A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable malonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E1 completely blocked both aggregoserpentin-induced aggregation and release reaction. Furthermore, platelet aggregoserpentin lowered basal and prostaglandin E1-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A2, resulting in the formation of platelet activating factor, but not of prostaglandins.     

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sepharyl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release action in rabbit platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable molonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E₁ completely blocked both aggregoserpentine-induced aggregation and release reaction. Furthermore, platelet aggregoserpentine lowered basal and prostaglandin E₁-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A₂, resulting in the formation of platelet activating factor, but not of prostaglandins.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.     

Inhibition of proliferation of ECV304 cells by a disintegrin from Chinese green tree viper

A novel disintegrin, stejnin, was purified from the Trimeresurus stejnegeri venom by gel filtration and reverse phase high performance liquid chromatography. The molecular weight of stejnin was determined to be 7428 Da by MALD-TOF MS analysis. The cDNA encoding the precursor of stejnin was cloned from the venom gland. From the deduced amino acid sequence, stejnin is composed of 71 amino acid residues contains the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Stejnin strongly inhibited ADP- and ristomycin-induced human platelet aggregation with IC50 of 45 and 50 nM, respectively. Stejnin also possessed potent inhibited cell proliferation of ECV304 cells.     

Conformational analysis of a toxic peptide from Trimeresurus wagleri which blocks the nicotinic acetylcholine receptor.

The 22-residue toxic peptide (WTX1) from the venom of the Southeast Asian snake Trimeresurus wagleri has multiple sites of action, but its lethal effect has been attributed to blocking the postsynaptic acetylcholine receptor at the neuromuscular junction. The 3-dimensional structure of WTX1 was studied using 2-dimensional nuclear magnetic resonance spectroscopy, circular dichroism, and computer simulations. In aqueous solution, WTX1 was shown to have extended and flexible "tails" defined by a short, rigid disulfide-bonded loop. The flexible regions can undergo structural rearrangement when moved from an aqueous to a less polar environment and may contribute to its effectiveness at different receptor sites. By substituting Gly or Phe for His at position 10, significant effects on the disulfide bond formation and, thereby, the activity of the peptide were observed. These results suggest that even subtle differences in single residues can have profound effects on the dynamics of folding, disulfide bond formation, and activity of this toxic peptide.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

A potent platelet aggregation inducer purified from Trimeresurus mucrosquamatus snake venom

A non-coagulant platelet aggregation inducer (called platelet 'aggregoserpentin') was isolated from Trimeresurus mucrosquamatus snake venom by CM-Sephadex chromatography and purified by gel filtration. It was homogeneous as judged by the ultracentrifugal analysis and electrophoresis on polyacrylamide gel and cellulose acetate membrane. The molecular weight was estimated to be 68 000 as judged by the SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-75. The ultracentrifugal analysis gave 3.19 Svedberg units. It was a protein-polysaccharide complex containing 340 amino acid residues and 50% carbohydrate per molecule. The isoelectric point was pH 5.4. It did not possess any of the hydrolase enzymatic properties which were found in the crude venom. The minimal concentration of 'aggregoserpentin' necessary to induce platelet aggregation was 10 ng/ml, about one four-hundredth of that of the crude venom. It did not cause lysis of platelets because lactate dehydrogenase was not found in supernatant after complete aggregation. An intravenous injection of 'aggregoserpentin' (35 microgram/kg) into rabbit ear marginal vein caused marked decrease of platelet number to approx. 10-20% of that of the control.     

Lipid peroxide makes rabbit platelet hyperaggregable to agonists through phospholipase A2 activation

Treatment of rabbit platelets with tert-butyl hydroperoxide and Fe2+ caused increasing arachidonic acid release, lysophosphatidylcholine formation, and aggregation with increasing concentrations of Fe2+. A combination of tert-butyl hydroperoxide and a low concentration of Fe2+, which by itself causes slight or no such activation, elicited synergistic release of arachidonic acid and aggregation under stimulation with a suboptimal concentration of collagen or arachidonic acid as an agonist. These responses were inhibited by pretreatment of the platelets with vitamin E or mepacrine in a concentration-dependent manner, but not by uric acid. The arachidonic acid release was dependent on the presence of Ca2+ in the medium. Synergistic formation of lysophosphatidylcholine, but not diacylglycerol, was also observed under this condition. The aggregation was also inhibited by indomethacin, a cyclooxygenase inhibitor. Cyclooxygenase activity was not affected by the oxidative treatment. These results suggest that lipid peroxide formed in membranes causes phospholipase A2 to become hypersusceptible to the agonist used, making the platelets hyperaggregable.     

Pathological alterations induced by neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom

The pathological alterations induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi, were studied in mice. Neuwiedase was devoid of hemorrhagic activity when tested in the skin up to a dose of 200 microgram, and also after intramuscular injection in the gastrocnemius. However, it induced bleeding when applied onto the mouse cremaster muscle in intravital microscopy experiments, and caused pulmonary hemorrhage when injected intravenously at doses higher than 5 microgram/g. Median lethal dose (LD(50)) by the intravenous route was 5 microgram/g, whereas LD(50) of crude venom was 0.47 microgram/g. After intramuscular injection, neuwiedase induced a mild myotoxic effect, evidenced histologically and by the increment in plasma creatine kinase activity, but it was devoid of hemorrhagic and thrombotic effects. In contrast, crude B. neuwiedi venom induced prominent hemorrhage and myonecrosis in gastrocnemius muscle. Both venom and neuwiedase induced an inflammatory reaction in muscle tissue characterized by abundant polymorphonuclear leukocytes. Moreover, a conspicuous edema developed in the foot pad after subcutaneous injection of neuwiedase. Anti-neuwiedase antibodies produced in rabbits were effective in the neutralization of hemorrhagic activity of crude venom, evidencing immunological cross-reactivity between neuwiedase and other hemorrhagic metalloproteinases present in the venom, and suggesting that metalloproteinases devoid of, or having low, hemorrhagic activity could be good immunogens to generate antibodies effective against high molecular mass metalloproteinasas having potent hemorrhagic activity. It is concluded that neuwiedase, despite its lack of hemorrhagic effect when injected in the gastrocnemius muscle, contributes to local tissue damage by inducing edema, inflammatory infiltrate and mild myotoxicity, and by degrading extracellular matrix components. In addition, large doses of neuwiedase may contribute to pulmonary bleeding     

Pharmacological Study of Edema and Myonecrosis in Mice Induced by Venom of the Bushmaster Snake (<Emphasis Type="Italic">Lachesis muta muta</Emphasis>) and Its Basic Asp49 Phospholipase A<Subscript>2</Subscript> (LmTX-I)

Previous in vitro studies show that Lachesis muta venom and its purified Asp49 phospholipase A₂, named as LmTX-I, display potent neurotoxic and myotoxic activities. Here, an in vivo study was conducted to investigate some pharmacological effects of the venom or its LmTX-I toxin, after intra-muscular injection in tibialis anterior (TA) and following subplantar injection in hind paws of mice. Findings showed that LmTX-I increased plasma creatine kinase activity and produced strong myonecrosis and inflammatory reactions in TA muscle. In addition to these effects, the venom also induced intense local hemorrhage. Pre-treatment of the venom with EDTA (5 mM) significantly inhibited the edema and hemorrhage. Histological examination showed that L. muta venom caused inner dermal layer thickening in the pad hind paw. In addition, there was marked inflammatory cell infiltration, particularly of neutrophils, and hemorrhage. LmTX-I also demonstrated edema-forming activity, which was inhibited by pretreatment with indomethacin.     

乌梢蛇血清的抗出血因子:一个有前途的抗蛇毒药物原料

用柱层析和聚丙烯酰胺凝胶盘状电泳法,从乌梢蛇血清中分离纯化了一个抗出血因子。用SDS-聚丙烯酰胺凝胶电泳法测得其分子量大约为65 kD;测定了五种蝮亚科蛇毒(尖吻蝮、竹叶青蛇、原矛头蝮、哈扑和短尾蝮)的最小出血剂量和乌梢蛇血清中抗出血因子对这五种蛇毒的抗出血活性;还测定了七种蛇毒(除上述五种毒蛇外,还包括圆斑蝰和银环蛇)的半数致死量,以及抗出血因子对中毒小鼠的治疗作用。结果显示:从乌梢蛇血清中提纯的抗出血因子的抗蛇毒活性,不仅可以抵抗它的捕食者尖吻蝮的蛇毒,而且还可以抵抗具出血活性的其它蛇毒;但它对不具出血活性的银环蛇毒的致死抑制作用不明显。该抗出血因子不仅在体外实验表现出强的中和出血毒素的活性,而且在体内实验中亦表现出对中毒小鼠良好的治疗作用,因而可能成为新的抗蛇毒药物的有前途的原料。乌梢蛇血清对血循毒的中和能力的获得,可能归因于尖吻蝮与乌梢蛇之间捕食与被捕食相互作用的关系。     

Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.     

Potent platelet aggregation inhibitor from Trimeresurus gramineus snake venom

Using DEAE-Sephadex A-50 column chromatography and gel filtration, a potent platelet aggregation inhibitor from Trimeresurus gramineus venom was purified. It was an acidic phospholipase a, rich in aspartic acid, glutamic acid and half-cystine, with an isoelectric point of 3.6. At a concentration of 10 μg/ml, the purified inhibitor showed a marked inhibitory effect on platelet aggregations induced by adenosine diphosphate, collagen, sodium arachidonate and ionophore A-23187 in rabbit platelet-rich plasma, washed platelet suspension, as well as in thrombin-degranulated platelet suspension. The ID₅₀ of this venom inhibitor was about 2.5–5 μg/ml in platelet aggregations induced by all these aggregation inducers. The action of this inhibitor could be partially antagonized by phosphatidylethanolamine. High concentration of Ca²⁺ (5 mM) did not reverse the inhibitory action even in the presence of ionophore A-238187. The [¹⁴C]serotonin release induced by sodium arachidonate and thrombin was unaffected. Malonic dialdehyde formation induced by these aggregation inducers remained unchanged. Basal and prostaglandin E₁-stimulated cAMP levels were not altered by this inhibitor. No lactate dehydrogenase was released even at a concentration of 62.5 μg/ml. Polylysine-induced platelet agglutination was not affected. β-Mercaptoethanol inactivated both its phospholiase A enzymatic and platelet inhibitory activities, while p-bromophenacyl bromide only inactivated the former activity. The possibility of acting on a common final step of platelet aggregation, i.e. the intercellular adhesion between the activated platelets, was proposed.     

竹叶青蛇毒血小板聚集剂的分离纯化及生化特性

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤和ＦＰＬＣＭｏｎｏＱ阴离子交换柱层析及Ｓｕｐｅｒｏｓｅ－１２凝胶过滤,从竹叶青蛇的蛇毒中纯化到一个能诱导人血小板聚集的均一组分．经ＳＤＳ－聚丙烯酰胺凝胶电泳测定其分子量为６８０００左右．等电点为４．３．测定了它的氨基酸组成,对它活化血小板的作用机理进行了初步研究,结果表明它是一种强的血小板激动剂．     

一种新的烙铁头蛇毒肌肉毒素

用分子筛和快速蛋白质液相色谱从烙铁头（ＴＲｒｉｍｅｒｅｓｕｒｕｓ ｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒中分离了一个新的碱性肌肉毒素,命名为ＴＭＰＢ。它的分子量为１６０００,等电点为９．２．用蛋白质序列仪测定了其Ｎ端２４个氨基酸残基,ＴＭＰＢ与其他两个从同种蛇毒中分离到的碱性磷酯酶Ａ２的同源性分别为４１．７％和５４．２％《     

Isolation of a venom factor devoid of proteolytic activity from Taiwan Habu (Trimeresurus mucrosquamatus): N-Terminal sequence homology and no functional similarity to factors IX/X-binding proteins and botrocetin

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated fromTrimeresurus flavoviridis and botrocetin fromBothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca²⁺ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated fromBothrops jararaca venom. We have therefore named this novel venom factortrimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.     

Identification of the disulfide bond pattern in albolabrin, an RGD-containing peptide from the venom of Trimeresurus albolabris: significance for the expression of platelet aggregation inhibitory activity

Albolabrin is a 73 amino acid peptide isolated from the venom of Trimeresurus albolabris. It contains an RGD sequence and 12 cysteines and is a potent inhibitor of both platelet aggregation and fibrinogen binding to the GPIIb/IIIa complex. This protein shows a high degree of analogy (primarily due to the alignment of all cysteines and the RGD) with a number of other viper venom proteins which inhibit cell adhesion and platelet aggregation and are referred to as disintegrins: rhodostomin, trigramin, flavoridin, applagin, elegantin, and batroxostatin. In this study, we found that the reduction and vinylpyridylethylation of albolabrin and flavoridin decreased their platelet aggregation inhibitory activity approximately 40-50 times. It can be postulated that the higher potency of native and reduced flavoridin as compared to albolabrin depends on the substitution of the Asp of albolabrin with a Phe at the C-terminal end of the RGD in flavoridin. The activity of a synthetic C-terminal peptide derived from flavoridin (residues 35-65) containing four cysteines was about 75 times lower than that of the original flavoridin. The substitution of a pair of cysteine residues with alanines in this peptide resulted in further loss of activity. In order to identify the disulfide bonds in albolabrin, the molecule was digested consecutively by trypsin and porcine pancreatic elastase. Peptides resulting from this digestion were isolated by reverse-phase HPLC and identified by amino acid composition and mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.