Blood coagulation mechanism in the snakes Waglerophis merremii and Bothrops jararaca

‘romp. Bto~hcm. Ph~vol. ‘rmted ,n Great Br,mn.

Vol. 69A. pp. 739 to 743. 1981 All nghts reserved

Copyght

0300-9629/81/080739-05SO2.OQ’O 0 1981 Pergamon Press Lfd

BLOOD COAGULATION MECHANISM IN THE SNAKES WAGLEROPHIS MERREMII AND BOTHROPS JARARACA LINDA NAHAS, AURA S. KAMIGUTI,FIXRUCIO BETTI,IDA and MARIA I. RODRIGUETS

Department of Physiopathology,

S. SANO MARTINS

Instituto Butantan, CP 65, 05504, Sio Paula, Brasil

(Received 3 December 1980) Abstract-l. The blood coagulation mechanism was investigated in the snakes Bothrops jararaca and Waglerophis merremii, using homologous and heterologous systems to study the clotting function. 2. Whereas the extrinsic pathway was efficient in both species, evidences for the intrinsic activation was found only in IV. merremii plasma. 3. The results obtained in several tests suggest that at least traces of Hageman factor are present in this plasma, providing the formation of a significant amount of contact product, besides the activation of prekallikrein- formed jn the same plasma.

INTRODUCUON An analytical review of the blood coagulation mechanism from lower vertebrates to mammals points out an increasing complexity which achieves its highest level in man. Research on human blood clotting was stimulated by pathological conditions and by discovery of haemostatic defects. Thus, in contrast with the massive literature on normal human coagulation and its disorder, very little information is available about this process in other vertebrates. However, studies on the evolutionary aspects of blood coagulation may help to elucidate the significance of this gradual complexity. Additionally, comparative studies may give evidences for alternate mechanisms of clot formation. The studies reported by several authors have shown that the clotting mechanism in snakes is slower than in mammals; that contact product formation is absent due to a deficiency in factor XII (Fantl, 1961; Lavras, 1979); that low concentration of factor VIII and IX are available (Fantl, 1961); and that powerful naturally occurring inhibitors are present (Hackett & Hann, 1967, Nahas et al., 1973). The purpose of the present study is to investigate the clot formation in two species of snakes in which the presence (Bothrops jararaca) or absence (Waglerophis merremii) of an inhibitor represents a natural condition (Nahas et ~1..1973). In addition, a comparative evaluation of contact activation is investigated in order to provide more information on the intrinsic and extrinsic prothrombin activation in these two snakes. MATERIALS AND METHODS Snakes. Two species Waglerophis merremii and Bothrops ,jararaca were obtained from Section of Herpetology of

Instituto Butantan, Sio Paulo. Blood collection: the snakes were bled from the aorta exposed by laparotomy, employing the two plastic syringes technique. Human and snake oxalated or titrated plasmas were obtained by mixing 9 parts of blood with 1 Dart of 1.34?/, sodium oxalate or 3.8”~;sodium citrate, respe&ively. ” Thrombin. Bovine thrombin (Roche) r500 NIH units,‘mll was dissolved in 50% glycerine and kept at -20°C. I%luI tions for tests were made in saline and used immediately. C.B.P. 69:4*-

I

Snake thrombin was prepared as previously described (Nahas et al., 1973). Thromboplastins. Specific thromboplastins were prepared from human snake brains, as saline extract (Biggs & Macfarlane, 1962). Fibrinogen. Human lyophilized fibrinogen (Behringwerk) was used and snake fibrinogen was prepared according to the method of Blomblck, as described by Biggs & Macfarlane (1972). Cephaline. A 1 in 100 suspension of chloroform extract of human and snake brain (Bell & Alton, 1954) was used. Kaolin. A saline suspension of 5 mg/ml of kaolin was obtained from British Drug Ltd, Poole. Dorset. Celite. Celite 512 obtained from Johnsmanville Co Ltd, 20 Albert Embankment. London SEl, England. Protamine. One per cent protamine sulphate Glaxc+ Evans, Brazil, appropriately diluted in buffered saline was used. BaSO, and AI(OI&. Treated plasmas were prepared as recommended by Biggs & Macfarlane (1962). Since an incomplete absorption was obtained with proportions indicated in the original technique, 400mg of BaSO, (Biggs, 1972) and 0.4 ml Al(OH)3 (Bertho-Grasman in Biggs, 1972) were added to each ml of plasma. To avoid plasma dilution the Al(OH), was previously centrifuged and the supernatant discarded. Polyestyrene tubes of 10, 5 and 2 ml capacity were used in tests in which a non-contact surface was required. Whole blood clotting time. This was determined in glass and plastic Lee-White tubes at a temperature of 37’C for human blood and 22 or 37°C for snake blood. Thrombin clotting time. This was determined by adding to 0.2 ml of human or snake plasma 3 units of bovine or snake thrombin, respectively. One stage prothrombin time was determined with human or snake saline brain thromboplastins according to the Quick’s method as described by Biggs (1972). Two stage prothrombin assay. This was determined by the method described by Biggs & Douglas (1953a) using human or snake fibrinogen. Prothrombin consumption test. As the concentration of the factors involved in the test is lower in snake blood than in human, the residual prothrombin in the serum was determined hourly until less than 20% was detected and results are reported as the mean time of consumption. The residual prothrombin concentration was determined by measuring the clotting times induced by the addition of tissue extract to whole blood.

740

LINDA NAHAS

Thromhoplustin yewration test. This was performed according to the method described by Biggs & Douglas (1953b) using homologous and heterologous incubation mixtures with respect to plasma. serum. cephalin and substrate plasma. Protamine sulphate 1 mg/ml was added to B. jurcrruca plasma to neutralize its natural inhibitor (Nahas et trl., 1973). Crlite rluute test for deficiency of XII and XI factors. The test according to the method of Nossel (1964) as described by Biggs (1972) was used. Km/in crphtrlin clotting time. This was determined according to the method described by Biggs (1972).

RESULTS

The clotting function in the snakes B. ,juraraca and M/: merremii was evaluated by clotting tests using homologous systems. When possible with plasma from B. .jararaca, the natural inhibitor was neutralized with protamine

time

1. Whole

blood clotting temperature,

Prothrombin

consumption

When prothrombin consumption was measured at 37’C in the absence of tissue factor and in the presence of contact surface (Table 2), no prothrombin was consumed during a period of 7 hr in B. jaruraca blood, whereas about 75”/:, was consumed in W. merremii blood. The addition of tissue extract did not increase the amount of consumed prothrombin, although some acceleration was observed in W. merremii blood and only a small amount of prothrombin (241,;) was slowly transformed in the B. ,jaruracu

One stuge prothrombin

The influence of temperature, contact surface and tissue extract was investigated. The data presented in Table 1 shows that W. merremii blood, in the absence of tissue extract, clotted with a mean of 28.4min (840min) at either 22 or 37°C when exposed to a glass surface. In contrast, when exposed to an inert surface such as polyestyrene no clot occurred at either temperature. With B. jararaca no clot formation took place after 24 hr in any conditions of temperature or contact surface when tissue extract was absent. In

Table

species the addition of tissue extract induced clot formation in a relatively short time (4-9 min) regardless of the contact surface or temperature. both

blood.

sulphate.

Whole blood clotting

et ~1.

time and kaolin cephalin

time

Table 3 shows the one stage prothrombin time performed in human, W. merremii and B. jararaca plasmas. using brain homologous and heterologous thromboplastins. As can be seen, in the homologous systems, short clotting times were obtained with human and W. merremii plasmas, whereas longer clotting times were recorded with all heterologous thromboplastins. Very long clotting times were observed with B. jararuca plasma either with homologous or heterologous thromboplastins, the longest clotting times being observed with human thromboplastin. The kaolin cephalin time also showed short clotting times with human and W. merremii plasmas when

time of W. merremii and B. jararaca: contact surface and tissue extract

influence

of

Blood clotting time (min) Contact surface Tissue extract -

W. merremii (N = IO)

+

-

B. jararaca (N = 10)

Table 2. Prothrombin

+

consumption

22°C Polyesterene

Glass

Glass

37’C Polyestyrene

28.4 (8-40) 5.1 (4-81

No clot 6.7 (G9)

4.7 (4-8)

6.9 (4-9)

No clot 7.8 (4-9)

No clot 7.8 (5-9)

No clot 7.9 (6-9)

No clot 7.7 (5-9)

31.3

No clot

( 13-40)

in W. merremii and B. jararaca blood:

influence

of tissue extract

9: Residual prothrombin (Hr from clot formation) Tissue extract II’.

mcrrrmii

4

5

6

7

1

2

100

95.4 (51-135) 95.8 (53-l 15)

95.5 (6@122) 84.6 (62-82)

14.5 (40-100) 70.4 (19-86)

56.8 (2&82) 46.1 (17-19)

42.6 (2%74) 28.1 (17-51)

38.5 (l&73) 24.0 (14-41)

28.4 (10-52) 24.0 (14-41)

100 I08 (64-146)

100 110 (51-156)

100

100

100

100

100

(44?30)

(33?30)

(32!:06)

(3Z90,

(N = 10)

B. jnraruca (N = 10)

3

0

+

100

+

100 100

,,?I

13)

Blood coagulation mechanism in snakes

741

Table 3. One stage prothrombin time and kaolin cephalin clotting time of human, W. merremii and B. jararaca plasma with homologous and heterologous thromboplastin and cephalin Clotting time (set) Cephalin

Thromboplastin Plasma (N = 10)

B. jararaca

Human

W. merremii

B. jararaca

114.6 (100-130)

109.8 (80-145)

47.3 (41-59)

78.1 (67-95)

63.1 (5>80)

81.8 (57-102)

14.1 (10-18)

14.8 (10-24)

46.1 (37-66)

47.9 (4&63)

50.4 (42-60)

365.5 (29&445)

99.8 (74125)

152.0 (120-192)

371.0 (256484)

329.0 (282-411)

319.0 (239-385)

Human

W. merremii

Human

14.1 (12-15)

W. merremii

B. jararaca

Table 4. Two stage prothrombin

time of human, W. merremii and B. jararaca plasmas in homologous systems Thrombin generation (units) Incubation time (min)

Plasma (N = 10)

1

2

3

4

5

6

Total units

Human

14.0 4.3 (9.0-20.0) (2.0-8.0)

1.6 (1.2-3.7)

0.4 (0.0-1.6)

0.0 -

0.0 -

20.3 (13.0~25.0)

W. merremii

1.7 (1.4-1.9)

(l.Z.5)

(O.lz.0)

0.0 -

0.0 -

0.0 -

3.0 (2.3-4.2)

14.4

B. jararaca

2.8 (1.0-4.2)

1.8 (1.0-2.7)

0.0 -

0.0 -

0.0 -

0.0 -

4.6 (2.8-6.9)

22.7

homologous cephalins were employed. Human plasma reacted best with human cephalin and poorly with W. merremii and B. jararaca cephalins. However W. merremii plasma reacted well with homologous, human and B. jararaca cephalins. The clotting times given by human, W. merremii and B. jararaca cephaIins with B. jararaca plasma were prolonged. Two stage prothrombin

The prothrombin remii

‘%

time

concentration

and B. jararaca

of human,

W. mer-

plasmas, estimated by two stage

Table 5. Comparative

prothrombin time using homologous incubation mixtures is illustrated in Table 4. As can be seen, the thrombin generation was very poor in plasma from both snakes, the best yield of thrombin units being found in the B. jararaca species. Thromboplastin

Serum

Cephalin

H H

H H H H

H H H H H H H

H H Wm Wm

Wm Wm Wm Wm

Wm

sys-

Activation product % Incubation time (min) Substrate

1

2

3

4

5

6

H H Wm H

6 1 2 2 3

40 6 13 15 5

104 34 13 18 6

103 40 12 16 5

103 34 10 13 4

102 31 9 11 3

Wm H

286

40 12

37 13

19 11

14 10

1;

Wm

40

92

80

62

40

27

Wm

% Activation as recommended by Biggs (1957). H = Human. Wm = W. merremii.

test

When thromboplastin generation test was performed in homologous and heterologous incubation mixtures and plasma substrates, the yield of intrinsic activator (Table 5), indicated that the W. merremii

Incubation mixture

Wm Wm

generation

thromboplastin generation in homologous and heterologous tems of human and W. merremii

Plasma

100

142

LINDANAHASer ul. Table 6. Comparative

thromboplastin generation in homologous and heterologous tems of human and B. jararaca

sys-

Activation product “1; Incubation time (min)

Incubation mixture Plasma

Serum

H H Bj Bj H H Bj Bj

H H H H Bj Sj Bj Bj

Cephalin

Substrate

I

2

3

4

5

6

H

H

36 1 6 1 1

84 1 14 1 5

103 1 36 8 8

104 9 36 13 16

99 8 34 13 16

96 10 31 12 14

Bj*

0

0

0

0

0

0

H

1 1

1 1

1 1

I 0.5

5 8

7 6.4

Bj*

H Bj*

Bj*

* 1 mg protamine sulphate added to ml of plasma. “, Activation as recommended by Biggs (1957). H = Human. Bj = B. jararaca.

serum and plasma in all heterologous systems produced very low concentration of activator. However. when the interactions occur in a completely homologous system, the level of clotting factors in the plasma and serum from W. merremii shows the formation of prothrombin activator equivalent to that found in the human. On the other hand, in the same test carried out with plasma. serum and substrates from human and B. jmwuco in heterologous systems (Table 6). no prothrombin activator was found in the incubation mixtures. even when homologous systems were used for B. jworucu. Contclct

phase

ccctirution

The contact activation system in human. W. merr’enrii and B. ,jwuraca plasma was estimated by the ability of a celite eluate (contact product) to correct the intact plasma in homologous and heterologous systems. As is shown in Table 7 a definite correction occurred only with W. merremii and human eluates on U’. mrrrrmii intact plasma. Human and W. merremii contact factors also provided some correction on B. juwuca intact plasma.

DISCUSSION In the present studies the coagulation systems in the blood of species of snakes, B. jararaca and W. merremii. have been compared. In the absence of tissue factor. only the blood from W’. merremii was capable of clot formation and prothrombin consumption and then only when a negative surface (glass) was also present. The coagulation time was slow (MOmin) but did suggest that an intrinsic coagulation pathway was available in this plasma. These results were unexpected, since absence of Hageman factor and consequently lack of an active intrinsic pathway, was first demonstrated in B. jararata by Lavras (Lavras et al., 1979) and Fantl (Fantl, 1951) in Nofechis scututus scutatus. in which also a low concentration of all other clotting factors interacting in the intrinsic system was found. In additional studies, the one stage prothrombin time, measured in the presence of homologous and heterologous reagents, showed that the clotting time of W. merremii plasma could be as rapid as that of human plasma provided that homologous thromboplastin was employed. With the kaolin cephalin clot-

Table 7. Contact product of human W. merremii and Source Intact plasma

Human

Celite eluate Human W. merremii

W. mrrremii

Human B. jararaca B. jararaca

B. jararacu

plasma

Clotting time (set)

B. jararuca

W. merremii

B. jararaca

Human W. merremii

Correction “A

Saline

Eluate

160.1 174.1 122.5

59.8 163.0 121.5

62 5 0

220.8 231.9 292.0

134.0 144.3 541.5

39 38 0

415.0 380.0 438.8

441.4 303.0 327.7

0 19 26

System: to 0.1 ml of intact plasma. 0.1 ml of eluate, 0.1 ml of homologous cephalin and 0.1 ml of 0.025 M calcium chloride were added and clotting time recorded.

743

Blood coagulation mechanism in snakes ting time, however, human cephalin was as potent as that from the snake plasma when snake plasma was employed but snake cephalin was less potent than human cephalin when human plasma was used. The prothrombin concentration of W. merremii plasma as measured by the two stage prothrombin time in a homologous system was only 14% of that found in the human. However, the yield of intrinsic prothrombin activator available by the thromboplastin generation test, was similar to that of the human, provided a homologous system was employed. When the contact phase of activation of W. merremii plasma was investigated with eluates, it was clear that either homologous or heterologous eluates were capable of shortening the clotting time. However, the celite eluates from W. merremii could not shorten the clotting times of human plasma. The failure of plasma from B. jararaca to induce clotting times equivalent to that found in human or W. merremii plasma in some test systems, could be explained at least in part by the presence in this plasma of a potent thrombin inhibitor to coagulation (Nahas et a/.. 1973). However, the inhibitor did not interfere with the extrinsic coagulation pathway, since the clotting times were similar in all three plasmas. Also, the inhibitor is not present in the celite eluate. Therefore, the most likely explanation for the failure of celite eluate from B. jararaca plasma to correct W. merremii or human plasmas (Table 7) would be to assume that the intrinsic coagulation pathway is absent in this snake. The results reported in this paper would indicate that the intrinsic coagulation system is present in W. merremii but absent in B. jararaca, as already described. Which factors are available in contact phase of W. merremii plasma remain to be determined. In preliminary studies (Webster, 1978) no Hageman factor, as measured by its ability to activate human prekallikrein, could be detected in this plasma. However, this plasma contained large amounts of an arginine esterase which was not inhibited by soybean trypsin inhibitor. In addition, further esterase activity could be generated when human active Hageman factor bound to supercel was added (Webster et al.. 1979a; Webster et al., 1979b). It appears possible, therefore that this plasma may and kallikrein bound to contain prekallikrein alpha-2-macroglobulin. B. jararaca plasma, on the other hand, contained much less direct arginine esterase and the addition of active Hageman factor did not increase its yield. However, the results obtained with tests (whole blood clotting time on glass, kaolin cephalin clotting time, thromboplastin generation test and contact product formation) give evidence for the presence of Hageman factor in M! merremii plasma in required concentration to activate the intrinsic system

in these tests. Its inability to activate human prekallikrein and correct intact human plasma could be explained by species specificity. On the other hand, kallikrein can directly activate factor IX (Osterud et a/., 1975) and activate factor VII either by its action as a plasminogen activator (Laake & Osterud. 1974) or by its formation of activated factor IX (Seligsohn ef al., 1979). It is possible, therefore, that only this alternative intrinsic coagulation pathway is operational in I%‘.merremii snake plasma. Acknowledyement-We would like to thank Dr Marion E. Webster for the careful revision of the manuscript and helpful suggestions. REFERENCES BELL W. N. & ALTONH. G. (1954) A brain extract as a

substitute for platelet suspensions in the thromboplastin generation test. Nature. Lond. 174, 88&881. BIGGSR. (1972) Human blood coagulation, haemostasis und thrombosis, 1st edn, pp. 589-671. Blackwells, Oxford. BIGGS R. & DOUGLASA. S. (1953a) The measurement of prothrombin in plasma. J. clin. Path. 6, 15-22. BIGGS R. & D~LJGLASA. S. (1953b) The thromboplastin generation test. J. c/in. Path. 6, 23-29. FANTLP. (1961) A comparative study of blood coagulation in vertebrates. Aust. J. exp. Biol. med. Sci. 39. 403412. HACKETT.E. & HANN C. (1967) Slow clotting of reptile blood. J. camp. Path. 77, 175-180. LAAKE K. & OSTERUD B. (1974) Activation of puritied human plasma factor VII by human plasmin. plasma kallikrein. and activated components of the human intrinsic blood coagulation system. Thromhos. Rrs. 5,

759-772. LAVRAS A. A. C., FICHMAN M., HIRAICHI E., B~CAULT M. A., TOBOT., SCHMUZIGER P., NAHAS L. & PICARELLI Z. P. (1979) Deficiency of kallikrein-kinin system and presence of potent kininase activity in the plasma of Bothrops jararaca 31(2), 168-174.

(Serpentes

Crotaline).

C&K.

Cult.

NAHAS L., BETTI F.. KAMIWTI A. S. & SATO H. (1973) Blood coagulation inhibitor in snake plasma (Borhrops jaruruca). Thromb. Diath. huemorrh. 30, IO&l 13. NOSSEL H. L. (1964) The contact phase of blood wagulution. pp. 138-141. Blackwells. Oxford. OSTERUD B.. LAAKE K. & PRYDZ H. (1975) Activation of factor IX. Thromb. Diath. haemorrh. 33, 553-563. SELIC;SOHNV.. OSTERUD B.. BROWNS. F.. GRIFFON J. H. & RAPAPORT S. I. (1979) Activation of human factor VII in plasma and in purified systems. Roles of activated factor IX, kallikrein and activated factor VII. J. c/in. Invest. 64. 1056-1065.

WEBSTERM. E. (1978) Personal communication. WEBSTERM. E., STELLAR. C. R., VILLA M. L. & TOFFOLETTO0. (1979a) Brasil factor-a new prekallikrein activator

in human

plasma.

Submitted

for publication.

WEBSTERM. E., STELLAR. C. R.. VILLA M. L. 8~ TOFFOLETTO0. (1979b) A new prekallikrein activator in human plasma which differs from Hageman factor and its fra8men&. Adc. exp. med. Biol. In press.