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The in vivo anticoagulant effects of Naja flava venom
Tnxicnrr, 1971, Vol . 11, pp . 419-422 . Perganron Prcsx. Printed in Great Britain
THE IN V1V0 ANTICOAGULANT EFFECTS OF NAJA FLA YA VENOM A. H. MoI-tAtt~trrn and M. M. HANNA Faculty of Medicine, Ain Shams University, Cairo, Egypt (Acceptedjor publication 27 March 1973)
Abedract-The in vlvn effects of Naja (lava venom on blood coagulation were studied in rabbits . In sublethal doses, it had no significant effects. In lethal doses, its anticoagulant activity can be attributed to interference with thromboplastin and thrombin generation as well as an increase of circulating heparin . The fibrinogenolytic effect which was evident in vitro could not be demonstrated In vivo . Ti-tE in vitro effects of Naja jlava venom on blood coagulation have been studied by MOHAMED and HANNA (1972) . In the present work, its in vivo actions were analysed. MA~R.IA S AND METHODS venom kindly provided by Prof. W. Deichman, Chairman of the Pharmacology Dept., Faculty of Medicine, Miami University, Florida, U.S.A. was used in a sublethal dose of 0~2 mg per kg body weight and a lethal dose of 1 ~0 mg per kg. Citrated plasma and serum were prepared as described by BItîGS and MACFAxLANE (1962). Thrombase LS.H. was used in a concentration of 5 units per ml saline. One thromboplastin tablet containing 10 mg of Thrombokinase (Geigy), with calcium, was used per 2~5 ml distilled water . Bovine Fibrinogen fraction I was used. Toluidine blue was employed in a concentration of 50 mg per 100 ml saline . Protamine sulphate `Evans', was used in a dose of 40 mg per kg body weight. Rabbits of about 1 kg body weight were divided into three groups of six rabbits each. Group I received sublethal doses of 0~2 mg venom per kg body weight. Group II received lethal doses of 1 ~0 mg venom per kg body weight . Group III received protamine sulphate (40 mg per kg body weight) before injection of the lethal dose of the venom. The venom was injected intramuscularly and blood samples were obtained 30 min later. Plasma recalcification time was determined as described by Btocs and MACFAxLANt=. (1962). Thrombin clotting time of plasma was performed by adding 0~1 ml thrombin solution to 0~1 ml plasma. One stage prothrombin time was determined by adding Thrombo kinase solution to an equal volume of plasma. Thrombin generation test was performed as described by PrI-tv~r and DACIE (1953). Thromboplastin generation test was carried out according to BIGGS and DOUGLAS (1953). Estimation of fibrinogen : Qtncx's technique (1951) was slightly modified, adding 30 NIH units of thrombin instead of calcium chloride to avoid the interference of heparin . Qualitative test for heparin is described by Blcos and MACFAR.LArrE (1962) . Quantitative estimation of heparin by the method of JACQvFs and CxAxLFS (1941) . All tests were carried out at 25°C. 419 Naja j~ava
71OXlCON l973 Vol. ll.
A. H. MOHAMED and M. M. HANNA
420
1.
RESULTS
Plasma recalc~cation, thrombin-clotting and the one-stage prothrombin time Sublethal doses of Naja ,(lava venom had no effect on the coagulability of the blood
(Table 1). Lethal doses caused a significant prolongation of the plasma recalcification time, thrombin-clotting time and the one-stage prothrombin time . Addition of toluidine blue to the reaction media, with appropriate saline controls, corrected the thrombin-clotting time and the prothrombin time : it decreased the effects of the venom on the recalcification time but did not abolish this effect (Table 1). Plasma from rabbits pretreated with protamine showed normal thrombin clotting and prothrombin times and a significant retardation of the plasma recalcification time, though the changes were significantly less than those obtained in absence of protamine. TABLE ] . THE HFPE(.TS OF Nqla ftaVa VBTVOM ON FLASrlA RECALCII~iCA1ION TII~, 1f~t0l~IN CLOTIIIdO TII~ AND 7HE ONE-ôrAOE FROTHROA~IIN TII~ IN RAHHTrS
Clotting time Test plasma Control Group I Group II Group II ~- tohridine blue Group III
Rocalcification time 233 f 26 251 f 18 392 f 21* 315 f 27* 300 f 24*
Thrombin clotting time 15 f 1 1S f 2 31 t 3* 18 f 3 16 f 2
Prothrombin time 12 f 1 13 f 1 25 f 3* 13 f 2 14 f 1
Results ere expt+eased as the mean f standard error of the mean. *Values significantly differrnt from control (p < 001).
2. Thrombin generation test
The results in Table 3 show that in sublethal doses, N. gava venom had no effect on generation of thrombin. In lethal doses, it retarded its generation . In protaminized rabbits, the venom had a similar effect but significantly less marked . 3. Thromboplastin generation test
The results shown in Table 2 demonstrate that sublethal doses did not affect thromboplastin precursors . Lethal doses interfere with its generation, probably through interference with one of the scrum factors. With the injected plasma and serum, the clotting TA~ 2. THB sFFBCrs
oF
Ngja Jlava vwoM oN rfntaa®ortastnv OHNSRA7ION
Source of reagents Al(OH), Plasma
Serum
Normal Normal Group I Normal Group II Normal Group III
Normal Group I Normal Group II Normal Group III Normal
Inwbation time 1 82 76 73 110 118 75 79
Results are expnaaed as mean f standard error. 'Values significantly different from control (p <0-01) . TOX7CON 7973 Vol. 77.
f3 f4 fS f 6* f 6* fS fS
2 65f 4 66 f 4 64 f 3 % f 4* 98 f S* 69 f S 56 f 4
3 Clotting time (~) 30 f 2 27 f 3 26 f 3 76 f 3* 72 f 4* 4S f 1* 28 f 3
4
S
12 f 1 12 f 2 12 f 2 53 f 3* 33 f 2* 41 f 3* 13 f 2
12 f 2 12 f 1 12 f 3 49 f 3* 30 f 2* 38 f 2* 14 f 3
Anticoagulant Action of Naja floua Venom
421
times were prolonged. In protaminized animals, there was still a significant prolongation of the clotting time when the serum of injected rabbits was used, but not when their plasma was supplemented with normal serum. 4. Fibrinogen concentrations Sublethal and lethal doses of the venom had no significant effect on fibrinogen concentration ofthe plasma in protaminized or non-protaminized animals. The fibrinogen values (mg/100 ml) for control and groups I, II and III were 279 ± 13, 265 ± 15, 282 ± 8 and 261 ± 12, respectively. Each value is the mean ± standard error based on svc determinations . Any increase of heparin in the plasma of injected rabbits could not affect the result of this test as the amount of thrombin added (30 units) was relatively large. 5. Heparin estimation There was no significant change with sublethal doses, while lethal doses produced a significant increase in circulating heparin. The heparin values (units per 100 ml) for control and groups I and II were 0"91 ± 0"08, 0"97 ± 0-12 and 272 ± 0~2, respectively . Each value is the mean + standard error based on six determinations . DISCUSSION
In lethal doses, Naja ,hfava venom prolonged the recalcification time of plasma, the thrombin clotting time and the one-stage prothrombin time . These effects were, in part, due to an increase in the concentration of circulating heparin, as confirmed by the partial correction of the defects by toluidine blue and protamine sulphate (Table 1) and by the demonstration of an increased blood concentration of heparin. However, failure of these two agents to restore the normal coagulability of the blood indicates that other mechanisms are also in action . Toluidine blue and protamine sulphate corrected the thrombin clotting time and the one-stage prothrombin time, indicating that the ren±ain_ing defect is related to the generation of thrombin and thromboplastin . The results of the thrombin generation test (Table 3) confirm its retardation with lethal doses of the venom. The results of the thromboplastin generation test (Table 2) show the presence of an anticoagulant in the blood. When this was neutralized in animals of group III, there was still a defect which could be corrected by normal serum but not by normal Al(OH) a-treated plasma . T~~ 3. Tm? ~crs oa NaJa flava Control
1 0
2 0
Group I
0
0
Group II
0
0
Group III
0
0
VENOM ON THROMBIN GfiNFRA7iON
(n~ ~nvrrs)
Tnm: (min) 10 11 4 S 6 7 8 9 12 13 14 15 2~2 7"4 ]0"1 9"2 8"2 4O 0~9 0"5 0 0 0 0 f0"2 fOß f0ß f0 "2 ~Oß +0"2 f0"1 t0~1 0 0 2~0 8" 1 lOß 7"8 6"4 4~2 1~6 0~4 0 0 0 f0~1 f0~2 f0"4 fOß f0 "2 f0 "1 f0~1 f0 "1 0 0 0 3~1 4~8 6ß 5~1 3~1 2"1 1~2 0"7 0 0 f0 "2 fOß f0 "4 fOß f0~2 f0"1 f0"2 f0"1 0 0 0 0~9 3"8 6"9 8"1 6"1 3"9 2"8 I"6 1~1 0"4 f0~2 f0"2 f0"4 fOß f0 "1 f0"1 f0~2 f0 "2 fOß f0"2 3 0
16 0 0 0 0
Results are expressed as mean f standard error (6 experiments for each vahu). The values for groupa lI and III are significantly different from the control (p <0 "Ol). For group I, the change are not significant. 710XICON (973 Yal. 11 .
422
A. H. MOHAMED and M. M. HANNA
Itt vitro studies by MoHnMeD and HANNA (1972) showed that the venom had a fibrinogenolytic effect . However, in the present work, fibrinogen concentration was not al%cted and apart from the effect of heparin, the thrombin clotting time and prothrombin time were not prolonged. Probably the concentration of the venom in the blood in the in vivo experiments was not sufficient to produce detectable fibrinogenolysis . Acknowledgement-This work was supported by a grant No . 03-006-1 from the National Institutes of Health, Hethesda, Md ., U.S .A . REFERENtvES Btaos, R. and Dotlaus, A. S. (1953) The thromboplastin generation test . .). clin . Path . 6, 23 . Btaos, R. and M~eFutrr~tve, R. G. (1962) Human Blood Coagulation and its Disorders. 3rd ed . Oxford : Blackwell. J~QuFS, I. B. and CHARLFS, A. F. (1941) The assay of heparin. Q. II. Pharm. l, 14 . Moll~t~, A. H. and HANNA, M. M. (1972) Theefforts of NaJa floua venom on blood coagulation . Toxicon 10, 619. PrrNt=.x, W. R. and D~cu¢, J . V. (1953) A simple method for studying the ~neration of thrombin in recalcified plasma . I. clin. Path. 6, 9. Qutcx, A. J. (1951) 711e Physiology andPathology ojHemostasis . London : Henry Kimpton.
7~OXICON 1973 Vol. II .
THE IN V1V0 ANTICOAGULANT EFFECTS OF NAJA FLA YA VENOM A. H. MoI-tAtt~trrn and M. M. HANNA Faculty of Medicine, Ain Shams University, Cairo, Egypt (Acceptedjor publication 27 March 1973)
Abedract-The in vlvn effects of Naja (lava venom on blood coagulation were studied in rabbits . In sublethal doses, it had no significant effects. In lethal doses, its anticoagulant activity can be attributed to interference with thromboplastin and thrombin generation as well as an increase of circulating heparin . The fibrinogenolytic effect which was evident in vitro could not be demonstrated In vivo . Ti-tE in vitro effects of Naja jlava venom on blood coagulation have been studied by MOHAMED and HANNA (1972) . In the present work, its in vivo actions were analysed. MA~R.IA S AND METHODS venom kindly provided by Prof. W. Deichman, Chairman of the Pharmacology Dept., Faculty of Medicine, Miami University, Florida, U.S.A. was used in a sublethal dose of 0~2 mg per kg body weight and a lethal dose of 1 ~0 mg per kg. Citrated plasma and serum were prepared as described by BItîGS and MACFAxLANE (1962). Thrombase LS.H. was used in a concentration of 5 units per ml saline. One thromboplastin tablet containing 10 mg of Thrombokinase (Geigy), with calcium, was used per 2~5 ml distilled water . Bovine Fibrinogen fraction I was used. Toluidine blue was employed in a concentration of 50 mg per 100 ml saline . Protamine sulphate `Evans', was used in a dose of 40 mg per kg body weight. Rabbits of about 1 kg body weight were divided into three groups of six rabbits each. Group I received sublethal doses of 0~2 mg venom per kg body weight. Group II received lethal doses of 1 ~0 mg venom per kg body weight . Group III received protamine sulphate (40 mg per kg body weight) before injection of the lethal dose of the venom. The venom was injected intramuscularly and blood samples were obtained 30 min later. Plasma recalcification time was determined as described by Btocs and MACFAxLANt=. (1962). Thrombin clotting time of plasma was performed by adding 0~1 ml thrombin solution to 0~1 ml plasma. One stage prothrombin time was determined by adding Thrombo kinase solution to an equal volume of plasma. Thrombin generation test was performed as described by PrI-tv~r and DACIE (1953). Thromboplastin generation test was carried out according to BIGGS and DOUGLAS (1953). Estimation of fibrinogen : Qtncx's technique (1951) was slightly modified, adding 30 NIH units of thrombin instead of calcium chloride to avoid the interference of heparin . Qualitative test for heparin is described by Blcos and MACFAR.LArrE (1962) . Quantitative estimation of heparin by the method of JACQvFs and CxAxLFS (1941) . All tests were carried out at 25°C. 419 Naja j~ava
71OXlCON l973 Vol. ll.
A. H. MOHAMED and M. M. HANNA
420
1.
RESULTS
Plasma recalc~cation, thrombin-clotting and the one-stage prothrombin time Sublethal doses of Naja ,(lava venom had no effect on the coagulability of the blood
(Table 1). Lethal doses caused a significant prolongation of the plasma recalcification time, thrombin-clotting time and the one-stage prothrombin time . Addition of toluidine blue to the reaction media, with appropriate saline controls, corrected the thrombin-clotting time and the prothrombin time : it decreased the effects of the venom on the recalcification time but did not abolish this effect (Table 1). Plasma from rabbits pretreated with protamine showed normal thrombin clotting and prothrombin times and a significant retardation of the plasma recalcification time, though the changes were significantly less than those obtained in absence of protamine. TABLE ] . THE HFPE(.TS OF Nqla ftaVa VBTVOM ON FLASrlA RECALCII~iCA1ION TII~, 1f~t0l~IN CLOTIIIdO TII~ AND 7HE ONE-ôrAOE FROTHROA~IIN TII~ IN RAHHTrS
Clotting time Test plasma Control Group I Group II Group II ~- tohridine blue Group III
Rocalcification time 233 f 26 251 f 18 392 f 21* 315 f 27* 300 f 24*
Thrombin clotting time 15 f 1 1S f 2 31 t 3* 18 f 3 16 f 2
Prothrombin time 12 f 1 13 f 1 25 f 3* 13 f 2 14 f 1
Results ere expt+eased as the mean f standard error of the mean. *Values significantly differrnt from control (p < 001).
2. Thrombin generation test
The results in Table 3 show that in sublethal doses, N. gava venom had no effect on generation of thrombin. In lethal doses, it retarded its generation . In protaminized rabbits, the venom had a similar effect but significantly less marked . 3. Thromboplastin generation test
The results shown in Table 2 demonstrate that sublethal doses did not affect thromboplastin precursors . Lethal doses interfere with its generation, probably through interference with one of the scrum factors. With the injected plasma and serum, the clotting TA~ 2. THB sFFBCrs
oF
Ngja Jlava vwoM oN rfntaa®ortastnv OHNSRA7ION
Source of reagents Al(OH), Plasma
Serum
Normal Normal Group I Normal Group II Normal Group III
Normal Group I Normal Group II Normal Group III Normal
Inwbation time 1 82 76 73 110 118 75 79
Results are expnaaed as mean f standard error. 'Values significantly different from control (p <0-01) . TOX7CON 7973 Vol. 77.
f3 f4 fS f 6* f 6* fS fS
2 65f 4 66 f 4 64 f 3 % f 4* 98 f S* 69 f S 56 f 4
3 Clotting time (~) 30 f 2 27 f 3 26 f 3 76 f 3* 72 f 4* 4S f 1* 28 f 3
4
S
12 f 1 12 f 2 12 f 2 53 f 3* 33 f 2* 41 f 3* 13 f 2
12 f 2 12 f 1 12 f 3 49 f 3* 30 f 2* 38 f 2* 14 f 3
Anticoagulant Action of Naja floua Venom
421
times were prolonged. In protaminized animals, there was still a significant prolongation of the clotting time when the serum of injected rabbits was used, but not when their plasma was supplemented with normal serum. 4. Fibrinogen concentrations Sublethal and lethal doses of the venom had no significant effect on fibrinogen concentration ofthe plasma in protaminized or non-protaminized animals. The fibrinogen values (mg/100 ml) for control and groups I, II and III were 279 ± 13, 265 ± 15, 282 ± 8 and 261 ± 12, respectively. Each value is the mean ± standard error based on svc determinations . Any increase of heparin in the plasma of injected rabbits could not affect the result of this test as the amount of thrombin added (30 units) was relatively large. 5. Heparin estimation There was no significant change with sublethal doses, while lethal doses produced a significant increase in circulating heparin. The heparin values (units per 100 ml) for control and groups I and II were 0"91 ± 0"08, 0"97 ± 0-12 and 272 ± 0~2, respectively . Each value is the mean + standard error based on six determinations . DISCUSSION
In lethal doses, Naja ,hfava venom prolonged the recalcification time of plasma, the thrombin clotting time and the one-stage prothrombin time . These effects were, in part, due to an increase in the concentration of circulating heparin, as confirmed by the partial correction of the defects by toluidine blue and protamine sulphate (Table 1) and by the demonstration of an increased blood concentration of heparin. However, failure of these two agents to restore the normal coagulability of the blood indicates that other mechanisms are also in action . Toluidine blue and protamine sulphate corrected the thrombin clotting time and the one-stage prothrombin time, indicating that the ren±ain_ing defect is related to the generation of thrombin and thromboplastin . The results of the thrombin generation test (Table 3) confirm its retardation with lethal doses of the venom. The results of the thromboplastin generation test (Table 2) show the presence of an anticoagulant in the blood. When this was neutralized in animals of group III, there was still a defect which could be corrected by normal serum but not by normal Al(OH) a-treated plasma . T~~ 3. Tm? ~crs oa NaJa flava Control
1 0
2 0
Group I
0
0
Group II
0
0
Group III
0
0
VENOM ON THROMBIN GfiNFRA7iON
(n~ ~nvrrs)
Tnm: (min) 10 11 4 S 6 7 8 9 12 13 14 15 2~2 7"4 ]0"1 9"2 8"2 4O 0~9 0"5 0 0 0 0 f0"2 fOß f0ß f0 "2 ~Oß +0"2 f0"1 t0~1 0 0 2~0 8" 1 lOß 7"8 6"4 4~2 1~6 0~4 0 0 0 f0~1 f0~2 f0"4 fOß f0 "2 f0 "1 f0~1 f0 "1 0 0 0 3~1 4~8 6ß 5~1 3~1 2"1 1~2 0"7 0 0 f0 "2 fOß f0 "4 fOß f0~2 f0"1 f0"2 f0"1 0 0 0 0~9 3"8 6"9 8"1 6"1 3"9 2"8 I"6 1~1 0"4 f0~2 f0"2 f0"4 fOß f0 "1 f0"1 f0~2 f0 "2 fOß f0"2 3 0
16 0 0 0 0
Results are expressed as mean f standard error (6 experiments for each vahu). The values for groupa lI and III are significantly different from the control (p <0 "Ol). For group I, the change are not significant. 710XICON (973 Yal. 11 .
422
A. H. MOHAMED and M. M. HANNA
Itt vitro studies by MoHnMeD and HANNA (1972) showed that the venom had a fibrinogenolytic effect . However, in the present work, fibrinogen concentration was not al%cted and apart from the effect of heparin, the thrombin clotting time and prothrombin time were not prolonged. Probably the concentration of the venom in the blood in the in vivo experiments was not sufficient to produce detectable fibrinogenolysis . Acknowledgement-This work was supported by a grant No . 03-006-1 from the National Institutes of Health, Hethesda, Md ., U.S .A . REFERENtvES Btaos, R. and Dotlaus, A. S. (1953) The thromboplastin generation test . .). clin . Path . 6, 23 . Btaos, R. and M~eFutrr~tve, R. G. (1962) Human Blood Coagulation and its Disorders. 3rd ed . Oxford : Blackwell. J~QuFS, I. B. and CHARLFS, A. F. (1941) The assay of heparin. Q. II. Pharm. l, 14 . Moll~t~, A. H. and HANNA, M. M. (1972) Theefforts of NaJa floua venom on blood coagulation . Toxicon 10, 619. PrrNt=.x, W. R. and D~cu¢, J . V. (1953) A simple method for studying the ~neration of thrombin in recalcified plasma . I. clin. Path. 6, 9. Qutcx, A. J. (1951) 711e Physiology andPathology ojHemostasis . London : Henry Kimpton.
7~OXICON 1973 Vol. II .