262
Biochimica et Biophysica Acta, 1160 (1992) 262-268 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34342
A novel a-type fibrinogenase from Agkistrodon rhodostoma snake venom Tur-Fu Huang, Mei-Chi Chang, Hui-Chin Peng and Che-Ming Teng Pharmacological Institute, Collegeof Medicine, National Taiwan University, Taipei (Taiwan) (Received 26 June 1992)
Key words: Fibrinogenase; Fibrinolysis; Fibrinogenolysis;Snake venom By means of CM-Sephadex C-50 column chromatography, gel-filtration on sephadex G-75 and Sephacryl S-200 columns, a purified fibrinogenase, kistomin, was obtained from venom of Agkistrodon rhodostoma. It was a single peptide-chain with a molecular mass of about 21800 Da containing about 202 amino-acid residues as revealed by amino acid analysis. Kistomin preferentially cleaved Aa- and subsequently the y-chain of fibrinogen, leaving the B/3-chain unaffected. Its fibrinogenolytic activity was estimated to be 36.6 +_4.5 mg/min per mg protein and was inhibited by the pretreatment of EDTA, suggesting that it is a metalloproteinase. Its fibrinogenolytic activity in platelet-poor plasma is much less potent as compared to that in purified fibrinogen solution. It inhibited ristocetin-induced aggregation of human platelets in a dose-dependent manner in the presence of von Willebrand factor.
Introduction
Snake venom constituents affect blood coagulation and platelet aggregation, an important cellular process in thrombosis and hemostasis [1]. Metalloproteinases are frequently found in snake venoms, especially from Crotalidae and Viperidae venoms [2-4]. Snake venom of Agkistrodon rhodostoma (Malayan pit viper) contains thrombin-like peptide [5-9], a-fibrinogenase [10], platelet aggregation inducer [11], fibrinogen receptor antagonist [12], phosphodiesterase, phospholipase A, 5'-nucleotidase and hyaluronidase [13]. The thrombinlike peptide has been used as a defibrinogenating agent in thrombosis [14,15]. Rhodostomin, a fibrinogen-receptor antagonist, is being evaluated for its antithromboric effect because fibrinogen binding to platelets is a common pathway for platelet aggregation [16]. In addition, it has been demonstrated that interaction of platelet-adhesive proteins may play a role in metastasis, since G R G D S and monoclonal antibodies directed against the common platelet-adhesive protein receptor for fibronectin, von Willebrand factor and fibrinogen, inhibited the metastases of tumor cells [17]. Many fibrinogenases have been purified from venoms of Agkistrodon acutus [18,19], Agkistrodon c. con-
Correspondence to: T.-F. Huang, Pharmacological Institute, College of Medicine, National Taiwan University, 1 Jen-Ai Road, Section 1, Taipei, Taiwan.
tortrix [20], Crotalus atrox [21-23] and Naja nigricollis [24], etc. An a-type fibrinogenase has been purified from venom of A. rhodostoma [10]. In this paper, we describe the discovery of a-type fibrinogenase from this venom and found that it was a single peptide-chain with a molecular weight of about 21800. It preferentially cleaved the A a - and subsequently the y-chain of fibrinogen without cleaving the B/3-chain. In addition, it inhibited ristocetin-induced aggregation of human platelets in the presence of von Willebrand factor. Materials and Methods
Materials The venom of A. rhodostoma was collected, centrifuged, lyophilized and stored at - 2 0 ° C . CM-Sephadex C-50, Sephadex G-75 and Sephacryl S-200 were purchased from Pharmacia, Sweden. Ampholytes were purchased from Bio-Rad. H u m a n thrombin, disodium E D T A , acrylamide, human fibrinogen, ristocetin, prostaglandin E I (PGE1), apyrase and heparin were purchased from Sigma. H u m a n von Willebrand factor (vWF) was prepared as previously described [37]. Other chemical reagents of analytical grade were obtained from Wako. Methods CM-Sephadex C-50 column chromatography. 2.0 g of the crude venom dissolved in 0.05 M ammonium acetate (pH 6.0) was applied to a column (4 x 90 cm) packed with CM-Sephadex C-50 preequilibrated with
263 0.05 M ammonium acetate. Gradient elution was carried out in three stages; (i), 0.05 M ammonium acetate (pH 6.0), 800 ml; (ii), 0.05 M ammonium acetate (pH 6.0), 600 ml vs. 0.4 M ammonium acetate (pH 8.0), 600 ml; (iii), 0.4 M ammonium acetate (pH 8.0), 600 ml vs. 0.8 M ammonium acetate (pH 8.0), 600 ml. The flow rate was adjusted to 22 m l / h , and eluates of 6 ml per tube were collected. The effluent was monitored continuously at 278 nm and 5°C with an LKB Uvicord. Gel filtration. Sephadex G-75 and Sephacryl S-200 were prepared in 0.01 M ammonium bicarbonate and packed in columns of various size according to the amount of the venom. The elution was carried out with 0.01 M ammonium bicarbonate. The flow rate was adjusted to 15-22 m l / h and eluates of 3 ml per tube were collected. Isoelectric focusing in the Rotofor cell. The sample was added to 2.5 ml ampholytes (pH range 3-10, 40% (w/v)) and its volume was brought to 50.0 ml. This material was applied to the Rotofor cell without further treatment. The sample was focused for 4 h at 12 W constant power. The initial condition was 465 V and 24 mA. At equilibrium, these parameters were 1020 V and 1 mA. 20 fractions were harvested and their pH values measured. Each fraction was assayed for protein concentration with the method of Bradford et al. [25] using bovine serum albumin (BSA) as the standard. Electorphoresis. The freshly prepared sample solution was added to an equal volume of sample buffer containing 10 M u r e a / 4 % SDS in the presence or absence of 4%/3-mercaptoethanol. A 7.5 or 12.5% gel was used. After electrophoresis, the gel was prefixed by the mixture of 50% methanol, 10% acetic acid and glutaraldehyde (10%) for 30 min. It was stained with Coomassie brilliant blue for 1 h, then destained. Mobility was plotted against the molecular weights of some standard proteins (myosin, 200000; /3-galactosidase, 116250; phosphorylase b, 97000; bovine serum albumin, 66200; ovalbumin, 42699; carbonic anhydrase, 29 000;/3-1actoglobulin, 18 400; lysozyme, 14 300; bovine trypsin inhibitor, 6200 and insulin A and B chains, 3000) on a semi-log scale and the molecular weights of samples were estimated by interpolation. Amino-acid analysis. Amino-acid analysis was carried out on a Beckman Model M121 analyzer using a two-column system. The hydrolysis was carried out in a l l0°C oven under a nitrogen vacuum atmosphere for 22 h. Preparation of human washed platelets. This was carried out according to the method described by Huang et al. [16]. Platelet aggregation was performed at 37°C using a LumioAggregometer (Chrono-Log). The extent of aggregation was expressed in light transmission unit. Fibrinogenolytic activity. A modified method of Ware et al. [26] was used. 0.2 ml of fibrinogen solution (10
m g / m l , in saline) and 50 /zl of venom solution were mixed and incubated at 37°C for various time intervals. The reation was stopped by adding 10 mM EDTA. The above incubation mixture was taken for the assay of clottable fibrinogen and for electrophoresis [32]. To the above mixture, 2.7 ml imidazole saline buffer ((pH 7.4), containing 2.5 mM Ca 2+) was added, then thrombin (2 U / m l in final concentration) was added to clot the remaining fibrinogen. The fibrin clot was removed carefully with a rod, blotted on filter paper and washed with saline. The fibrin was put in a tube containing 1 ml of 10% N a O H and boiled for 30 min, then 1 ml water and 3 ml 20% NazCO 3 was added, mixed and finally 1 ml of phenol reagent. After mixing and standing at room temperature for 20 min, the absorbance was measured at 540 nm. Fibrinolytic activity. The method of Astrup and Mullertz [27] was employed. 200 p.l of thrombin (100 U / m l ) was added to a plate containing 10 ml of 2 m g / m l fibrinogen solution to form fibrin. Test samples (50 #1) were applied onto fibrin surface and plates were incubated for 24 h at 37°C. Fibrinolytic activities were expressed as dimension of lyzed zone. Hemorrhagic activity. Hemorrhagic activity was determined by the method reported previously [28]. The method consists of an intracutaneous injection of 50 p.1 of the test solution (1 m g / m l ) into the back skins of mice, measurement of the sizes of the hemorrhagic lesion of the removed skin were made after 20 h treatment.
The inhibitory effects of EDTA on fibrinogenolytic activity. The test samples were pretreated with 10 mM E D T A at 37°C for 30 min and dialysed. Then the fibrinogenolytic activity of the EDTA-treated sample was measured as mentioned previously. Results
Fractionation of A. rhodostoma venom Using CM-Sephadex C-50 column chromatography, the venom was separated into 12 fractions (Fig. 1). Five fractions were obtained in the first stage elution and seven fractions in the third stage elution. Thrombin-like activity and fibrinolytic activity were concentrated at Fraction VIII. This fraction was desalted by dialysis and refractionated into three fractions on a Sephadex G-75 column. Fraction 2 possessed fibrino(geno)lytic and antiplatelet activities (Fig. 2) 3 mg of this subfraction was further separated by isoelectric focusing using the Rotofor cell. Two protein peaks with isoelectric points of pH 7.6 and 8.2, respectively, were found to possess fibrinolytic activity. The fibrinolytic activity of the fraction with p I 7.6 was more potent than that of the other fraction (pI 8.2). However, as shown in Fig. 3 only the fraction with p I 8.2 possessed the inhibitory activity on ristocetin (0.6 mg/ml)-induced platelet ag-
264 0.05M
0.05M
0.4M
0.SM
pH6.0
pH8.0
plI 8.0
I
I
0.500Pit 6.0
A A
VII VIII
o+oo[
L
400
A"
3o0 ~
i
Biochimica et Biophysica Acta, 1160 (1992) 262-268 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34342
A novel a-type fibrinogenase from Agkistrodon rhodostoma snake venom Tur-Fu Huang, Mei-Chi Chang, Hui-Chin Peng and Che-Ming Teng Pharmacological Institute, Collegeof Medicine, National Taiwan University, Taipei (Taiwan) (Received 26 June 1992)
Key words: Fibrinogenase; Fibrinolysis; Fibrinogenolysis;Snake venom By means of CM-Sephadex C-50 column chromatography, gel-filtration on sephadex G-75 and Sephacryl S-200 columns, a purified fibrinogenase, kistomin, was obtained from venom of Agkistrodon rhodostoma. It was a single peptide-chain with a molecular mass of about 21800 Da containing about 202 amino-acid residues as revealed by amino acid analysis. Kistomin preferentially cleaved Aa- and subsequently the y-chain of fibrinogen, leaving the B/3-chain unaffected. Its fibrinogenolytic activity was estimated to be 36.6 +_4.5 mg/min per mg protein and was inhibited by the pretreatment of EDTA, suggesting that it is a metalloproteinase. Its fibrinogenolytic activity in platelet-poor plasma is much less potent as compared to that in purified fibrinogen solution. It inhibited ristocetin-induced aggregation of human platelets in a dose-dependent manner in the presence of von Willebrand factor.
Introduction
Snake venom constituents affect blood coagulation and platelet aggregation, an important cellular process in thrombosis and hemostasis [1]. Metalloproteinases are frequently found in snake venoms, especially from Crotalidae and Viperidae venoms [2-4]. Snake venom of Agkistrodon rhodostoma (Malayan pit viper) contains thrombin-like peptide [5-9], a-fibrinogenase [10], platelet aggregation inducer [11], fibrinogen receptor antagonist [12], phosphodiesterase, phospholipase A, 5'-nucleotidase and hyaluronidase [13]. The thrombinlike peptide has been used as a defibrinogenating agent in thrombosis [14,15]. Rhodostomin, a fibrinogen-receptor antagonist, is being evaluated for its antithromboric effect because fibrinogen binding to platelets is a common pathway for platelet aggregation [16]. In addition, it has been demonstrated that interaction of platelet-adhesive proteins may play a role in metastasis, since G R G D S and monoclonal antibodies directed against the common platelet-adhesive protein receptor for fibronectin, von Willebrand factor and fibrinogen, inhibited the metastases of tumor cells [17]. Many fibrinogenases have been purified from venoms of Agkistrodon acutus [18,19], Agkistrodon c. con-
Correspondence to: T.-F. Huang, Pharmacological Institute, College of Medicine, National Taiwan University, 1 Jen-Ai Road, Section 1, Taipei, Taiwan.
tortrix [20], Crotalus atrox [21-23] and Naja nigricollis [24], etc. An a-type fibrinogenase has been purified from venom of A. rhodostoma [10]. In this paper, we describe the discovery of a-type fibrinogenase from this venom and found that it was a single peptide-chain with a molecular weight of about 21800. It preferentially cleaved the A a - and subsequently the y-chain of fibrinogen without cleaving the B/3-chain. In addition, it inhibited ristocetin-induced aggregation of human platelets in the presence of von Willebrand factor. Materials and Methods
Materials The venom of A. rhodostoma was collected, centrifuged, lyophilized and stored at - 2 0 ° C . CM-Sephadex C-50, Sephadex G-75 and Sephacryl S-200 were purchased from Pharmacia, Sweden. Ampholytes were purchased from Bio-Rad. H u m a n thrombin, disodium E D T A , acrylamide, human fibrinogen, ristocetin, prostaglandin E I (PGE1), apyrase and heparin were purchased from Sigma. H u m a n von Willebrand factor (vWF) was prepared as previously described [37]. Other chemical reagents of analytical grade were obtained from Wako. Methods CM-Sephadex C-50 column chromatography. 2.0 g of the crude venom dissolved in 0.05 M ammonium acetate (pH 6.0) was applied to a column (4 x 90 cm) packed with CM-Sephadex C-50 preequilibrated with
263 0.05 M ammonium acetate. Gradient elution was carried out in three stages; (i), 0.05 M ammonium acetate (pH 6.0), 800 ml; (ii), 0.05 M ammonium acetate (pH 6.0), 600 ml vs. 0.4 M ammonium acetate (pH 8.0), 600 ml; (iii), 0.4 M ammonium acetate (pH 8.0), 600 ml vs. 0.8 M ammonium acetate (pH 8.0), 600 ml. The flow rate was adjusted to 22 m l / h , and eluates of 6 ml per tube were collected. The effluent was monitored continuously at 278 nm and 5°C with an LKB Uvicord. Gel filtration. Sephadex G-75 and Sephacryl S-200 were prepared in 0.01 M ammonium bicarbonate and packed in columns of various size according to the amount of the venom. The elution was carried out with 0.01 M ammonium bicarbonate. The flow rate was adjusted to 15-22 m l / h and eluates of 3 ml per tube were collected. Isoelectric focusing in the Rotofor cell. The sample was added to 2.5 ml ampholytes (pH range 3-10, 40% (w/v)) and its volume was brought to 50.0 ml. This material was applied to the Rotofor cell without further treatment. The sample was focused for 4 h at 12 W constant power. The initial condition was 465 V and 24 mA. At equilibrium, these parameters were 1020 V and 1 mA. 20 fractions were harvested and their pH values measured. Each fraction was assayed for protein concentration with the method of Bradford et al. [25] using bovine serum albumin (BSA) as the standard. Electorphoresis. The freshly prepared sample solution was added to an equal volume of sample buffer containing 10 M u r e a / 4 % SDS in the presence or absence of 4%/3-mercaptoethanol. A 7.5 or 12.5% gel was used. After electrophoresis, the gel was prefixed by the mixture of 50% methanol, 10% acetic acid and glutaraldehyde (10%) for 30 min. It was stained with Coomassie brilliant blue for 1 h, then destained. Mobility was plotted against the molecular weights of some standard proteins (myosin, 200000; /3-galactosidase, 116250; phosphorylase b, 97000; bovine serum albumin, 66200; ovalbumin, 42699; carbonic anhydrase, 29 000;/3-1actoglobulin, 18 400; lysozyme, 14 300; bovine trypsin inhibitor, 6200 and insulin A and B chains, 3000) on a semi-log scale and the molecular weights of samples were estimated by interpolation. Amino-acid analysis. Amino-acid analysis was carried out on a Beckman Model M121 analyzer using a two-column system. The hydrolysis was carried out in a l l0°C oven under a nitrogen vacuum atmosphere for 22 h. Preparation of human washed platelets. This was carried out according to the method described by Huang et al. [16]. Platelet aggregation was performed at 37°C using a LumioAggregometer (Chrono-Log). The extent of aggregation was expressed in light transmission unit. Fibrinogenolytic activity. A modified method of Ware et al. [26] was used. 0.2 ml of fibrinogen solution (10
m g / m l , in saline) and 50 /zl of venom solution were mixed and incubated at 37°C for various time intervals. The reation was stopped by adding 10 mM EDTA. The above incubation mixture was taken for the assay of clottable fibrinogen and for electrophoresis [32]. To the above mixture, 2.7 ml imidazole saline buffer ((pH 7.4), containing 2.5 mM Ca 2+) was added, then thrombin (2 U / m l in final concentration) was added to clot the remaining fibrinogen. The fibrin clot was removed carefully with a rod, blotted on filter paper and washed with saline. The fibrin was put in a tube containing 1 ml of 10% N a O H and boiled for 30 min, then 1 ml water and 3 ml 20% NazCO 3 was added, mixed and finally 1 ml of phenol reagent. After mixing and standing at room temperature for 20 min, the absorbance was measured at 540 nm. Fibrinolytic activity. The method of Astrup and Mullertz [27] was employed. 200 p.l of thrombin (100 U / m l ) was added to a plate containing 10 ml of 2 m g / m l fibrinogen solution to form fibrin. Test samples (50 #1) were applied onto fibrin surface and plates were incubated for 24 h at 37°C. Fibrinolytic activities were expressed as dimension of lyzed zone. Hemorrhagic activity. Hemorrhagic activity was determined by the method reported previously [28]. The method consists of an intracutaneous injection of 50 p.1 of the test solution (1 m g / m l ) into the back skins of mice, measurement of the sizes of the hemorrhagic lesion of the removed skin were made after 20 h treatment.
The inhibitory effects of EDTA on fibrinogenolytic activity. The test samples were pretreated with 10 mM E D T A at 37°C for 30 min and dialysed. Then the fibrinogenolytic activity of the EDTA-treated sample was measured as mentioned previously. Results
Fractionation of A. rhodostoma venom Using CM-Sephadex C-50 column chromatography, the venom was separated into 12 fractions (Fig. 1). Five fractions were obtained in the first stage elution and seven fractions in the third stage elution. Thrombin-like activity and fibrinolytic activity were concentrated at Fraction VIII. This fraction was desalted by dialysis and refractionated into three fractions on a Sephadex G-75 column. Fraction 2 possessed fibrino(geno)lytic and antiplatelet activities (Fig. 2) 3 mg of this subfraction was further separated by isoelectric focusing using the Rotofor cell. Two protein peaks with isoelectric points of pH 7.6 and 8.2, respectively, were found to possess fibrinolytic activity. The fibrinolytic activity of the fraction with p I 7.6 was more potent than that of the other fraction (pI 8.2). However, as shown in Fig. 3 only the fraction with p I 8.2 possessed the inhibitory activity on ristocetin (0.6 mg/ml)-induced platelet ag-
264 0.05M
0.05M
0.4M
0.SM
pH6.0
pH8.0
plI 8.0
I
I
0.500Pit 6.0
A A
VII VIII
o+oo[
L
400
A"
3o0 ~
i
