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1 197 J. Physiol. (I958) I42, I THE COAGULATION OF HORSE BLOOD By P. FANTL AND A. G. MARR From the Baker Medical Research Institute, Alfred Hospital, Melbourne, Australia (Received 9 September 1957) Available information about the coagulation of horse blood is contradictory. Soulier & Larrieu (1953) found that horse blood contained clotting factors in concentrations not markedly different from those of human blood. However, Bell, Archer & Tomlin (1955) reported that in horse blood prothrombin and clotting times were prolonged and clot retraction was poor, in spite of platelet counts within normal human limits. They (Bell, Tomlin & Archer, 1955) found that the thromboplastin activity produced by horse blood is far less than that obtained from human blood, and concluded that the blood of normal horses of both sexes was relatively deficient in antihaemophilic factor. On the other hand, from results of thrombin generation tests, Sj0lin (1956) claims to have shown a relative lack of Christmas factor in normal horse plasma, and later he observed (Sj0lin, 1957) that the defective thrombin generation in horse plasma could be improved by either horse or human platelets and concluded that the slow clotting of horse plasma was due to a lack of Hageman factor. Some of the published results would thus indicate that normal horses are bleeders, but an inquiry among those who professionally observe horses during work or sport did not indicate that horses suffered from haemorrhage more than other animals when subjected to comparable trauma. In view of these apparent contradictions it was of interest to investigate the blood coagulation mechanism of the horse, and compare it with that of man. METHODS Blood was obtained from seven horses. One was normally used for the production of diphtheria antitoxin, four were draught horses and two were race horses. Their ages ranged from 2 to 17 years. According to veterinary examination the horses did not suffer from haemorrhagic tendencies or any other disabilities. Blood was obtained from the external jugular vein through a trocar, and was collected with the addition of 'th volume of 0-1 M sodium oxalate. Three horses were several miles from the laboratory and the specimens were transported in an ice box. The four draught horses were bled a few yards from the laboratory and the tests carried out immediately after collection. 'I3 PHYSIO. CXLII

2 198 P. FANTL AND A. G. MARR In view of the multiplicity of names for the various clotting factors their synonyms are given in brackets as follows: prothrobmin accelerator (labile factor, factor V, Ac-globulin, pro-accelerin); S.P.C.A. (proconvertin, stable factor, factor VII, cothromboplastin, autoprothrombin I); a-prothromboplastin (antihaemophilic factor, thromboplastinogen A, factor VIII, platelet cofactor I); fl-prothromboplastin (P.T.C., Christmas factor, thromboplastinogen B, factor IX, platelet cofactor II, autoprothrombin II). The whole blood clotting time was measured at 370 C in glass tubes using 1-5 ml. of blood according to the technique of Lee & White (1913). The thromboplastin generation test used is that of Biggs & Douglas (1953) as modified by Bell & Alton (1954), except that aluminium hydroxide-treated citrated plasma was replaced by oxalated plasma treated with barium sulphate (Ba-plasma). Phospholipids (P-lipids) were prepared from human, horse and sheep brain essentially according to Eagle (1935). The rate of formation and the yield of thrombin in freshly drawn horse blood were measured according to Fantl (1954 a, b). Platelets were counted by the method of Feissly & Ludin (1949). The activity of the a- and fi-prothromboplastins was determined in the partial thromboplastin test of Langdell, Warner & Brinkhous (1953). Blood samples deficient in a- or,b-prothromboplastin were taken from patients with a- and,b-haemophilia respectively and collected in a solution containing 0-104M trisodium citrate and 0-053M citric acid. The plasmas were stored at C until used. One-stage prothrombin tests were carried out: (a) with horse or human brain extracts according to Quick (1938); (b) with Russell viper venom and brain phospho-lipid similar to that described by Hobson & Witts (1941). Prothrombin assays were carried out according to Fantl (1954a). Prothrombin accelerator activity was determined in Ba-plasma with a prothrombin preparation isolated from stored human plasma as described by Fantl & Marr (1956). S.P.C.A. was measured with the method of de Vries, Alexander & Goldstein (1949). One of the thrombin preparations used was of bovine origin (Parke, Davis, 'Thrombin Topical') and others were prepared from horse and human euglobulins according to Fantl & Ebbels (1953). But in the case of horse plasma only 15vol M citric acid was required for maximum precipitation of the euglobulins. The determination of thrombin activity and the relationship between clotting time and thrombin units are given by Fantl (1954 a). Antithrombic activity of serum was measured by the rate of disappearance of thrombin. Fibrinogen was determined as fibrin N (micro-kjeldahl) converted by a factor of 5-92 (Fantl & Marr, 1956). RESULTS Thromboplastin generation tests The formation of thromboplastin from Ba-plasma and serum obtained from both horse and human blood, and phospholipid and calcium ions, was measured with the thromboplastin generation test. Results of six independent experiments are given in Table 1. It is apparent that the maximum thromboplastin activity was obtained in each case after the same incubation time. Both human and horse thromboplastin gave similar clotting times with human plasma; however, longer clotting times were obtained with the horse plasma substrate. The thromboplastin

3 THE COAGULATION OF HORSE BLOOD 199 generation tests were also carried out with modifications of the substrate. These included variations of the calcium ion concentration between 0-02 and 0-03M, dilution of substrate plasma with 0415M-NaCl solution and with defibrinated human or horse plasma, and the use of the euglobulin fraction of horse and human plasma. The results, however, were similar to those given in Table 1. Horse and human blood components showed the same rate of thromboplastin formation, but thromboplastin action appeared to be delayed in horse plasma when compared with that of human plasma. TABLE 1. A comparison of thromboplastin formation with human and horse blood components* Incubation Shortest clotting time with time for maximum substrate plasma of thromboplastin A Ba-plasma activity Man Horse and serum of (min) (sec) (sec) Man 4 (3-5) 11 (10-12) 17 (16-19) Horse 3-5 (2-5) 12 (11-13) 18 (17-20) * The figures are average results, variations are given in parentheses. TABLE 2. The rate of formation and yield of thrombin in dilute horse blood* Incubation time for maximum thrombin Shortest clotting formation time Composition of reaction mixture (min) (sec) Whole blood 0 04 ml. plus: 0-15M-NaCl 0-1 ml. 24 (21-28) 16-5 (15-18) Human Ba-plasma ml. 22 (21-24) 16 (14-19) Human stored plasma ml. 20 (17-21) 15 (12-18) Brain P-lipid 36,ug 9 (7-10) 14 (13-16) * The figures are average results, variations are given in parentheses. Human Ba-plasma was used as the substrate. Thrombin formation in horse blood The rate of formation and yield of thrombin in freshly drawn horse blood were determined. Results are given in Table 2. From Table 2 it will be noticed that diluted horse blood produced a maximum amount of thrombin in 24 min with shortest clotting time of 16-5 sec. The comparative figures for human blood are min with a clotting time of sec. The horse blood therefore showed a delay in maximum thrombin formation. It should be added that if horse Ba-plasma is used instead of human Ba-plasma as substrate for this test a similar delay in the thrombin clotting time as has been noted before (Table 1) is observed. It will be further noticed that the addition to dilute horse blood of Ba-plasma as a source of ao-prothromboplastin or the addition of stored plasma as a source of f-prothromboplastin did not markedly reduce the incubation time, nor influence the thrombin yield. But the addition of phospholipids greatly reduced the incubation time required for maximum thrombin formation without affecting the thrombin yield. The horse blood specimens used for the experiments in Table 2 contained between 60,000 and 13-2

4 200 P. FANTL AND A. G. MARR 100,000 platelets/mm3 blood. From the results given in the two tables it is apparent that no deficiency of a plasma factor could be detected. However, the platelet counts were on the lower limits of normal, and therefore a delayed formation of thrombin was observed which was readily corrected by the addition of phospholipids as a substitute for platelets. TABLE 3. A comparison of the a- and,-prothromboplastin activities of human and horse plasmas in the partial thromboplastin test Clotting time (sec) obtained with Source of ml ml. a-prothromboplastin: Horse plasma Human plasma ,B-Prothromboplastin: Horse plasma Human plasma TABLE 4. One-stage prothrombin tests Clotting time (sec) with Russell viper Composition of Horse Human venom + reaction mixture (ml.) brain brain P-lipids Horse plasma* (12-19) Human plasma (12-18) 16 Horse plasma 0-08t horse serum 0-02 Human plasma horse serum 0*021 Horse plasma 0-08t human serum 0-02 Human plasma 0-08t human serum * All plasma and serum oxalated. The o- and /3-prothromboplastin activities of horse plasma The activity of ac- and of /3-prothromboplastin in plasma was determined by the correction of the deficiency of human plasma taken from patients with a complete deficiency in one or other of the two components. The results obtained with five different specimens are given in Table 3. From Table 3 it is apparent that the plasma of the investigated horses showed similar activity of both thromboplastin precursors to that of normal human plasma. These results confirm the normal thromboplastin generation in horse blood. Prothrombin and prothrombin conversion factors One-stage prothrombin tests were carried out on oxalated horse plasma with the use of homologous and human brain extract, and also with Russell viper venom and phospholipid (Table 4). It can be seen that the homologous brain extract gave shorter clotting times than did heterologous extract. But Russell

5 THE COAGULATION OF HORSE BLOOD 201 viper venom gave identical clotting times with both plasmas. It will be noticed that the addition of serum, homologous to the brain, gave a shorter clotting time than was obtained with heterologous combinations. One-stage results give no quantitative information of the components of the prothrombin complex. Prothrombin assays were therefore carried out in diluted horse plasma, and the results are given in Table 5. The prothrombin activity of horse plasma is in the same range as has been observed for human plasma (Fantl, 1954 a). Similar results were obtained with either human or horse brain extract. It should be pointed out again that longer clotting times were noticed when horse Ba-plasma was used instead of TABLE 5. Prothrombin assays of horse plasma* Incubation time Shortest Prothrombin for maximum clotting (u./ml. oxalated thrombin yield (min) time (sec) plasma) 2 (1-3) 17 ( ) 510 ( ) * Means, range in brackets. Human Ba-plasma was used to measure thrombin. TABLE 6. The S.P.C.A. activity of horse serum Composition of reaction mixture: horse plasma ml., horse Ba.plasma ml., plus Clotting time (ml.) (sec) 0-15M.NaCl Horse serum Horse serum O015M-NaCl Horse serum M-NaCl 0-045J human Ba-plasma as a source of fibrinogen for the testing of thrombin. It will be noted that the maximum yield of thrombin was obtained in horse plasma in 1-3 min, whereas it takes 4-5 min for maximum thrombin formation to occur in normal human plasma. This probably indicates a higher activity of prothrombin conversion factors in horse plasma. For the determination of prothrombin accelerator activity a prothrombin preparation isolated from stored human plasma was used, which in the assay procedure gave, after 13 min incubation, 116 u. thrombin/ml. The addition of ml. human Ba-plasma or ml. horse Ba-plasma resulted in a yield of 660 u. thrombin/ml. after 7 min incubation. From this experiment it appears that the prothrombin accelerator activity of horse plasma was at least twice that of human plasma. The method of de Vries et al. (1949) was used to measure the effect of horse serum on shortening the one-stage prothrombin time (Table 6). From the results in Table 6 it is apparent that horse serum has a shortening effect on the one-stage prothrombin clotting time, which is of the same order as that observed with human serum in an homologous system.

6 202 P. FANTL AND A. G. MARR The thrombin-fibrinogen reaction in horse plasma Varying concentrations of thrombin preparations of horse, human and bovine origin were tested on oxalated horse and human plasmas (Fig. 1). The results indicate that, independent of the source of thrombin, oxalated human plasma gave shorter clotting times than oxalated horse plasma. In order to produce the same clotting time horse plasma requires 3-5 times more thrombin than does human plasma ~~~ 0 E ,_30 0 -= E* _ o *0 0 I,I Clotting time of human plasma (sec) Fig. 1. Clotting times of oxalated human and horse plasmas with mammalian thrombins. Reaction mixtures consisted of: 0-2 ml. oxalated human or horse plasma, 01 ml. veronal buffer ph 7-2, and 0 05 ml. of dilutions of human, horse, or bovine thrombin preparations in veronal buffer. Temperature 280 C. Other factors being constant, thrombin clotting times are dependent upon fibrinogen concentration. Therefore the thrombin clotting times of horse and human plasma as well as a euglobulin fraction isolated from horse plasma were measured. The influence of fibrinogen concentration is shown in Fig. 2. It is apparent that horse plasma gave a longer clotting time than did human plasma with the same fibrinogen and thrombin concentrations. When the fibrinogen concentration was above 0-2 mg/l. reaction mixture (100 mg/100 ml. plasma) its variation caused little change in clotting time. Lowering the concentration below this level markedly prolonged clotting time. The fibrinogen concentration of oxalated horse plasma varied between 200 and 350 mg/100 ml.

7 THE COAGULATION OF HORSE BLOOD 203 The prolonged thrombin clotting time of horse plasma must be due therefore to causes other than fibrinogen concentration. It will be noted that the euglobulin fraction isolated from horse plasma showed shorter clotting times than the original plasma. 50 A 40~ v.e3 -A 20A 20 ~ ~ ~ ~ Fibrinogen concentration (mg/mi. reaction mixture) Fig. 2. Influence of fibrinogen concentration on thrombin clotting times. Reaction mixtures consisted of: 02 ml. substrate in 015m-NaCl, 0.1 ml. veronal buffer ph 7X2, and 0 05 ml. horse thrombin. Temperature 28 C. Substrates were oxalated: horse plasma (A- A); horse plasma euglobulin (U-,u); and oxalated human plasma (0-0). In order to find whether the longer thrombin clotting time of horse plasma was due to increased antithrombic activity, a human thrombin preparation was incubated with human and with horse serum and the residual activities were determined at intervals (Fig. 3). After 1 min incubation time no difference between the two sera was observed, but after 7 min the horse serum had a greater antithrombic activity than the human serum. It would appear that the antithrombic activity cannot be the cause of the prolonged thrombin clotting times of horse plasma (Figs. 1 and 2), which were all shorter than 1 min. The thrombin clotting times of horse and human plasma were unaltered by treatment with a barium sulphate suspension. This excludes the presence of heparin, which is removed by barium sulphate (Nilsson & Wenckert, 1954). Further, the addition of an equal volume of defibrinated horse plasma to human plasma was without significant effect on the thrombin clotting time. Therefore horse defibrinated plasma does not contain an inhibitor of clotting.

8 204 P. FANTL AND A. G. MARR It is possible that the prolonged thrombin clotting time is characteristic of horse fibrinogen. A number of substances, including phenols, shorten the thrombin clotting time (Gerber & Blanchard, 1945). This phenomenon is partly due to the influence of phenols on the surface structure of fibrinogen (Fantl & Ebbels, 1953). Therefore the effect of catechol on horse and human plasma and their isolated euglobulin fractions was determined (Table 7). 1 *6.~0.8 E 0 O \ I Incubation time (min) Fig. 3. Antithrombic activity of human and horse serum. Reaction mixtures consisted of (ml.) serum 0 4; veronal buffer ph 7-2, 0-8; human thrombin, ml. portions were added to 04 ml. oxalated human plasma. Temperature 28 C. Mixtures contained either human serum (0-0) or horse serum (U-U). TABLE 7. The influence of catechol on the apparent thrombin activity Apparent thrombin activity Substrate (u.) Horse plasma 0-27 ( ) Horse plasma + catechol 1-15 ( ) Human plasma 1-18 ( ) Human plasma + catechol 2-71 ( ) Horse euglobulin 0-5 Horse euglobulin + catechol 1-6 Human euglobulin 1.5 Human euglobulin + catechol 3-0 Composition of the reaction mixture the same as that used in Figs. 1 and 2. The catechol conconcentration was 0045M in the reaction mixture. The figures are average results, variations are given in parentheses.

9 THE COAGULATION OF HORSE BLOOD 205 It is apparent that catechol accelerated the thrombin-fibrinogen reaction in both horse and human plasmas and euglobulin fractions. In the presence of catechol the reactivity of horse fibrinogen towards thrombin was, however, still less than that of human fibrinogen. DISCUSSION These experiments show that all the recognized clotting factors in human blood are active in that of the horse. However, marked quantitative differences between the two species are apparent. Thromboplastin generation tests with horse blood components indicated a low activity of the thromboplastin when tested with horse plasma, but an activity equal to that of human blood when tested on human plasma. This indicates that thromboplastin formation in horse blood proceeds at a similar rate to that in human blood but that subsequent reactions in horse blood are delayed. Further, the rate of thrombin formation in horse blood was slower than in human blood. This may have been due to inadequate numbers of platelets and also to the fact that horse platelets showed less tendency to liberate phospholipids during clotting, since very rapid clotting could be induced by the addition of brain phospholipids. With regard to plasma factors required for blood clotting it is evident that o- and /3-prothromboplastin and S.P.C.A. of horse plasma had similar activity to those of human plasma. In order to get maximum activity in a one-stage prothrombin test it was essential to use homologous brain extract. This is in agreement with the rule that brain thromboplastins show some degree of species specificity in conversion of prothrombin. The addition of serum homologous to the brain abolished species specificity. This is in accord with observations made by Burstein (1950) and by Mann & Hurn (1952). Prothrombin assays carried out with horse plasma gave results within the same range as found for human plasma. The high activity of prothrombin accelerator in horse plasma is in agreement with findings of Soulier & Larrieu (1953) and of Bell, Archer & Tomlin (1955). The prolonged thrombin clotting time of horse plasma when compared with that of human plasma is not unique. Guinea-pig, rat, and marsupial plasma also show lengthened thrombin clotting time (Fantl & Ebbels, 1953; Fantl & Ward, 1957). Evidence given in this paper supports the previous assumption that thrombin isolated from mammalian blood acts according to potency independent of source. It is therefore possible that the differences in the rates of conversion of the fibrinogens into fibrin are due to differences in the structure of the native fibrinogens. The change in horse fibrinogen produced by catechol did not completely abolish the delay in the formation of fibrin. The different isoelectric points and solubility of human and horse fibrinogen

10 206 P. FANTL AND A. G. MARR (Astrup & Darling, 1942) also support the assumption of structural differences in the native fibrinogens. It is noteworthy that human fibrinogen has given the shortest thrombin clotting time of all mammals investigated so far. The comparatively long thrombin clotting time is the main reason for an apparent reduced coagulability of horse plasma. It may be mentioned that the coagulation time of horse blood was min. Using the same technique human blood clots in min. Horse blood had a higher sedimentation rate than human blood as noted by Hammersland, Herrin & Haynes (1938), so that gelling of horse plasma occurred usually after the cells had settled. Nevertheless, clot retraction occurred in all instances. Although the results of some of the tests suggest that the coagulability of normal horse blood is less than that of normal human blood, it should be pointed out that coagulation forms only part of the haemostatic process and that there is no evidence that this is impaired in horses. SUMMARY 1. Thromboplastin formation from plasma and serum components takes place at the same rate in human and horse blood. 2. oc-prothromboplastin (antihaemophilic factor) and fl-prothromboplastin (P.T.C., Christmas factor) and prothrombin of horse plasma have similar activities to those of human plasma. 3. Horse as well as human brain extracts show some degree of species specificity in one-stage prothrombin tests. 4. Prothrombin accelerator (proaccelerin) of horse plasma is more active than that of human plasma. 5. S.P.C.A. (factor VII) in horse serum is as active as that of human serum when acting in homologous systems. 6. Thrombins isolated from mammalian plasmas give shorter clotting times with human than with horse plasma. It is desired to acknowledge the assistance of G. D. Rudduck M.B., B.S., B.V.Sc.,of the Prahran City Council and the Commonwealth Serum Laboratories, Melbourne. Part of the expenses of this investigation were defrayed by a grant from the National Health and Medical Research Council, Australia. REFERENCES AsTRuP, T. & DARuNG, S. (1942). Blood coagulation and species specificity of fibrinogen. Acta physiol. 8cand. 3, BELL, W. N. & ALTON, H. G. (1954). A brain extract as a substitute for platelet suspension in the thromboplastin generation test. Nature, Lond., 174, BELL, W. N., ARCHER, R. K. & TOMLIN, S. C. (1955). Coagulation mechanism of the horse. Nature, Lond., 175, BELL, W. N., ToMLN, S. C. & ARCHER, R. K. (1955). The coagulation mechanism of the blood of the horse with particular reference to its 'haemophilioid' status. J. comp. Path. 65,

11 THE COAGULATION OF HORSE BLOOD 207 BIGGS, R. & DOUGLAS, A. S. (1953). The thromboplastin generation test. J. clin. Path. 6, BURSTEIN, M. (1950). Mise en evidence dans le s6rum des mammiferes d'un accelerateur de la conversion de la prothrombin des oiseaux. C.R. Acad. Sci., Paris, 230, DE VRIES, A., ALEXANDER, B. & GOLDSTEIN, R. (1949). A factor in serum which accelerates the conversion of prothrombin to thrombin. Blood, 4, EAGLE, H. (1935). Studies of blood coagulation. 1. The role of prothrombin and of platelets in the formation of thrombin. J. gen. Physiol. 18, FANTL, P. (1954 a). The use of substances depressing antithrombin activity in the assay of prothrombin. Biochem. J. 57, FANTL, P. (1954 b). Thrombin formation and yield in shed blood in relation to thromboplastins and prothromboplastins. Aust. J. exp. Biol. med. Sci. 32, FANTL, P. & EBBELS, L. (1953). The interaction of thrombin with the plasma components of man and experimental animals. Aust. J. exp. Biol. med. Sci. 31, FANTL, P. & MARR, A. G. (1956). The preservation of coagulation factors in human plasma. AUst. J. exp. Biol. med. Sci. 34, FANTL, P. & WARD, H. A. (1957). Comparison of blood clotting in marsupials and man. AWst. J. exp. Biol. med. Sci. 35, FEISSLY, R. & LUDIN, H. (1949). Microscopie par contrastes de phases (III applications & l'hematologie). Rev. Htmat. 4, GERBER, C. F. & BLANCHARD, E. W. (1945). The effect of certain substances on clotting timc, in vitro. Amer. J. Physiol. 1464, HAMMERSLAND, H. L., HERRIN, H. S. & HAYNES, C. F. (1938). A study of the blood in horses infected with infectious anaemia. J. Amer. vet. med. Ass. 93, N.S., 46, HoBSON, F. & WITTS, L. J. (1941). Venom-lecithin reagent for the accelerated clotting test (prothrombin time). J. Path. Bact. 52, LANGDELL, R. D., WARNER, R. H. & BRIuKHOUS, K. M. (1953). Effect of antihemophilic factor on one-stage clotting tests. J. Lab. clin. Med. 41, LEE, R. I. & WHITE, P. D. (1913). A clinical study of the coagulation time of blood. Amer. J. med. Sci. 145, MANN, F. D. & HUIEN, M. M. (1952). Species specificity of thromboplastin: role of the cothromboplastin reaction. Proc. Soc. exp. Biol., N.Y., 79, MELLANBY, J. (1909). The coagulation of the blood. J. Physiol. 38, NILSSON, I. M. & WENCKERT, A. (1954). Demonstration of a heparin-like anticoagulant in normal blood. I. Human blood. Acta med. 8cand., Suppl. i50, QUICK, A. J. (1938). The nature of the bleeding in jaundice. J. Amer. med. Ass. 110, SJ0LIN, K. (1956). Lack of Christmas factor in horse plasma. Nature, Lond., i78, 153. SJ0LIN, K. (1957). Coagulation defect in horse plasma. Proc. Soc. exp. Biol., N.Y., 94, SOULIER, P. & LARRIEU, M. J. (1953). Measurement of thromboplastic factors and profactors in plasma. J. Lab. clin. Med. 41,