diazo-bis-8-amino-1-naphthol-5-7-disulphonic acid: the sample used contained

Transcription

1 377 J. Physiol. (I937) 90, I2. I I5.3 THE ACTION OF ANTICOAGULANTS BY J. 0. WAKELIN BARRATT From the Department of Bacteriology, Serology and Experimental Pathology, Lister Institute of Preventive Medicine (Received 23 April 1937) IN the present paper an attempt is made to present anticoagulant action in a quantitative aspect. METHOD The anticoagulants (1) dealt with are NaCI, chlorazol sky blue and Chlorazol sky blue is the sodium salt of dimethoxy-diphenyl- heparin. diazo-bis-8-amino-1-naphthol-5-7-disulphonic acid: the sample used contained 78-6 p.c. of dye, 11-7 p.c. of sodium chloride and 10-5 p.c. water. Ordinary commercial heparin was employed, the active principle of which is chondroitin-trisulphuric acid [Jorpes, 1935]. For use in experiment chlorazol and heparin were dissolved, in suitable concentrations, in 0-85 p.c. solution of sodium chloride. As coagulant system citrated blood plasma (2) and a solution of thrombin in the form of the venom of Echis carinatus (3) [Barratt, 1913] were employed. The former was obtained by adding to human blood, immediately after withdrawal from the median basilic vein into a paraffined glass syringe, an amount of sterile p.c. solution of trisodium citrate sufficient to produce a concentration of 0 3 p.c. in the volume of blood removed. The venom employed was dissolved in 0-85 p.c. NaCl solution in a concentration of 1 in 160,000. Anticoagulant action was investigated by noting the increase of coagulation period following addition of the anticoagulant (1) to the coagulant system (2 and 3), experiments being made by mixing together: (1) 0x2 ml. of the anticoagulant in suitable concentration; (2) 0-2 ml. of plasma; and (3) 0-2 ml. of thrombin solution, proper admixture being immediately obtained by shaking. Control experiments were made by

2 378 J. 0. W. BARRATT using 0-85 p.c. NaCL solution or distilled water in place of (1). No difference in respect of the coagulation period was observed if the anticoagulant were mixed first with plasma and then thrombin added, or if anticoagulant and thrombin were mixed and then plasma added; nor was any marked alteration of the coagulation period obtainable by allowing the anticoagulant to act upon one or other reagent in the coagulant system for periods up to 30 min. or more before adding the remaining reagent. When admixture was judged to be complete a drop of the mixture of (1), (2) and (3) was placed on a glass slide, ringed with vaseline and a coverslip applied. The preparation was then observed under the microscope, with darkground illumination and the coagulation time taken as the period elapsing between admixture and the first appearance of visible fibrils of fibrin. This was determined without difficulty for periods up to about 100 min. With somewhat longer periods it is difficult, owing to the extreme fineness and very scanty number of the fibrils observed, to ascertain the exact time of visible fibril formation, and for periods exceeding 3 hours very accurate determinations cannot be made. The onset of "setting" generally preceded the first appearance of visible fibrils for periods up to about 30 mi. for longer periods " setting " (as also sluggishness of movement on slightly tilting the containing tube) appears later than visible fibril formation. When with further addition of anticoagulant visible fibrils become difficult to recognize, soft "setting", later becoming firm, is observed. Even when visible fibrils cease to be formed "setting", soft or firm, still occurs but, if sufficient anticoagulant is added, "setting" ceases to be recognizable. All experiments were carried out at room temperature (16-18 C.). RESULTS The nature of the action of the anticoagulants employed. In all experiments citrated plasma was used. There were, therefore, only two substances upon which the anticoagulants could act in delaying coagulation, namely fibrinogen, present in the blood plasma employed, and thrombin (snake venom): owing to the addition of sodium citrate any possible action of anticoagulant upon prothrombin or thrombokinase would be without effect upon coagulation. It is therefore to be expected that the action of the anticoagulant would be to diminish the concentration of fibrinogen or of thrombin and would thus be equivalent to the effect of dilution of one or other of these coagulant substances. Now

3 ACTION OF ANTICOAGULANTS simple dilution of either thrombin or of fibrinogen causes retardation of coagulation, but while dilution of thrombin (the concentration of fibrinogen remaining unchanged) causes diminution of the number of fibrin fibrils, which at the same time increase in length and thickness, dilution of fibrinogen, on the contrary (the concentration of thrombin remaining unchanged), results in the fibrils becoming very fine and soon ceasing to be visible, though " setting " still occurs: in both cases, as the coagulation period is prolonged, the clot becomes increasingly soft [Barratt, 1920]. The addition of NaCl, chlorazol and heparin causes, in addition to delay of onset of coagulation, diminution in number and increase in length of fibrin fibrils: thinning of the fibrils does not occur. This is exhibited in Exps. 1-13, Table I. It follows, therefore, that the action of these anti- TABLE I. Experiments exhibiting the anticoagulant action of varying concentrations (x) of NaCl, chlorazol and heparin upon a mixture of citrated plasma (0-32 ml. of plasma being contained in 1 ml. of the coagulant mixture) and venom (1 in 480,000). x is given in g. per ml. of the mixture. The observed coagulation times (t) are given in minutes: the figures in brackets represent calculated coagulation times Anti- x t Exp. coagulant g. per ml. min. Fibrin fibrils 1 NaCl [140] Scanty, fine, ,u long [29.6] [6.5] Numerous, fine, 25-50,u long [5.5] 5 Chlorazol [70] Moderatelynumerous, fine, , long [20] [10-2] [6.5] Numerous, fine, 25-50, long 9-40 [55] 10 Heparin [161] Moderately numerous, fine, /L long [73] [6.5] Numerous, fine, 25-50,t long [5 5] coagulants is directed to thrombin and not to fibrinogen. The anticoagulant apparently enters into combination with part of the thrombin added, the remainder being free. The combination, if it occurs, resembles a dissociable combination, for, although the greater part of the thrombin disappears, when relatively large amounts of anticoagulant are added, nevertheless, a small fraction still remains uncombined: thus a simple stoichiometric relation between the anticoagulant added and the amount of thrombin disappearing is not observed. 379 The experiments given in Table I are exhibited graphically in Fig. 1, in which the abscissa represent concentration of anticoagulant and the

4 380 J. 0. W. BARRATT ordinates coagulation time. It will be noted that there is a general similarity between the three curves, which would be still closer if the scale of abscissse for the two curves to the left were suitably increased. The action of the anticoagulant in all three curves is obviously similar in character ~00 o o.o0 0*b2 0:03 Anticoagulant: g. per ml. Fig. 1. The curve to the right exhibits the anticoagulant action ofnacl (Exps. 1-4, Table I); the middle curve that of chlorazol (Exps. 5-9); and the curve to the left heparin (Exps ). When NaCl is used as anticoagulant the effect of increasing concentration is to reduce the number of fibrils appearing until at length fibrils are difficult to find, only one being present in several fields of the microscope, while at the same time the length of the fibrils undergoes considerable increase, extending over one-third to half the diameter of the field. It has not, however, been found possible under the conditions of experiment followed to obtain a considerable increase in thickness of fibrils, such as is observed with simple dilution of thrombin [Barratt, 1920]. At concentrations of NaCl exceeding 0*0400 g. per ml. of the coagulant mixture, with the concentration of plasma and venom given in Table I, visible

5 ACTION OF ANTICOAGULANTS fibrils cease to appear and at somewhat higher concentrations "setting" also ceases. The clot in Exps. 3 and 4. Table I, was firm, exhibited marked retraction, and serum was readily expressed on pressure with a glass rod: in Exp. 1, the clot was much less firm and was very friable, no retraction occurring and serum not being expressible. When chlorazol and heparin are used as anticoagulants the same diminution in number and increase in length of fibrin fibrils is observed as with NaCl, though the conditions under which observations are made are not so favourable owing to the deep colour of the dye and the presence of very fine granules when heparin is employed. Each of the three anticoagulants studied may therefore be regarded as an antithrombin. It is of interest to note that Huggett & Silman [1932] state that chlorazol sky blue FF (Chicago blue) "is an antithrombokinase". "It had no effect, however, on the action of thrombase itself." Later Hugget t [1934] observed that "thrombase was found to be inhibited by chlorazol sky blue FFS" as was also "the action of thrombokinase (with calcium ions) in activating prothrombase to thrombase". Howell & Holt [1918] state that "heparin inhibits clotting mainly by preventing the activation of prothrombin to thrombin. It acts as an antiprothrombin rather than as an antithrombin". In other words heparin produces its anticoagulant effect "by a reaction with prothrombin"; it does not prevent clotting when added to mixtures of thrombin and fibrinogen. Mellanby [1935] finds that "heparin prevents the coagulation of oxalated plasma by thrombase. This action depends upon the neutral salt content of the plasma since heparin does not inhibit the coagulation of dialysed oxalate plasma by thrombase". Measurenent of anticoagulant action An attempt was now made to measure the degree of anticoagulant action exhibited by the three anticoagulants studied. Consideration will first be given to the simpler case of NaCl. When chlorazol and heparin are used as anticoagulants, NaCl being also present (in a concentration of 0*0105 g. per ml. in all experiments except 9 and 13 where the concentration is g. per ml.), a double anticoagulant effect has to be investigated. Any equation representing the NaCl curve in Fig. 1 must fulfil two conditions. The first condition is that the coagulation time (t) increases with increase of anticoagulant (x). A glance at Table I, however, shows that t is 381

6 382 J. 0. W. BARRATT not simply proportional to x, but is represented by a function of x which increases much more rapidly than does x itself. A simple function of this type would be xp where p is a constant greater than 1. Furthermore, as x increases, t also increases, but no matter how much x is increased, t does not reach a maximum limit, in other words thrombin (y), although it becomes less and less with increase of x, does not disappear. It has been pointed out above that t is dependent not upon the concentration of thrombin added, but upon the amount of thrombin remaining free in presence of the anticoagulant. Thus, if in Exps. 1-4, Table I, the concentration of NaCl were g. per ml. in all experiments, the concentration of thrombin which would give the values of t obtaining in Exps. 1, 2 and 3 would be approximately one-hundredth, one-tenth and two-thirds respectively of the amount of thrombin added: these values representing approximately the concentrations to which y has been reduced by the action of the anticoagulant. The second condition, which must be fulfilled by any equation representing the NaCl curve, is that as the anticoagulant (x) diminishes in concentration the coagulation time (t) also diminishes, but it does not become zero, reaching a minimum depending upon the concentration of thrombin (y) added, the minimum value of t becoming greater as the concentration of thrombin added in each series of experiments is diminished, so that there is a separate curve for each concentration of thrombin employed. It may be pointed out that the concentration of NaCl cannot be reduced to zero, fibrinogen being insoluble in water: nevertheless, the point at which further diminution of concentration of NaCl ceases to cause any appreciable shortening of the coagulation time can be determined graphically or calculated as described below. The relation between concentration of thrombin (y) and the corresponding coagulation time (t), the concentration of fibrinogen being the same in all experiments, has been found [B a rra t t, 1934] to be represented by t= B t1.43~< (1 t-(-) 7or t@... 3 where B is a constant. When NaCl is allowed to act upon thrombin it is found experimentally that when the amount of thrombin added is represented by y the amount remaining free is y (Ax)"', where eexrpeetx represents the h concentration ocnrto offnc NaCl and A and p are constants. Substituting this value in equation (1) we have t*43_=y [I + (AX)P....(2)

7 ACTION OF ANTICOAGULANTS 383 If the amount of thrombin added (y) is the same in all experiments equation (2) becomes tl43 = C [1+ (Ax)P],... (3) where C is a constant. Many series of experiments of the type indicated in Exps. 1-4, Table I, showed that, using equation (3), a fair agreement was met with between observed and calculated coagulation times for periods not exceeding 100 min. Above this limit it is, as already mentioned, difficult, owing to the fineness and diminished number of the fibrils appearing, to determine with accuracy the time at which visible fibrils may be regarded as making their appearance. Even so, however, experiments made above this limit do not appear to afford any ground for contesting the validity of equation (3). The values of p obtained in different series of experiments were in the neighbourhood of 6X0. The values of A were usually not far removed from 80. The value of C depends upon the concentration of thrombin added. The actual values of p, A and C for Exps. 1-4, Table I, were 5 9, 76-7 and 11 0 respectively; the calculated values of t are given in brackets. When chlorazol and heparin are used as anticoagulants NaCl is also of necessity present. Thus in Exps. 5-13, Table I, NaCl was present to the extent of 0*0105 g. per ml., except in Exps. 9 and 13, where the amount was 0*0077 g. per ml. In these experiments it seemed likely, judging from the appearance of the curves in Fig. 1, that for these two anticoagulants an equation similar to equation (3) would also be applicable. The simplest assumption to make would be that the amounts (x') of chlorazol and heparin added were equivalent in their anticoagulant effect to proportional amounts (ax', where a is a constant) of NaCl, in which case equation (3) would become t143 = C [1 + AP (ax'+ x)p].... (4) If x were negligible compared with ax', as would be the case with the higher concentrations of anticoagulant, equation (4) becomes tl43=-c [1+ (Aax')P].... (5) Putting Aa =A' this assumes the form of equation (3). The applicability of equation (4) was tested in numerous series of experiments and a fair agreement between calculated and observed values of t obtained. This is illustrated in Exps. 5-9 and in Table I. In these experiments the values of p, A and C remained the same as in Exps. 1-4, and the values of a for chlorazol and heparin were 7*1 and 11-3

8 384 J. 0. W. BARRATT TABLE II. Constants employed in calculating values of coagulation time (t) Exp. Anticoagulant p A C a 1-4 NaCl Chlorazol Heparin respectively (cp. Table II). The calculated values of t are given in brackets. It would, therefore, appear that equations (3) and (4) are applicable to the three anticoagulants tested. The constants A and Aa represent the anticoagulant power of NaCl (76.7) and of chlorazol (545) and heparin (867), the relative values being 1, 741 and 113 respectively. The values of A and a for NaCl and chlorazol are probably approximately correct but, since the degree of purity of the sample of heparin employed is unknown, the value of a given for this anticoagulant in Table II is doubtful: all that can be asserted is that the anticoagulant power of heparin is greater than that of chlorazol. SUMMARY 1. The action of the anticoagulants NaCl, chlorazol and heparin in delaying the coagulation of a mixture of fibrinogen and thrombin is exerted upon thrombin: no evidence of any action upon fibrinogen was observed. 2. An equation is given by means of which the coagulation time in presence of varying amounts of anticoagulant can be calculated. 3. Anticoagulant power is indicated in the equation by a constant. REFERENCES Barratt, J. 0. W. (1913). Proc. Roy. Soc. B, 87, 177. Barratt, J. 0. W. (1920). Biochem. J. 14, 189. Barratt, J. 0. W. (1934). J. Phy8iol. 80, 422. Howell, W. H. & Holt, E. (1918). Amer. J. Physiol. 47, 328. Huggett, A. St G. & Silman, H. (1932). J. Physiol. 74, 9P. Huggett, A. St G. (1934). Ibid. 82, 21P. Jorpes, E. (1935). Biochem. J. 29, Mellanby, J. (1935). Proc. Roy. Soc. B, 116, 1.