation of Inhibitor Activity Each tube contains 0.03 cc. of inhibitor solution, 0.1 cc. of chicken plasma, and 0.02 cc. of activator solution. Experiment I, 1.6 mg. per cc. (= 1: 1) of Heparin R; Experiment II, 2.2 mg. per cc. (= 1: 1) of Jnhibitor C-L. + = clotted; f = not completely clotted; - = not clotted.

Downloaded from www.jbc.org by guest, on January 14, 2010

Tube

I nhibitor dilution I

Control I
I 8 min. +

Experiment I Clotting after

I 30 min.

Experiment II

--

7 min.

30 min. --

60 min.

180 min.

60 min.

180 min.

A B 1 2 3 4 5 6 7 8 II,

I + 1:l 1:2 1:4 1:8 1:16 1:32 164 1:128

Estimation: Experiment I, 1100 inhibitor 250 inhibitor units per mg.

units

per mg.;

Experiment

Measurewzent of Inhibition-The measurements are carried out in a water thermostat at 30 f 0.1. A number of metal stands are suspended in the thermostat in such a manner that they can be freely turned over. These stands, which are similar in design to those described by Fischer (7), carry small Pyrex tubes (10 X 75 mm.) tightly closed by a cover that is screwed to the stands. In each experiment a fresh strip of waxed paper is placed between the rubber lining of the cover and the tubes.

152

Chemistry

of Blood Coagulation.

A normal clotting time of between 6 and 10 minutes appeared to give the best results and was chosen in our experiments. In our experience, the addition of 0.02 cc. of an approximately 0.03 per cent solution of the activator to 0.1 cc. of plasma almost invariably led to a clotting time within this range. The actual concentration necessary has to be ascertained for each batch of plasma. Each tube contains a glass bead of 4 mm. diameter, 0.03 cc. of the solution of the inhibitor at various dilutions in physiological saline, and 0.1 cc. of plasma. After all the tubes have been filled, 0.02 cc. of the activator solution is added, the tubes are closed, inverted once, and suspended in the thermostat. The first reading is carried out after a period corresponding to 4 times the normal clotting value, which has been determined in a preliminary experiment with 0.03 cc. of saline instead of the inhibitor. The complete immobility of the glass bead is taken as the criterion of clotting. As a check on the results the readings are repeated after 8 and 12 times the normal clotting time. It sometimes occurs in the first reading that at a certain inhibitor dilution the tube contents are viscous but not completely coagulated. If at this dilution the sample is found to be clotted in the subsequent reading, the inhibitor activity (expressed in inhibitor units per mg.) is taken as 20 per cent lower than actually calculated (see Table I, Experiment 1). All determinations are carried out in duplicate. Two typical experiments are given in Table I.
DISCUSSION

Downloaded from www.jbc.org by guest, on January 14, 2010

It is obvious that the method here described can only have the accuracy of a biological test and not that of a quantitative chemical procedure. The chief reason is the fact that the end-point, namely the formation of a clot, is comparatively ill defined. It nevertheless seems preferable to determine this end-point rather than some other property related to the clotting in an unknown manner, e.g. the transmission of light through plasma. Attempts were made to increase the precision of the method by examining intermediate concentrations of the inhibitor. When in a given case the dilution 1: 4 had been found active and the dilution 1:s inactive, four intermediate dilutions were examined. Another method is to start from two solutions of the same in-

Chargaff,

Bancroft,

and Stanley-Brown

153

hibitor in which the relative concentrations are 2: 3 and to compare the activities in both dilution series. Thus, an intermediate value can be found which in general will be more accurate than the one found in a single series. It has proved useful to establish an inhibitor standard with which to control the freshly obtained plasma preparations in order to be independent of individual variations. In these experiments a heparin preparation from beef lungs was chosen, the activity of which had repeatedly been found to be between 800 and 1000 inhibitor units. 1 inhibitor unit determined by the method described here appears to correspond roughly to 0.07 Howell unit. The activity of two heparin samples which are on the market was estimated. A heparin preparation of 15 Howell units per mg. obtained from the Connaught Laboratories, Toronto, Canada, was found to have 220 inhibitor units per mg.; another sample of 5 Howell units per mg., obtained from Hynson, Westcott and Dunning, Inc., Baltimore, contained about 70 inhibitor units per mg. The accuracy of the method could be increased if it were possible to establish an inhibition curve. We have not been able, however, to determine such curves, contrary to the observations of Fischer and Schmitz (3). While it is perfectly easy to obtain a characteristic activation curve, the action of the inhibiting substance seems to have a different mechanism. A large number of experiments showed that there exists a minimum dose of the inhibitor which will keep the plasma liquid for many hours. Two typical experiments are given in Table I in which an inhibitor dilution of 1: 64 and 1:32 respectively did not prevent clotting within 30 minutes, whereas the samples containing the next higher concentration stayed liquid for 3 hours and more. Whether this phenomenon is connected with the softening effect exerted by heparin on the fibrin clots, as mentioned before, or whether we are dealing here with a special poisoning effect, we do not know. In the case of heparin the animal organism seems to be able to destroy its inhibiting properties in a comparatively short time. It may be different with some of the synthetic anticoagulants. It will be a matter of further work to determine whether this minimum dose coincides with the toxicity level of certain inhibitors. We are indebted to Mrs. Charlotte the course of these experiments. Breitung for assistance in

Downloaded from www.jbc.org by guest, on January 14, 2010

154

Chemistry

of Blood Coagulation.
SUMMARY

1. The conditions are discussed under which the inhibition effect of heparin and similar substances on plasma clotting can be measured. 2. A method is described for the estimation of inhibitor activity, and a definition is given of the inhibitor unit.
BIBLIOGRAPHY

1. Howell, W. H., Bull. Johns Hopkins Hosp., 42, 199 (1928). 2. Charles, A. F., and Scott, D. A., J. Biol. Chem., 102, 425, 431 (1933); Tr. Roy. Sot. Canada, sect. 5,28,55 (1934). Scott, D. A., and Charles, A. F., J. Biol. Chem., 102, 437 (1933). 3. Fischer, A., and Schmitz, A., 2. physiol. Chem., 210, 129 (1932). 4. Schmits, A., Z. physiol. Chem., 234, 216 (1935). 5. Jorpes, E., Biochem. J., 29, 1817 (1935). 6. Fischer, A., Biochem. Z., 249, 357 (1931). 7. Fischer, A., Arch. ges. Physiol., 226, 737 (1930).

Downloaded from www.jbc.org by guest, on January 14, 2010