Pathology, Lister Institute of Preventive Medicine.)

Transcription

1 422 6I2. II5 ESTIMATION OF FIBRINOGEN AND THROMBIN. BY J. 0. WAKELIN BARRATT. (From the Department of Bacteriology, Serology and Experimental Pathology, Lister Institute of Preventive Medicine.) (Received November 3, 1933.) THE objective of the experiments about to be described is the estimation of fibrinogen and thrombin. The former is estimated in terms of the amount commonly present in human blood plasma to which p.c. of trisodium citrate has been added: this amount will be shown later to be remarkably constant. The source of thrombin is the venom of Echis carinatus, a small Indian viper; the unit of concentration of thrombin is taken as that concentration which will completely convert into fibrin the fibrinogen contained in normal human plasma. The methods employed for estimation are based upon the effect on fibril formation of diminution of one or other component of the coagulant mixture [Barratt, 1915]. The term "coagulation time" is used in this investigation to signify the time of onset of coagulation indicated by the first appearance, under dark-ground illumination, of fibrils of fibrin. For dilution of plasma or venom 0*85 p.c. NaCl solution was employed. The temperature of experiment was 16-18' C. In the observations given in Tables II and III equal parts of citrated plasma and venom solution were taken, in all other observations the proportion was nine of the former to one of the latter. The actual concentration of plasma and venom present in the mixed liquid in each observation is given in the tables in the second and third columns respectively. Estimation offibrinogen. If equal amounts of thrombin are added to a series of increasing dilutions of fibrinogen, contained in citrated human plasma, the coagulation time is found to increase with each successive dilution, as is ilustrated by the experiment set forth in Table I and by the curve to the

2 ESTIMATION OF FIBRINOGEN AND THROMBIN. 423 TABLE I. Observation The effect of dilution of fibrinogen upon coagulation time (cp. Table IV). Concentrations used for coagulation Plasma (z) p.c. Venom (y) 88 1 in 640, ,, 91 22,. 11,... Coagulation time (t) in min. Calculated Observed for t=5-15/z * * Fig. 1. The effect upon coagulation time of dilution of fibrinogen is shown in the curve to the left (Table I) and of thrombin to the right (Table IV). Scale: abscissie, periods of 100 min.; ordinates, concentration of plasma, 39 p.c. (curve to left), or venom, 1 in 1,540,000 (curve to right). Obs. 1-7, Tables I and IV, form a single series. left in Fig. 1. In this and other experiments the relation between coagulation time (t) and relative concentration of fibrinogen, i.e. percentage of plasma (z), is given by the equation a zp )... (1) where a is a constant varying in individual experiments and having the value 5-15 for that given in Table I. The constant p lies within a limited range of values: its average value in numerous experiments was 0-5. Relative values of the content of fibrinogen in different solutions of fibrinogen may be readily obtained by equation (1) from the coagulation

3 424 J. 0. W. BARRATT. times observed when the same amount of thrombin has been added to each solution. If the concentration of fibrinogen is very low some difficulty may, however, be experienced in ascertaining the exact moment at which fibrils first appear, the thickness of the fibrils becoming exceedingly small so as to render rapid recognition impossible. In a dilution containing 075 p.c. of normal human plasma fibrils cease to appear and "setting" is difficult to detect or no longer recognizable (Table II). The value of p, it may be noted, was found to range in different experiments between 035 and Another method of estimation which avoids the difficulty of exact determination of coagulation time in high dilutions of fibrinogen, when fibrils are of liminal visibility, is the following, based upon ascertainment of the point at which visible fibril formation ceases. A series of dilutions of the liquid whose content of fibrinogen is to be determined is made, and to each the same amount of thrombin is added, a sample of each being then placed in a glass cell, sealed with vaseline, for examination under TABLE II. The dilution of plasma at which addition of thrombin fails to give rise to visible fibrils of fibrin. Concentrations used Description of for coagulation plasma Condition of clot Observab Plasma A, Age tion p.c. Venom Consistence Fibrils Sample (days) 1 0*75 1 in 320,000 Very soft Absent a 0 2,, I in 1,280,000 Soft,, b 0 3, 1in 320,000 Very soft,, c 1 4,, 1 in 1,280,000 Remained,, d 1 liquid 5,, 1in 320,000 Soft,, a 7 6,, 1 in 5,120,000 Very soft,, b 43 dark-ground illumination at the end of 24 hours. The dilution at which visible fibrils cease to be formed is in this way ascertained. By a comparison of determinations thus made for different solutions, an estimate of the corresponding relative concentrations of fibrinogen is obtained. It will be noted that, in this method, only the presence or absence of fibrils is noted, no attempt being made to determine the time at which fibrils become visible. The dilution at which visible fibril formation ceases corresponds, as already mentioned, to a concentration of normal human plasma of 0-75 p.c. By the use of these two methods relative estimations of fibrinogen may be made. In order to obtain absolute determinations a standard value of fibrinogen is required. This cannot be given in terms of a definite amount of fibrinogen owing to the circumstance that it is not at present

4 ESTIMATION OF FIBRINOGEN AND THROMBIN. 425 possible to obtain fibrinogen in a state of purity or even in a stable form, but a natural unit of concentration is furnished by human blood plasma, which in health appears to exhibit very small variation of its content of fibrinogen. The limited range of variaton is shown by the appearance of nearly identical coagulation times when simultaneous determinations are TABLE III. Experiment illustrating the extent of variation of content of fibrinogen in human plasma. Concentrations used for coagulation Obserra- Coagulation Source Observa- Plasma time of tion p.c. Venom (min.) plasma in 1,767, A 2,,,,,, 15*0 B 3,, 1 in 2,580,000 19*5 A 4,,,,,, 19-5 B 5,, 1 in 394, A (1) 6,,,,,, 53 A (14) 7,,,,,, 5*2 (66) 8,,,,,, 5-3 A (98) 9,,,,,, 5*5 (182) 10 Pt,,,, 5-2 A (198) made with citrated plasma from different individuals, equal amounts of thrombin being added in each case. The same is indicated still more strikingly when comparative tests are made with citrated plasma obtained at different times from the same individual, such determinations being rendered possible by the use of venom, deterioration of which at 0 C. is very slow [Barratt, 1932]. Illustrative observations are shown in Table III. In the first four, two samples of citrated plasma obtained from different individuals are found to have identical coagulation times when tested shortly after collection (1 and 2) and again 2 days later (3 and 4). The next six observations (5-10) are made with samples of human plasma from the same subject, taken at intervals (given by the figures in brackets in column 5) during a period of 198 days. Since the coagulation time is practically identical in these experiments, it follows that neither the content of thrombin in the solution of venom employed, which was made up two years before the first experiment, nor the content of fibrinogen in the samples of plasma, varied in any marked degree during the period of experiment. The plasma in Obs. 5-10, it may be observed, was examined shortly after collection of blood except in Obs. 10, in which it was 24 hours old when used. The content of fibrinogen in normal human plasma thus furnishes a standard, which may be taken to represent unit concentration. Estimations in terms of this unit may be made in liquids containing fibrinogen by either of the two methods given above. PH. Lxxx. 28

5 426 J. 0. W. BARRATT. Estimation of thrombin. If thrombin is added to plasma in diminishing amounts, coagulation takes place with increasing slowness. The relation between the time required for "setting" to occur (t) and the amount of thrombin added (y) was found by Fuld [1902] to be given by the equation log log 2... (2) t2 i Martin [1905], using snake venom, observed the relation ty=constant... (3) when the quantity of thrombin added was small: with large amounts of thrombin the observed values of t were greater than the calculated periods. Rettger [1909] also noted that equation (3) failed if a maximum amount of thrombin was employed, further increase of thrombin no longer causing a diminution of the time of coagulation. Similar results were obtained by Barratt [1915]. If, however, the plasma employed is citrated and the coagulation period taken as the time required for the formation of visible fibrils of fibrin (this being capable of more exact measurement than the occurrence of "setting") the relation b t yq...(4) is obtained, where b is constant only for individual experiments, while q varies within narrow limits, ranging in different experiments between 0-65 and 0*75. This is illustrated by the experiment detailed in Table IV TABLE IV. The effect of dilution of thrombin upon coagulation time (op. Table I). Concentrations used for Coagulation time (t) coagulation in min. Observa- Plasma (z) Calculated for tion p.c. Venom (y) Observed q= /yo in 640, ,, 1 in 5,760, ,, 1 in 51,840, ,, 1 in 155,520, and shown in the curve to the right in Fig. 1. In this experiment the constant b has the value , and the exponent q is taken at 0 7, the coagulation times calculated in the fifth column agreeing, it will be seen, fairly closely with the observed periods. Experiments of this type afford a valuable method of making comparative estimations of thrombin in venom dilutions.

6 ESTIMATION OF FIBRINOGEN AND THROMBIN. 427 Absolute estimations may also be made. These are based upon the relation observed between the amounts of fibrinogen and thrombin taking part in the production of fibrin. If thrombin is added to citrated plasma it is usually found that the fluid expressed from the resulting clot at the end of several days, when the interaction between fibrinogen and thrombin may be regarded as complete, contains thrombin if a relatively large amount of thrombin has been added, while, on the other hand, fibrinogen is present if only a relatively small amount of thrombin is used. This is shown by the addition of citrated plasma to the expressed fluid in the first case, and of venom in the second, when clotting occurs. If, however, fibrinogen and thrombin are mixed in suitable proportions, the expressed fluid is found to be free from either, that is to say, the further addition of plasma or thrombin no longer causes clotting. This is illustrated in Table V: in Obs. 1, 2 and 3 fibrinogen is absent from the expressed fluid, TABLE V. Determination of the amount of thrombin required to convert fibrinogen into fibrin. Concentrations used for Content of fluid excoagulation Approximate pressed from clot A A ratio of Observa- Plasma fibrinogen to Fibrinotion p.c. Venom thrombin gen Thrombin in 21,500,000 50,000: in 41,000, ,000: in 71,000, ,000: in 144,000, ,000: in 144,000,000 72,000: in 331,000, ,000: in 568,000, ,000: in 1,135,000, ,000: 1 + while in Obs. 4 it is present, the converse being true of thrombin. If it is assumed that fibrinogen and thrombin are in corresponding amounts midway between Obs. 3 and 4, the relation between fibrinogen and thrombin would be in the neighbourhood of 384,000: 1, the plasma employed being assumed to contain 0 5 p.c. of fibrinogen and venom being regarded as pure thrombin. This proportionality, which does not seem to be altered by dilution-in Obs. 5-8, in which the concentrations used are about one-eighth of those in Obs. 1-4, a similar relation is indicated-renders possible the estimation of thrombin in terms of a unit, for it is obvious that if normal human plasma is taken as representing unit concentration of fibrinogen, then the concentration of thrombin just sufficient to convert all the fibrinogen present in the plasma into fibrin must be taken to represent unit concentration of thrombin. It is, however, preferable to indicate this concentration by the corresponding coagulation time rather 28-2

7 428 J. 0. W. BARRATT. than to attempt to express it in terms of thrombin. Experiments made to determine this period-that is to say, the coagulation time when thrombin is added in amount just sufficient to convert all the fibrinogen present. in undiluted normal human plasma into fibrin-were found to give a mean value of approximately 135 mi., corresponding to a 1 in 77,500,000 concentration of the sample of venom used in Obs. 1-7, Tables I and IV. Using this value of t for unit concentration, the following illustration of the determination of titre of a solution of thrombin may be given. In Obs. 1, Table I, the observed value of t for 88 p.c. plasma is 5 min. The value of t for undiluted plasma is by equation 1, p. 423, 5 X min. The concentration of thrombin employed in Table I is, therefore, by equation 4, p. 426, (7) = 121 times unit concentration. SUMMARY. 1. Methods of estimation of fibria and thrombin, based upon the observed effect of dilution, are described. 2. As units the following are emplbyed: (a) Unit concentration of fibrinogen: the concentration present in normal human plasma. (b) Unit concentration of thrombin: the concentration which is just sufficient to convert all the fibrinogen present in normal human plasma into fibrin. REFERENCES. Barratt, J. 0. W. (1915). Biochem. J. 9, 511. Barratt, J. 0. W. (1932). J. Phy8iol. 75, 428. Fuld, E. (1902). Beitr. chem. Phy8iol. 2, 514. Martin, C. J. (1905). J. Physiol. 32, 207. Rettger, L. J. (1909). Amer. J. Phy8iol. 24, 406.