Toxlcon, 1976, VoL 14, pp . 49-34. Paysmon Drew. Printed is ChestBritain.
THE EFFECTS OF THE PURIFIED THROMBIN-LIKE AND ANTICOAGULANT PRINCIPLES OF AGKISTRODON ACUTUS VENOM ON BLOOD COAGULATION IN YIVO CHAOIiO Ouxaxa and CIS-Mu1a T>3rra Pharmacological Institute, College of Medicine, National Taiwan University, Taipoi, Taiwan, Republic of China
(Acceptedforpublication 12 August 1975) CHA0130 OvxArio and (~-Mnia T>~ra. The effects of the purified thrombin-like and antiooagulant principles of AgMstrodon acutus venom on blood coagulation in vivo. Toxtcon 14, 49-54, 1976.-T'he effects of the purified thrombin-1>7ce and anticoagulant principles of the snake venom of Agkittrodon acutus on blood coagulation of rabbits to vtvo were studied. The thrombin-like principle caused a marked prolongations of whole blood coagulation time and ono-stage plasma prothrombin time and a marked decc+easo of the fibrinogen concentration, while no significant change in the two-stage plasma prothrombin level was detected . It is concluded that the retardation of blood clotting by the thrombin-h7ce principle was chiefly due to the daiease of plasma fibrinogen level. The anticoagulant principle caused a marked, but transient prolongation of whole blood coagulation time and one-stage plasma prothrombin time with no significant change in the two-stage plasma prothrombin level or plasma fibrinogen level . Combining these results with our previous to vitro findings, it is concludedthat the retardation of bloodclotting by the anticoagulant principlemight be due to the interference in the interaction between prothrombin and its activation factors.
INTRODUCTION
(1957) reported that in vitro the venom of Agkistrodon acutus had a coagulant action at high concentrations and an anticoagulant action at low concentrations . He also demonstrated that the coagulant effect of the venom was due to a thrombin-like action on fibrinogen and the anticoagulant action was due to the inactivation of prothrombin, thromboplastin and Ao-globulin and also due to fibrinolysis . G~mNG and OUYANC3 (1967) succeeded in isolating the coagulant (thrombin-like) and anticoagulant principles from the same venom. OUYAN(3 et al. (1971) further purified the thrombin-like principle of the venom and studied its properties in comparison with those of bovine thrombin. They (OUYANG and TBrrG, 1972) further purified the anticoagulant principle of the venom and studied its physico-chemical properties . OvxANO and TaNG (1973) also demonstrated that the inhibition of prothrombin activation by the anticoagulant principle was due to an interference in the interaction between prothrombin and its activation factors because of the reversible binding of these factors with the anticoagulant principle of the venom. However, the effects of the purified thrombin-like and anticoagulant principles of the venom on blood coagulation in vivo have not yet been elucidated, hence it was considered of interest to study these problems . OUYANG
MATFRiAi C AND METHODS The venom of Agkistrodon acutus was collected in our laboratory, centrifuged, lyophilized and stored in a desiccator containing anhydrous CaCI,. Albino rabbits weighing 1"5-25 kg were used as test animals. Food was withheld for 1fr20 hr before each experiment. The thrombin-17ce principle of Agkistrodon acutus 49 TOXICON 1976 Vot. Is
50
trHAOHO OUYANG and CIiUMING TENG
venom was purified by the procedure of Ovv.~xa et al. (1971) . Tho anticoagulant principle of the venom was purified by the procedure of OUYANa and Taxa (1972). The two principles were homogeneous as judged by electrophoresis on cellulose acetate membrane, disc electrophoresis in polyacrylamide gel and ultracentrifugal analysis. Rabbit brain thromboplastin was prepared as described by Bicas and MncF~va (1962). Aooel~ator globulin was prepared in the manner described by Jo~orr and $EHCiSRa (Techrdques in Blood Coagulation used at Wayne State University, 1953). Fibrinogen was purchased from Pentea Co ., U.S A. Bovine thrombin topical was generously supplied by Park, Devis & Co ., U.S .A. It was dissolved in S0~ glycerol saline to give a stock solution containing 1000 NIH Units per ml (S~asxs and S~rx, 1942). Qotting time of the whole blood was determined by the method of Lss and WFUZS (1913) . For the titration of plasma prothrombin level, both the one-stage method of Quccg (1938) and the two-stage method of W~uea and Ssca~es (1949) were used . For titration of plasma fibrinogen level, the method of W~Re et al. (1947) was used . RESULTS The effects on the clotting time of whole blood
An i.v. dose of 1 mg of the crude venom of Agkistrodon status, per kg of body weight, slightly prolonged the normal clotting time of whole blood. The blood samples obtained during 1-6 hr after the injection showed incomplete clots (the blood did not completely solidify in the tube). A dose of 2 mg of the crude venom per kg caused marked prolongation of the whole blood coagulation time immediately after the injection, and the blood became incoagulable for several hours starting within 1 hr after the injection. The coagulation time returned to its initial values within 48 hr. With higher doses, the effects were more pronounced. With a dose of 10 mg per kg three rabbits died immediately after the injection. An i.v. dose of 0"05 mg of the thrombin-like principle of Agkistrodon status per kg did not significantly change the normal clotting time of whole blood. However, the blood samples obtained 1~ hr after the injection showed incomplete clots. A dose of 0"1 mg of the principle per kg did not change the whole blood coagulation time significantly immediately after the injection, but the blood became incoagulable 1 hr after the injection. The coagulation time returned to its initial values within 24 hr. With greater doses, the effects were more pronounced . With doses of 0"5 and 1 mg per kg of body weight, the animals died 17 and 8 min after the injection, respectively. No shortening of the whole blood coagulation time was found after the injection of the thrombin-like principle of the venom. An i.v. dose of 0~5 mg of the anticoagulant principle per kg did not change the normal clotting time of whole blood significantly. An i.v. dose of 1 mg of the anticoagulant principle per kg caused a marked prolongation of the whole blood coagulation time starring within 5 min after the injection. However, the coagulation time returned to its initial value within approximately 4 hr (Table 1). . The effects on one-stage plasma prothrombin time
An i.v. dose of 2 mg of the crude venom per kg caused a marked prolongation of onestage plasma prothrombin time within 15 min after the injection, and the maximum effect was usually reached during 2~ hr after the injection. The plasma prothrombin time returned to its initial values within 24 hr after the injection. An i.v. dose of 0"1 mg of the thrombin-like principle per kg caused a marked prolongation of one-stage prothrombin time within 5 min after the injection and the maximum effect was usually reached during 2~ hr after the injection. The plasma prothrombin time returned to its initial value within 24 hr after the injection. An i.v. dose of 1 mg of the anticoagulant principle per kg caused only a slight prolongation of one-stage prothrombin time within 5 min after the injection. The plasma prothrombin time returned to its initial value within 30 min after the injection (Table 2). TOXICON 1976 Vnl. I~t
A. acutus Venom on Blood Coagulation
Sl
TABLE 1. WHOIB HLAOD CL011iN0 TII~S IN RABHTIS AFTER THE INTRAVBNOUS IIQJECITONS OF THB CRUDE VENOM, TEB 1'SROMBIPi-IIYH AND ANTi00A0ULAN1" PRINCIPIES OF AgkLstrodon acutus v~toM
Crude venom Time
Control S min 13 30 i hr 2 4 6 24 2 days 3 4 S 6 7 8
2"0 mg/kg (n=4) 2"3 10" 0 9"6 7" 0
S"8 4"0 3"3 2"9 23
f 0"2 f 2"8 f 3"2 f 0"S' I I I f i"0' f 0~6' f 0"4 f 0"2 f 0"2
S"0 mg/kg (n=3)
Thrombin-like principle 0~1 mg/kg (n=3)
0"2 mg/kg (n=5)
Whole blood clotting time (min) f 2" S f 0" 3 2"8 0"2 2" 0 ~ 0"2 24"3 f 4"2 3"3 f 0"2 3"0 f 0"4 I 2"S f 0"3 3" 2 f 0"2 I 3"0 f 0" S' 2"S ~ 0"2' I I I I I I I I I I 4"3 ~ 0"4' I S"8 f 0"S' 2"7 f 0"2' 3"4 ~ 0"2' S"2 ~ 0"4' 2"3 f 0"4 3"2 f 0"3' S"0 ~ 0"3 2"S f 0" 3 3"0 f 0"2 4"7 f 0"4 3"7 f 0"2 4"S ~ 0"3 4"2 f 0"4 22 f 0"2 3"7 f 02 3"3 f 0"2
Anticoagulant principle i"0 mg/kg (n=4) 2"8 f 0"3 >6 hr
THE EFFECTS OF THE PURIFIED THROMBIN-LIKE AND ANTICOAGULANT PRINCIPLES OF AGKISTRODON ACUTUS VENOM ON BLOOD COAGULATION IN YIVO CHAOIiO Ouxaxa and CIS-Mu1a T>3rra Pharmacological Institute, College of Medicine, National Taiwan University, Taipoi, Taiwan, Republic of China
(Acceptedforpublication 12 August 1975) CHA0130 OvxArio and (~-Mnia T>~ra. The effects of the purified thrombin-like and antiooagulant principles of AgMstrodon acutus venom on blood coagulation in vivo. Toxtcon 14, 49-54, 1976.-T'he effects of the purified thrombin-1>7ce and anticoagulant principles of the snake venom of Agkittrodon acutus on blood coagulation of rabbits to vtvo were studied. The thrombin-like principle caused a marked prolongations of whole blood coagulation time and ono-stage plasma prothrombin time and a marked decc+easo of the fibrinogen concentration, while no significant change in the two-stage plasma prothrombin level was detected . It is concluded that the retardation of blood clotting by the thrombin-h7ce principle was chiefly due to the daiease of plasma fibrinogen level. The anticoagulant principle caused a marked, but transient prolongation of whole blood coagulation time and one-stage plasma prothrombin time with no significant change in the two-stage plasma prothrombin level or plasma fibrinogen level . Combining these results with our previous to vitro findings, it is concludedthat the retardation of bloodclotting by the anticoagulant principlemight be due to the interference in the interaction between prothrombin and its activation factors.
INTRODUCTION
(1957) reported that in vitro the venom of Agkistrodon acutus had a coagulant action at high concentrations and an anticoagulant action at low concentrations . He also demonstrated that the coagulant effect of the venom was due to a thrombin-like action on fibrinogen and the anticoagulant action was due to the inactivation of prothrombin, thromboplastin and Ao-globulin and also due to fibrinolysis . G~mNG and OUYANC3 (1967) succeeded in isolating the coagulant (thrombin-like) and anticoagulant principles from the same venom. OUYAN(3 et al. (1971) further purified the thrombin-like principle of the venom and studied its properties in comparison with those of bovine thrombin. They (OUYANG and TBrrG, 1972) further purified the anticoagulant principle of the venom and studied its physico-chemical properties . OvxANO and TaNG (1973) also demonstrated that the inhibition of prothrombin activation by the anticoagulant principle was due to an interference in the interaction between prothrombin and its activation factors because of the reversible binding of these factors with the anticoagulant principle of the venom. However, the effects of the purified thrombin-like and anticoagulant principles of the venom on blood coagulation in vivo have not yet been elucidated, hence it was considered of interest to study these problems . OUYANG
MATFRiAi C AND METHODS The venom of Agkistrodon acutus was collected in our laboratory, centrifuged, lyophilized and stored in a desiccator containing anhydrous CaCI,. Albino rabbits weighing 1"5-25 kg were used as test animals. Food was withheld for 1fr20 hr before each experiment. The thrombin-17ce principle of Agkistrodon acutus 49 TOXICON 1976 Vot. Is
50
trHAOHO OUYANG and CIiUMING TENG
venom was purified by the procedure of Ovv.~xa et al. (1971) . Tho anticoagulant principle of the venom was purified by the procedure of OUYANa and Taxa (1972). The two principles were homogeneous as judged by electrophoresis on cellulose acetate membrane, disc electrophoresis in polyacrylamide gel and ultracentrifugal analysis. Rabbit brain thromboplastin was prepared as described by Bicas and MncF~va (1962). Aooel~ator globulin was prepared in the manner described by Jo~orr and $EHCiSRa (Techrdques in Blood Coagulation used at Wayne State University, 1953). Fibrinogen was purchased from Pentea Co ., U.S A. Bovine thrombin topical was generously supplied by Park, Devis & Co ., U.S .A. It was dissolved in S0~ glycerol saline to give a stock solution containing 1000 NIH Units per ml (S~asxs and S~rx, 1942). Qotting time of the whole blood was determined by the method of Lss and WFUZS (1913) . For the titration of plasma prothrombin level, both the one-stage method of Quccg (1938) and the two-stage method of W~uea and Ssca~es (1949) were used . For titration of plasma fibrinogen level, the method of W~Re et al. (1947) was used . RESULTS The effects on the clotting time of whole blood
An i.v. dose of 1 mg of the crude venom of Agkistrodon status, per kg of body weight, slightly prolonged the normal clotting time of whole blood. The blood samples obtained during 1-6 hr after the injection showed incomplete clots (the blood did not completely solidify in the tube). A dose of 2 mg of the crude venom per kg caused marked prolongation of the whole blood coagulation time immediately after the injection, and the blood became incoagulable for several hours starting within 1 hr after the injection. The coagulation time returned to its initial values within 48 hr. With higher doses, the effects were more pronounced. With a dose of 10 mg per kg three rabbits died immediately after the injection. An i.v. dose of 0"05 mg of the thrombin-like principle of Agkistrodon status per kg did not significantly change the normal clotting time of whole blood. However, the blood samples obtained 1~ hr after the injection showed incomplete clots. A dose of 0"1 mg of the principle per kg did not change the whole blood coagulation time significantly immediately after the injection, but the blood became incoagulable 1 hr after the injection. The coagulation time returned to its initial values within 24 hr. With greater doses, the effects were more pronounced . With doses of 0"5 and 1 mg per kg of body weight, the animals died 17 and 8 min after the injection, respectively. No shortening of the whole blood coagulation time was found after the injection of the thrombin-like principle of the venom. An i.v. dose of 0~5 mg of the anticoagulant principle per kg did not change the normal clotting time of whole blood significantly. An i.v. dose of 1 mg of the anticoagulant principle per kg caused a marked prolongation of the whole blood coagulation time starring within 5 min after the injection. However, the coagulation time returned to its initial value within approximately 4 hr (Table 1). . The effects on one-stage plasma prothrombin time
An i.v. dose of 2 mg of the crude venom per kg caused a marked prolongation of onestage plasma prothrombin time within 15 min after the injection, and the maximum effect was usually reached during 2~ hr after the injection. The plasma prothrombin time returned to its initial values within 24 hr after the injection. An i.v. dose of 0"1 mg of the thrombin-like principle per kg caused a marked prolongation of one-stage prothrombin time within 5 min after the injection and the maximum effect was usually reached during 2~ hr after the injection. The plasma prothrombin time returned to its initial value within 24 hr after the injection. An i.v. dose of 1 mg of the anticoagulant principle per kg caused only a slight prolongation of one-stage prothrombin time within 5 min after the injection. The plasma prothrombin time returned to its initial value within 30 min after the injection (Table 2). TOXICON 1976 Vnl. I~t
A. acutus Venom on Blood Coagulation
Sl
TABLE 1. WHOIB HLAOD CL011iN0 TII~S IN RABHTIS AFTER THE INTRAVBNOUS IIQJECITONS OF THB CRUDE VENOM, TEB 1'SROMBIPi-IIYH AND ANTi00A0ULAN1" PRINCIPIES OF AgkLstrodon acutus v~toM
Crude venom Time
Control S min 13 30 i hr 2 4 6 24 2 days 3 4 S 6 7 8
2"0 mg/kg (n=4) 2"3 10" 0 9"6 7" 0
S"8 4"0 3"3 2"9 23
f 0"2 f 2"8 f 3"2 f 0"S' I I I f i"0' f 0~6' f 0"4 f 0"2 f 0"2
S"0 mg/kg (n=3)
Thrombin-like principle 0~1 mg/kg (n=3)
0"2 mg/kg (n=5)
Whole blood clotting time (min) f 2" S f 0" 3 2"8 0"2 2" 0 ~ 0"2 24"3 f 4"2 3"3 f 0"2 3"0 f 0"4 I 2"S f 0"3 3" 2 f 0"2 I 3"0 f 0" S' 2"S ~ 0"2' I I I I I I I I I I 4"3 ~ 0"4' I S"8 f 0"S' 2"7 f 0"2' 3"4 ~ 0"2' S"2 ~ 0"4' 2"3 f 0"4 3"2 f 0"3' S"0 ~ 0"3 2"S f 0" 3 3"0 f 0"2 4"7 f 0"4 3"7 f 0"2 4"S ~ 0"3 4"2 f 0"4 22 f 0"2 3"7 f 02 3"3 f 0"2
Anticoagulant principle i"0 mg/kg (n=4) 2"8 f 0"3 >6 hr
