Mechanism of action of the platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom

The platelet aggregation inhibitor purified from Agkistrodon halys snake venom inhibited rabbit platelet aggregations induced by thrombin, sodium arachidonate, collagen or ionophore A-23187. The ic₅₀ was about 11 μg/ml in platelet aggregation regardless of which aggregation inducer was used. β-Mercaptoethanol abolished both the phospholipase A enzymatic and platelet aggregation inhibitory activities of this venom inhibitor. p-Bromophenacyl bromide-treated venom inhibitor lost almost completely its phosphilipase A enzymatic activity, but retained its platelet aggregation inhibitory effect. In the presence of EGTA, the venom inhibitor still showed the same inhibitory activity on thrombin-, sodium arachidonate-, collagen- or ionophore A23187-induced platelet aggregations triggered by successive addition of Ca²⁺. The activation of platelet phospholipase A and the serotonin release reaction triggered by Ca²⁺ influx were unaffected by this venom inhibitor. It also inhibited the clot retraction of platelet-rich plasma. It is concluded that the inhibitory effect of the venom inhibitor on platelet aggregation is independent of its phospholipase A enzymatic activity. Its mode of action is different from those of other known platelet inhibitory drugs. This venom inhibitor possibly acts on a common step subsequent to platelet shape change, leading to inhibition of platelet aggregation.     

A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom

Y.-S. Li, K.-F. Liu, Q.-C. Wang, Y.-L. Rpan and G.-C. Tu. A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom. Toxicon23, 895–903, 1985. — By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G — 75, a potent platelet function inhibitor was purified from Vipera russelli siamensis venom. It appeared as a single protein band on polyacrylamide gel electrophoresis in the presence or absence of SDS, and consists of 123 amino acid residues. Its NH₃-terminal residue is serine. It showed the following characteristics: molecular weight, 13,800; isoelectric point, 10.4; ld(In50), 0.5 ± 0.12 mg/kg (i.v.). The platelet inhibitor exhibited phospholipase A₂ activity with a specific activity of 35 μmoles/min/mg. From 2 g of the venom, 70 mg of the purified inhibitor was obtained. Inhibition of human platelet aggregation induced by ADP or adrenaline was dose-dependent, with id(In50) of 1.14μg/ml or 0.37 μg/ml, respectively. The platelet aggregation induced by thrombin or collagen was also inhibited and the inhibitory activity on platelet aggregation was heat stable (at 100°C, 20 min) in an acidic medium (pH 5.8), while its phospholipase A₂ activity was relatively heat labile under the same condition. The release of ³H-serotonin in platelets stimulated by ADP was also inhibited and this was positively correlated with inhibition of platelet aegregation induced by ADP (r = 0.998, P < 0.002).     

Expression and characterization of a novel plasminogen activator from Agkistrodon halys venom

A venom gland cDNA library of Agkistrodon halys was constructed and screened with a probe based on the consensus sequence of venomic serine proteases. Next, we determined the sequences of the entire open reading frames of two selected positives which were found to encode novel serine proteases of 234 and 233 amino acids in length and named as Haly-PA and Haly 2, respectively. Upon protein data base search, Haly-PA showed the highest similarity of 82% to the previously characterized plasminogen activator, TSV-PA (Zhang et al. 1995, J. Biol. Chem., 270, 10246–10255). Haly 2 displayed a 78% similarity to β-fibrinogenase (Hung et al. 1994, B. B. R. C., 205, 1707–1715). Haly-PA was successfully expressed using the baculovirus system and secreted into the culture media as a 32 kDa glycoprotein. In the western analysis of snake venom, anti-Haly-PA antibody detected the same size of band indicating that this enzyme is a component of snake venom. Recombinant Haly-PA was purified to homogeneity using the combination of anion exchange and gel filtration column. In the fibrino(geno)lytic assay, recombinant Haly-PA displayed an indirect fibrino(geno)lytic activity depending on the presence of plasminogen and cleaved the plasminogen to generate the active plasmin. These results indicate that Haly-PA is a plasminogen activator and displays fibrino(geno)lytic activity through conversion of plasminogen to plasmin.     

In vivo effects of the purified thrombin-like and anticoagulant principles of Agkistrodon acutus (Hundred pace snake) venom

The thrombin-like enzyme of Agkistrodon acutus venom caused a marked prolongation of whole blood coagulation time and one-stage plasma prothrombin time and a marked decrease of fibrinogen level, while no significant change in the two-stage plasma prothrombin level was detected. The retardation of blood clotting by the thrombin-like enzyme was chiefly due to the decrease of plasma fibrinogen level. The thrombin-like enzyme did not cause significant change in blood pressure, heart rate, EKG or respiration at a defibrinating dose (0·1 mg/kg, i.v.). It also did not induce platelet aggregation. Fibrin formed by the thrombin-like enzyme was not cross-linked. The thrombin-like enzyme could further digest the -chain of fibrin and the fibrin formed by this enzyme was more susceptible to plasmin degradation than fibrin formed by thrombin.

The anticoagulant principle of Agkistrodon acutus venom 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 in vitro findings, it is concluded that the retardation of blood clotting by the anticoagulant principle might be due to the interference in the interaction between prothrombin and its activation factors. The anticoagulant principle did not affect platelet aggregation induced by ADP. It also did not cause significant change in blood pressure, heart rate, EKG or respiration at an i.v. dose of 1–10 mg/kg.     

Purification and partial characterization of hyaluronidase from five pace snake (Agkistrodon acutus) venom

—A hyaluronidase (EC 3.2.1. 35) was isolated and purified from Agkistrodon acutus venom. The purification procedure included CM-Sephadex C-50 chromatography, gel-filtration on Sephadex G-75 and CM-Sephadex C-25 chromatography. The purified preparation of the enzyme was homogeneous on polyacrylamide gel electrophoresis at pH 4.3, a 45-fold purification being obtained. The hyaluronidase was a glycoprotein (positive PAS staining) with a molecular weight of 33,000 and a pI of 10.3. No hemorrhagic activity was found. The hyaluronidase had an optimum pH of 3.5–5.0 and an optimum temperature of 37°C. It was heat sensitive, was more stable in the acidic than in the neutral region, and lost its activity in the alkaline region. Fe²⁺, Cu²⁺ and heparin inhibited the venom hyaluronidase. The K_m value for hyaluronic acid was 6.2 × 10⁻³ mg/ml.     

Inhibition of platelet aggregation by 5′-nucleotidase purified from Trimeresurus gramineus snake venom

By means of DEAE-Sephadex A-50 column chromatography and gel filtrations on Sephadex G-75, Sephacryl S-300 and Sephadex G-100, successively, a potent 5′-nucleotidase was purified from Trimeresurus gramineus venom. The venom 5′-nucleotidase is a single polypeptide chain and homogeneous as judged by SDS-polyacrylamide gel electrophoresis. It is a thermostable glycoprotein consisting of 589 amino acid residues. Its molecular weight was estimated to be 74,000 by SDS-polyacrylamide gel electrophoresis. It possessed nucleotidase activities toward adenosine monophosphate and adenosine diphosphate. The specific activities toward AMP and ADP were 504 ± 28 and 101 ± 8 μg P_i/min per mg, respectively. Pre-incubation of this venom's 5′-nucleotidase with ADP resulted in the cleavage of ADP and formation of adenosine. The 5′-nucleotidase activity was inhibited by EDTA. Both Zn²⁺ and Co²⁺ reversed the inhibitory effect of EDTA.In rabbit platelet-rich plasma, it inhibited completely the ADP (2 × 10^-5 g/ml)-induced platelet aggregation. It also inhibited the platelet aggregations induced by sodium arachidonate (100 μM), collagen (20 μg/ml) and ionophore A-23187 (5 μM). In rabbit platelet suspensions, it inhibited the platelet aggregation induced by ADP (2 × 10^-5 g/ml), sodium arachidonate (100 μM) and low concentration of thrombin (0.03 U/ml). The collagen (20 μg/ml)- and ionophore A-23187 (5 μM)-induced platelet aggregations were not affected significantly by this venom 5′-nucleotidase. In ADP-refractory platelet-rich plasma, the venom 5′-nucleotidase inhibited the platelet aggregations induced by collagen (20 μg/ml) or sodium arachidonate (100 μM). The venom 5′-nucleotidase showed a more pronounced inhibitory effect on sodium arachidonate-induced platelet aggregation than creatine phosphate/creatine phosphokinase and apyrase did. No lactate dehydrogenase was released by this venom 5′-nucleotidase, indicating that no platelet lysis occurred. It is concluded that removal of ADP, which is released by these platelet aggregation inducers, and the subsequent accumulation of adenosine are responsible for the inhibitory effect of the venom 5′-nucleotidase on platelet aggregations.     

The effect of Trimeresurus mucrosquamatus snake venom on platelet aggregation

Trimeresurus mucrosquamatus venom decreased the platelet number in rabbits to about 10–20% of the control after i.v. injection (1 mg. kg body weight). The venom induced platelet aggregation of rabbit platelet-rich plasma and washed platelets at concentrations between 1 and 1000 μg/ml, with the maximum at 30 μg/ml. Venom-induced platelet aggregation was calcium dependent and was accompanied by release of nucleotides. This aggregation was potentiated by potassium cyanide, and inhibited by N-ethylmaleimide. The venom also induced aggregation of ADP-refractory platelets.     

Fibrinolytic enzyme from Agkistrodon halys brevicaudus (Korean mamushi) snake venom.

By means of DEAE-Sepharose CL-6B ion exchange chromatography and TSK-GEL G2000 SW high-performance gel filtration, a purified protein with fibrinolytic activity was obtained from the venom of Agkistrodon halys brevicaudus (Korean mamushi). The protein was homogeneous as judged by isoelectric focusing electrophoresis and high-performance gel filtration. Its mol. wt is 39,200 and its isoelectric point 4.12. The specific fibrinolytic activity of the protein was 3.2 times higher than that of the crude venom. The fibrinolytic activity of the purified principle was 33 units/mg protein (units of standard urokinase activity).     

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI approximately 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an approximately 13.7kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 1SLWQFGKMINYVM-GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0A resolution. These crystals are monoclinic and have unit cell dimensions of a=33.9, b=63.8, c=49.1A, and beta=104.0 degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 times more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation. Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies.     

Cardiotoxin from Naja naja atra snake venom: A potentiator of platelet aggregation

Cardiotoxin, isolated from Naja naja atra snake venom, potentiates platelet aggregation induced by ADP, thrombin, collagen and venom phospholipase A₂. The malondialdehyde formation caused by ADP, thrombin and venom phospholipase A₂ were also increased in the presence of cardiotoxin. Both potentiation of aggregation and increase in malondialdehyde were blocked by indomethacin or Ca²⁺ (5 mM or 0.05 mM). Cardiotoxin did not potentiate thrombin-induced aggregation of p-bromophenacyl bromide-modified platelets. Thromboxane B₂ formation induced by thrombin or collagen was also increased by cardiotoxin, while that by arachidonate was not affected. As a membrane-active polypeptide, cardiotoxin might augment the Ca²⁺-flux during the activation of the platelet membrane by aggregation inducers and then increase the activation of endogenous phospholipase A₂.     

Inhibition of human platelet aggregation by -amino acid oxidase purified from Naja naja kaouthia venom

-Amino acid oxidase (LAO) widely exists in snake venoms. Purification of LAO from the Naja naja kaouthia (monocellate cobra) venom has been reported (Tan and Swaminathan, 1992), but its structural characterization and physiological function remained to be determined. The function of snake venom LAOs in hemostasis, especially their effect on platelet aggregation, has been controversial. We determined the N-terminal amino acid sequence of the N. n. kaouthia LAO named K–LAO to be DDRRSPLEECFQQNDYEEFLEIAKNGLKKTxNPKHVXxV (38 residues). The protein data base search revealed that the enzyme had high similarities with other snake venom LAOs. Further, platelet aggregation studies revealed that K–LAO functionally did not induce platelet aggregation in a platelet-rich plasma system, but that it inhibited platelet aggregation induced by agonists such as ADP, collagen and ristocetin in a dose-dependent manner. K–LAO diminished platelet aggregation more intensely under low than high shear stress. This inhibitory activity of K–LAO on either ristocetin-induced or shear-induced platelet aggregation was quenched by addition of catalase. These results indicate that K–LAO functions as an inhibitor to platelet aggregation through the formation of hydrogen peroxide. The enzyme may contribute to the development of a severe hematological disorder due to cobra envenomation.     

Distribution and pathology of copperhead (Agkistrodon contortrix) venom

Rats injected i.p. or i.m. with varying doses of tagged Agkistrodon contortrix venom showed the largest amounts of venom in the kidney and liver at 24 hr, with a slightly different distribution for other organs, and the smallest amounts in the brain and spinal cord. However, the rats injected i.m. showed the highest level at the site of the injection. Edema was seen in both the s.c. and muscle tissues of the rats given the venom i.m., and focal hemorrhage was observed in rats receiving 25 mg/kg or more of venom.     

Purification and characterization of a new platelet aggregation inhibitor with dissociative effect on ADP-induced platelet aggregation, from the venom of Protobothrops elegans (Sakishima-habu)

A platelet aggregation inhibitor, named snake venom platelet aggregation dissociator (SV-PAD)-1, with a dissociative reaction of ADP-induced platelet aggregation, was purified from the venom of Protobothrops elegans (Sakishima-habu) by gel-filtration employing Sephadex G-100, and ion-exchange chromatographies using DEAE-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Mono S. By this procedure, about 1.5 mg of purified protein was obtained from 1.0 g of P. elegans venom. The purified protein showed a single protein band and the molecular weight was about 110 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed four-bands of 7.7, 7.8, 7.95, and 8.15. This protein strongly inhibited ADP-induced platelet aggregation in rabbit platelet-rich plasma (PRP), and its IC₅₀ was about 58 nM. It inhibited ristocetin-induced platelet aggregation in rabbit PRP (IC₅₀: 100 nM), but hardly blocked collagen-induced platelet aggregation. This protein promptly dissociated platelet aggregation in rabbit PRP stimulated by high-concentration ADP.     

Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom

J. J. Zhang, Z. X. Chen, Y. Z. He and X. Xu. Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom. Toxicon22, 931–935, 1984. — AaHI, a proteolytic hemorrhagic toxin from A. acutus venom, contains one g-atom Zn per mole protein and 2 g-atoms Ca per mole protein. AaHI is activated by calcium and slightly inhibited by zinc. When treated with EDTA, AaHI is completely inactivated. When dialyzed against 1,10-O-phenanthroline, 50–80% of the metal content and activities are lost, while 90% of the hemorrhagic and proteolytic activities are retained when dialyzed against 1,10-O-phenanthroline containing 5mM Ca²⁺. The results suggest that calcium is essential for the hemorrhagic and proteolytic activities. The circular dichroism spectra show that calcium may play an important role in maintaining a proper structure for AaHI. The function of zinc is not clear.     

Inhibition of moccasin (Agkistrodon piscivoris) venom proteolytic activity by the serum of the Florida king snake (Lampropeltis getulus floridana)

King snake serum appears to be an effective antivenin for mice against moccasin venom. Venom proteases were inhibited by serum on gelatin and casein substrates. Serum heated at 70°C for 30 min did not lose its effectiveness as an antivenin or its protease-inhibiting activity, but serum heated at 75°C for 30 min did not inhibit the digestion of gelatin by venom. The ring test, immunodiffusion, and immunoelectrophoresis demonstrated that king snake serum contained at least one antibody for antigens in moccasin venom. King snake serum proteins were separated by acrylamide gel electrophoresis at pH 8·9 with ten bands being resolved. The γ-globulin portion of king snake serum, when eluted from the first cm fraction of acrylamide gels, inhibited casein digestion by venom. No other fractions inhibited proteolytic activity. When a serum-venom mixture in which an antigen-antibody reaction had occurred was subjected to electrophoresis, the pattern of serum proteins was altered. None of the fractions eluted from this gel inhibited the digestion of casein by venom, indicating that antibodies in the serum had combined with antigenic venom proteases prior to electrophoresis. King snake serum that was fractionated by ammonium sulfate precipitation was separated electrophoretically. All three fractions contained a γ-globulin band and inhibited the digestion of casein by venom.     

Jerdonuxin, a novel snaclec (snake C-type lectin) with platelet aggregation activity from Trimeresurus jerdonii venom

Serious clinical symptoms of Trimeresurus jerdonii bite are mainly caused by abnormalities of blood system. We have previously identified and characterized several bioactive components affecting human blood system, such as serine proteases, metalloproteinases and disintegrins. But few snaclec was characterized in the T. jerdonii venom. In this study, a novel snaclec, named jerdonuxin, was isolated, molecular cloned and characterized as a human platelet agonist. On SDS-polyacrylamide gel electrophoresis, jerdonuxin showed a single band with an apparent molecular weight of 120 kDa under non-reducing conditions and two distinct bands with apparent molecular weights of 18 kDa (α-subunit) and 14 kDa (β-subunit) under reducing conditions. The cDNA sequence of each subunit of jerdonuxin was identified. The precursors of both subunits contain a 23-amino acid residue signal peptide and the mature proteins are composed of 135 and 125 amino acids for α- and β-subunits, respectively. The N-terminal amino acid sequences of each subunit determined by Edman degradation were consistent with deduced amino acid sequences of cDNA. Jerdonuxin dose-dependently induced human platelet aggregation. The phosphorylation profile pattern induced by jerdonuxin showed similar with mucetin (a platelet agonist via glycoprotein Ib), but different from stejnulxin (an agonist via glycoprotein VI). The jerdonuxin-induced platelet aggregation was inhibited by the anti-GPIbα or anti-GPIIb polyclonal antibodies, but not by anti-GPVI polyclonal antibodies. In summary, a novel snaclec of platelet agonist was purified and characterized from the T. jerdonii venom and our data also suggested that GPIb was involved in jerdonuxin-induced platelet aggregation.     

Purification, characterization and potent lung lesion activity of an l-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom

L-amino acid oxidases (LAOs) are one of the major components of snake venoms, which possess numerous biological functions. However, little is known of the influence of LAOs on organ lesions. In the present study, a unique LAO from Agkistrodon blomhoffii ussurensis snake venom named ABU-LAO was purified by Heparin-Sepharose FF chromatography followed by an ion-exchange chromatography procedure. The purified ABU-LAO appears a dimer with a molecular mass of approximately 108.8kDa. Kinetics studies showed that ABU-LAO is very active towards its substrates L-Asn, L-Phe, L-Tyr, L-Leu, L-Ile and L-Trp. The most striking observation in the present study is that ABU-LAO causes severe pneumorrhagia, pulmonary interstitial edema, fusion of pulmonary alveoli, cardiac interstitial edema and bleeding when being intravenously injected into BALB/c mice. ABU-LAO also induces liver cell necrosis and release of cytokines including IL-6, IL-12 and IL-2 from highly purified human peripheral blood monocytes and T cells, respectively. In conclusion, ABU-LAO potently induces lesions in lungs and livers. The ability of ABU-LAO will contribute to the understanding of the pathogenesis of snakebite wound.     

Purification of a capillary permeability increasing-enzyme from the venom of Agkistrodon caliginosus (kankoku-mamushi)

A capillary permeability increasing-enzyme was purified from the venom of Agkistrodon caliginosus by gel-filtration on Sephadex G-100, ion exchange chromatographies on CM-Sephadex C-50 and DEAE-Sephadex A-50 and affinity chromatography on p-aminobenzamidine-ε-aminocaproic acid-Sepharose 4B. By this procedure, 6.2 mg of the purified enzyme was obtained from 4 g of the venom. The purified enzyme was homogeneous by disc electrophoresis at pH 8.3 and pH 4.5, and did not show any caseinolytic, clotting or bradykinin-releasing activities. The enzyme hydrolyzed $\text{N-α-}\text{tosyl}\text{-}\text{l}\text{-}\text{arginine}$ methylester and the specific activity of the enzyme was 7.3 $\text{N-α-}\text{tosyl}\text{-}\text{l}\text{-}\text{arginine}$ methylester units per mg of protein. When 3 μg of the enzyme was injected into the depilated skin of the back of a rabbit, capillary permeability was increased.     

Purification and characterization of a platelet aggregation inducer from Calloselasma rhodostoma (Malayan pit viper) snake venom

A potent platelet aggregation inducer was purified from Calloselasma rhodostoma snake venom by Sephadex G-75, CM-Sephadex C-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight estimated to be 28,160 +/- 1280. It was devoid of phospholipase A2, fibrino(geno)lytic and thrombin-like activities. The venom inducer elicited platelet aggregation and the serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its platelet activation. Creatine phosphate/creatine phosphokinase and indomethacin did not inhibit the venom inducer-induced aggregation and release reaction. Mepacrine and verapamil preferentially inhibited aggregation, while PGE1 completely blocked both aggregation and release reaction. It is concluded that the venom inducer activates platelets through the activation of endogenous phospholipase A2 or C, leading to intracellular calcium mobilization, but independent of the ADP release reaction or thromboxane A2 formation.     

Properties of NAD glycohydrolase purified from five-pace snake (Agkistrodon acutus) venom

NAD glycohydrolase (NADase) (E.C. 3.2.2.5) from five-pace snake (Agkistrodon acutus) venom was purified to electrophoretic homogeneity through a 4-step isolation procedure, including column chromatography using DEAE-Sephadex A-50, Sephadex G-75, CM Sephadex C-50 and Sephadex G-100. The final product was 11.8-fold purified with a 3.9% yield. The pure enzyme showed maximal activity at about 40 degrees C with optimal pH at 7.5. It was a glycoprotein with a pI of 7.6. Its mol. wt was respectively 98,000 as measured by gel filtration and 50,000, by SDS-PAGE. There was only one N-terminal residue, proline. NADase is thus composed of two identical subunits in each molecule. The enzyme contained copper ions. NADase activity was lost when the copper enzyme complex was treated with EDTA. The Km of the enzyme for beta-NAD, NADP and beta-NGP were 0.50 mM, 0.13 mM and 0.16 mM respectively.