Antihemorrhagic Activity Research Articles

Antihemorrhagin in the blood serum of king cobra ( Ophiophagus hannah): purification and characterization

King cobra ( Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS–PAGE. Its apparent molecular weight determined by SDS–PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.

Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum.

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.

Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40 318 AMU for DM40 and 42 373 and 43 010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS–PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.

The antihemorrhagic factor, erinacin, from the European hedgehog ( Erinaceus europaeus), a metalloprotease inhibitor of large molecular size possessing ficolin/opsonin P35 lectin domains

From muscle extracts of the European hedgehog, Erinaceus europaeus, an antihemorrhagic factor, erinacin, was purified by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, hydroxylapatite and gel filtration columns. A purification of approx. 1400-fold was achieved with an overall yield of 21% in antihemorrhagic activity. The molecular weight of erinacin determined by gel filtration was approx. 1000 kDa. SDS-PAGE of erinacin under reducing conditions indicates that it consists of two types of subunits, α and β, with molecular weights of 37 and 35 kDa, respectively, in a ratio of 1:2. In the presence of 6 M guanidine–HCl, erinacin dissociates into α-subunits and β-subunit decamers. From these results the subunit assembling of erinacin has been formulated as α 10·2 β 10. The molecular weight of the subunits and of the β-subunit decamer was confirmed by MALDI-TOF mass spectrometry. Erinacin inhibits the hemorrhagic and proteolytic activity of the major hemorrhagic metalloprotease from the venom of Bothrops jararaca. Complete inhibition was achieved in an equimolar mixture of inhibitor and enzyme suggesting an equimolar complex. Erinacin is not inhibiting serine proteases such as trypsin and chymotrypsin, it was characterized to be a metalloprotease inhibitor. In electronmicroscopy, flower bouquet-like structures characteristic for some animal lectins were observed. Amino acid sequence analysis indicated that both subunits are almost identical and are composed of common amino terminal, collagen- and fibrinogen-like domains homologous to proteins of the ficolin/opsonin P35 lectin family.

The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against virginia opossum ( Didelphis virginiana) serum

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occuring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum ( Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon™-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat ( Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake ( Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake ( C. viridis lutosus), Prairie rattlesnake ( C. viridis viridis), Tancitaran dusky rattlesnake ( C. pusillus), Northern Mojave rattlesnake ( C. scutulatus scutulatus type B) and Northern Pacific rattlesnake ( C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH, reacted with all the hemorrhagic venoms except for the venom of the King cobra ( Ophiophagus hannah) and did not react with the non-hemorrhagic venoms. The hemorrhagic binding site of CAH monoclonal antibody and the antihemorrhagin in Virginia opossum are different binding sites. The five-step western blot will be a very useful assay for determining hemorrhagic activity without using live animals.

Comparison of antihemorrhagic activities in skeletal muscle extracts from various animals against Bothrops jararaca snake venom

T. Omori-Satoh, M. Takahashi, Y. Nagaoka and D. Mebs. Comparison of antihemorrhagic activities in skeletal muscle extracts from various animals against Bothrops jararaca snake venom. Toxicon 36, 421–423, 1998.—Antihemorrhagic activities of skeletal muscle extracts from various animals were compared in inhibiting the hemorrhagic activity of Bothrops jararaca venom. The muscle extracts of the European hedgehog ( Erinaceus europaeus) exhibited the strongest activity, followed by those of other insectivores such as the shrew ( Crocidura russula) and mole ( Talpa europaea). The antihemorrhagic activities of muscle extracts from experimental animals such as mice, rats, guinea-pigs, hamsters and rabbits were negligible.

Crystal structure of the von Willebrand factor A1 domain in complex with the function blocking NMC-4 Fab.

The presence of one or more copies of von Willebrand factor type A domains identifies a superfamily of proteins usually involved in biological processes controlled by specific molecular interactions, often adhesive in nature. We have solved the crystal structure of the prototypic von Willebrand factor A1 domain, essential for the antihemorrhagic activity of platelets, in complex with the function blocking antibody, NMC-4, at 2.2 A resolution. This has led to the recognition of a putative binding groove for the platelet receptor, glycoprotein Ib alpha, formed by two adjacent alpha-helices and a beta-strand. The structure also shows a contact interface between A1 domain pairs, suggesting a hypothetical mechanism for the regulation of protein assembly and heterologous ligand binding mediated by homophilic interactions of type A domains.

Inhibition of Toxic Activities of Bothrops asperVenom and Other Crotalid Snake Venoms by a Novel Neutralizing Mixture

The majority of snake bites in Central America are caused by Bothrops asper,whose venom induce complex local effects such as myonecrosis, edema and especially hemorrhage. These effects are only partially neutralized by the clinically used antivenom, even when administered rapidly after envenomation. Recently we screened 49 substances for antihemorrhagic activity and found that a mixture composed of CaNa 2EDTA, a B. asperserum fraction (natural antidote), and the currently used horse polyvalent antiserum is highly effective in the neutralization of local and systemic hemorrhage developing after B. asperenvenomation (Borkow et al., Toxicon 35, 865–877, 1997). In the present study we screened the best six antihemorrhagic compounds for their capacity to neutralize the lethal activity in mice and the proteolytic, hemolytic, and antiattachment activities in vitroof the venom. The compounds tested included the currently used horse antivenom, rabbit antiserum against whole B. aspervenom or against heated venom, B. asperand Natrix tessellataserum fractions, and CaNa 2EDTA. The constituents of the antihemorrhagic mixture were also the best inhibitors of the other examined toxic activities. Importantly, the mixture effectively neutralized toxic activities of an additional nine venoms from snakes abundant in Central America. This work suggests that the polyvalent antivenom used in Central America could be enriched with a B. asperserum fraction producing a more effective antivenom. In addition, the local application of CaNa 2EDTA to neutralize hemorrhagic toxins, immediately after a snake bite, may provide rapid inhibition of local damage caused by the venoms.

Targeted delivery of drugs and proteins to the liver via receptor-mediated endocytosis

Targeting of drugs and proteins to the liver via the asialoglycoprotein receptor was investigated in mice. Carboxymethyl-dextran (CMD), carboxymethyl-amylose (CMA), and poly-l-glutamic acid (PLGA) were modified with 2-imino-2-methoxyethyl (IME)-thiogalactosides to obtain galactosylated derivatives as carriers of drugs with low-molecular weights. Proteins were targeted to the liver by direct attachment of galactose moieties. Pharmacokinetic analysis clearly showed that galactosylated derivatives were taken up by the liver depending on the molecular weight and configuration of macromolecules, the number of galactose residues, and the administered dose. Based on the obtained results, we attempted to selectively deliver vitamin K5, which acts as a coagulant in the liver. Galactosylated PLGA (Gal-PLGA) possessing 18 galactose residues was selected as a hepatotropic carrier since it was efficiently accumulated and gradually degraded in the liver after intravenous injection. The attachment of vitamin K5 did not alter the distribution properties of Gal-PLGA, and vitamin K5 was successfully delivered to the liver by the conjugation. The anti-hemorrhagic activity of the conjugate was assayed after intravenous injection in mice treated with warfarin. Vitamin K5 conjugated with Gal-PLGA showed coagulant activity at any periods studied after intravenous injection, while free vitamin K5 only showed the activity at 4 h after administration. These results indicate the usefulness of galactosylated macromolecules as hepatotropic carriers of drugs whose site of action is in the liver.

511 Effects of nociceptin on spinal reflexes in newborn rats

An antihemorrhagic protein has been isolated from the serum of Chinese mamushi (Gloydius blomhoffi brevicaudus) by using a combination of ethanol precipitation and a reverse-phase high-performance liquid chromatography (HPLC) on a C8 column. This protein—designated Chinese mamushi serum factor (cMSF)—suppressed mamushi venom-induced hemorrhage in a dose-dependent manner. It had no effect on trypsin, chymotrypsin, thermolysin, and papain but inhibited the proteinase activities of several snake venom metalloproteinases (SVMPs) including hemorrhagic enzymes isolated from the venoms of mamushi and habu (Trimeresurus flavoviridis). A similar protein (Japanese MSF, jMSF) with antihemorrhagic activity has also been purified from the sera of Japanese mamushi (G. blomhoffi). The N-terminal 70 and 51 residues of the intact cMSF and jMSF were directly analyzed; a similarity between the sequences of two MSFs to that of antihemorrhagic protein (HSF) from habu serum was noticed. To obtain the complete amino acid sequences of MSFs, cDNAs encoding these proteins were cloned from the liver mRNA of Chinese and Japanese vipers based on their N-terminal amino acid sequences. The mature forms of both MSFs consisted of 305 amino acids with a 19-residue signal sequence, and a unique 17-residue deletion was detected in their His-rich domains.

1820 Ultrastructure of the LTTD of the HABU, trimeresurus flavoviridis

King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS–PAGE. Its apparent molecular weight determined by SDS–PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.

Electronic structure-activity relationship for derivatives of propenone. Communication II. 2-cinnamoylbenzimidazoles and 2-(β-benzoylvinyl)-1-methylbenzimidazoles

Our systematic investigations of some regularities of the electronic structure-activity relationship (ESAR) in a series of chalcone derivatives and the corresponding heterocyclic analogs substantiated the structural characteristics that determine the antioxidant, antiallergic, antiinflammatory, and other types of activity [1, 2]. In earlier communications we described the results of antiallergic activity studies [1] and the analysis of ESAR for derivatives of 4-carboxyvinylenechalcones and 3-einnamoylcoumarins [3]. As a continuation of these studies, we considered it reasonable to obtain heterocyclic analogs of chalcone, in which one of the aromatic rings was replaced by the benzimidazole fragment. Benzimidazole derivatives are known to process more than 25 types of pharmacological action, including antiallergic [4] and immunomodulating activity (dibazole). We synthesized two types of benzimidazole analogs, 2cinnamoylbenzimidazoles and 2-(13-benzoylvinyl)-l-methylbenzimidazoles (see Table I). Compounds I a n were synthesized by condensation of 2-acetylbenzimidazole with aromatic aldehydes in the presence of a basic or acidic catalyst; compounds I I a n were synthesized by the reaction of 2-formyl-l-methylbenzimidazole with corresponding acetophenones. Biological testing of the compounds obtained revealed pronounced antiallergic and sedative activities (AA and SA, respectively) as well as moderate hypotensive and antihemorrhagic activities. The outlined conclusions [3] about the decisive contribution of electronic parameters of the ct,IB-unsaturated carbonyl fragment to the biological activity of chalcone and coumarin derivatives served as a basis for the search for ESAR in a series of synthesized benzimidazole analogs of chalcone.

Isolation, characterization and mode of neutralization of a potent antihemorrhagic factor from the serum of the snake Bothrops asper

A potent antihemorrhagic factor (BaSAH 1) was isolated from the serum of the snake Bothrops asper by ammonium sulfate precipitation at 40–60%, Sephacryl S-200 and Sephadex G-50 gel filtration, DEAE-Sepharose, and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 5.2 and a molecular weight of 66 kDa. 4 μg of the purified factor BaSAH were needed to neutralize the hemorrhagic dose of B. asper whole venom compared to 60 μg of the clinically used horse polyvalent immunoglobulins. Moreover, 0.35 μg of BaSAH were sufficient to achieve complete neutralization of the main hemorrhagic toxin (BaH1), with a molar ratio of 2:1. The antihemorrhagic activity was stable between pH 1.5–9 and up to 60°C but lost activity completely after 30 min of heating at 70°C. BaSAH did not digest the hemorrhagic toxin BaH1 or formed a precipitin line with it, nor with the whole venom. Both ELISA experiments and chromatography of BaSAH after incubation with the 125I-labeled hemorrhagic toxin BaH1 demonstrated that the mechanism of the neutralization involves a formation of an inactive soluble complex between the natural antihemorrhagin and the main hemorrhagin of B. asper venom.

A potent antihemorrhagin in the serum of the non-poisonous water snake Natrix tessellata: isolation, characterization and mechanism of neutralization

The main natural antihemorrhagic factor (NtAH), which inhibits the hemorrhagic activity of Bothrops asper snake venom, was isolated from the serum of the non-poisonous water snake Natrix tessellata by ammonium sulfate precipitation at 35–55%, Sephadex G-75 gel filtration, ion exchange chromatography on DEAE-Sepharose and CM-Sepharose and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 4.5 and a molecular mass of about 880 kDa. The antihemorrhagic activity was stable between pH 5.5–11.7 and up to 50°C, but lost activity after 20 min at 60°C. It did not form a precipitin line with the main hemorrhagin of Bothrops asper snake venom (BaH1), nor with the whole venom, which suggests that the antihemorrhagic factor is not an immunoglobulin. The mechanism of neutralization by the isolated antihemorrhagic factor NtAH did not include digestion of the hemorrhagic toxin BaH1. Chromatography of NtAH with active 125I-labeled BaH1 toxin as well as ELISA experiments demonstrated that the mechanism of neutralization involves formation of an inactive soluble complex between the natural NtAH of the non-poisonous water snake and the main hemorrhagin of Bothrops asper venom.

Muscle extract of hedgehog, Erinaceus europaeus, inhibits hemorrhagic activity of snake venoms

The antihemorrhagic activity of muscle extract of hedgehog, Erinaceus europaeus, was tested on various snake venoms with hemorrhagic activity. The extract inhibited strongly hemorrhagic activity of venoms from Bitis arietans, Bothrops jararaca and Vipera latastei gaditana, and remarkably that of venoms from Agkistrodon halys blomhoffi, Bitis gabonica rhinoceros, Bitis nasicornis, Bothrops atrox asper, Crotalus horridus horridus and Vipera berus. The antihemorrhagic activity against eight other snake venoms was below the detection level.

Primary structure of the antihemorrhagic factor in serum of the Japanese Habu: a snake venom metalloproteinase inhibitor with a double-headed cystatin domain.

The complete amino acid sequence of an antihemorrhagic factor, HSF, in the serum of the Japanese Habu snake, Trimeresurus flavoviridis, has been determined. The protein is composed of 323 amino acid residues and contains three asparagine-linked oligosaccharide chains at positions 123, 185, and 263. The molecule contains two copies of the cystatin domain in the N-terminal portion up to position 240, and these domains show a remarkable sequence homology (about 50%) to those of plasma glycoproteins such as alpha 2-HS (human) and fetuin (bovine) and to a lesser extent to that of HRG (human). The amino acid sequence of the noncystatin region towards the C-terminus is unique, showing no significant homology with those of the corresponding regions of alpha 2-HS and fetuin. In spite of the presence of cystatin domains, HSF does not inhibit cysteine proteinases such as papain and cathepsin B but does inhibit several metalloproteases in Habu venom. The results suggest that HSF is the first protein found to be functionally related to metalloproteinase inhibitors among the structurally homologous proteins with a double-headed cystatin domain, and is a member of a novel family (family 4) with divergent functions of the cystatin superfamily proteinase inhibitors. Although HSF possesses similar physicochemical properties to those of oprin, a snake venom metalloproteinase inhibitor with antihemorrhagic activity isolated from opossum serum [Catanese & Kress (1992) Biochemistry 31, 410-418], its primary structure is strikingly different from that of oprin.

Antihemorrhagic factors from the blood serum of the western diamondback rattlesnake Crotalus atrox

Several antihemorrhagic factors isolated from C. atrox serum are glycoproteins with mol. wt ranging from 65,000 to 80,000. The antihemorrhagic activity of these factors was stable at a pH range of 1.3–11.5 and at temperatures up to 85°C, for 30 min. The isolated antihemorrhagins also neutralized the proteolytic activity of C. atrox venom, as tested with azocollagen and gelatin, and formed a complex with hemorrhagic toxin e isolated from the same venom. The neutralization capacity of the isolated antihemorrhagins was six times as high as the commercial polyvalent antivenom produced for clinical use.

Studies on the anti-hemorrhagic substances in herbs classified as hemostatics in Chinese medicine. X. On hemostatic activities of the parched herbs for hemostatics

The effect of heat treatment on the anti-hemorrhagic action of seven hemostatic herbs employed especially after parching in order to work effectively as an anti-hemorrhagic agent in traditional Chinese medicine, was examined. It was found that the anti-hemorrhagic activities of the following 5 herbs are apparently increased by parching: Kaika (Sophorae immaturus Flos), Renbo.(Nelumnbins Receptaculum), Gusetsu (Nelumnbins Rhizomatis Nodus), Chiyu (Sanguisorbae Radix) and Gaiyou (Artemisiae argyi Folium).

Studies on the antihemorrhagic substances in herbs classified as hemostatics in Chinese medicine. IX. On the antihemorrhagic principles in Typha lactifolia L.

By following the antihemorrhagic activity according to Tajima's method, two antihemorrhagic principles, a new flavonol glucoside (1) and an unknown compound (2), were isolated from the dried pollen of Typha lactifolia L., which is one of the most important herbs for treatment of stagnant blood and bloody stools in traditional Chinese medicine.The structure of 1 was established as isorhamnetin 3-rutinoside-7-rhamnoside (3-[6-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl] Oxy-5-hydroxy-7-[β-L-mannopyranosyl] oxy -2-(4-hydroxy-3 -methoxyphenyl)-4H-1-benzopyran-4-one), based on chemical transformations and spectral data.

Proteolytic, hemorrhagic and hemolytic activities of snake venoms

Proteolytic, hemorrhagic and hemolytic acitivites were tested on 47 different venoms from the Crotalidae, Viperidae, Elapidae, and Hydrophiidae families. Antihemorrhagic activity of crude opossum ( Didelphis virginiana) and woodrat ( Neotoma micropus) serum was tested against the venoms that presented hemorrhagic activity. All venoms showed proteolytic activity when non-specific substrates such as hide powder and collagen were used. Members of the Crotalidae family had the highest hide powder, chymotrypsin-like and hemorrhagic activity. However, members of the Elapidae family had the highest collagen activity. Hemolytic activity was present in 85% of the snake venoms tested. The crude opossum and woodrat serum neutralized the hemorrhagic activity of all the hemorrhagic venoms. Of particular interest is the poor correlation between the venom activities measured here and the phylogenetic position of the snake that possess them. This is particularly true at the genus and species level. Differences in activities were found among individuals of the same genus. The significance of these differences among venoms of closely related snakes is unknown. They do not seem to be adaptive, however little is known of the physiology and habits of most venomous snakes.