Diversity and antimicrobial activity of culturable fungi associated with sea anemone Anthopleura xanthogrammica

Background: The main objective of this study was to isolate fungi associated with Anthopleura xanthogrammica and measure their antimicrobial and enzymatic activities. A total of 93 fungal strains associated with A. xanthogrammica were isolated in this study, of which 32 isolates were identified using both morphological characteristics and internal transcribed spacer (ITS) sequence analysis. The antibacterial activities of 32 fungal isolates were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Edwardsiella tarda, Vibrio harveyi, Fusarium oxysporum, and Pyricularia oryzae by agar diffusion assay. Extracellular hydrolytic enzyme activities of the fungal isolates were determined by agar diffusion assays. Enzyme activities were detected from clear halo size. Results: The isolated fungi belonged to 18 genera within 7 taxonomic orders of 1 phylum. The genera Aspergillaceae were the most diverse and common. The antimicrobial activities of 32 isolates were evaluated, and 19 (59.4%) of fungi isolate displayed unique antimicrobial activities. All fungal strains displayed at least one enzyme activity. The most common enzyme activities in the fungi isolates were amylase and protease, while the least common were pectinase and xylanase. Conclusions: This is first report on the sea anemone-derived fungi with antimicrobial and enzyme activities. Results indicated that sea anemone is a hot spot of fungal diversity and a rich resource of bioactive natural products.

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 µg), goat (HU50 = 13.2 ± 0.3 µg), rabbit (HU50 = 34.7 ± 0.5 µg), and human (HU50 = 25.6 ± 0.6 µg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 µg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.(AU)

Cnidae sizes in the two morphotypes of the giant Caribbean anemone Condylactis gigantea (Actiniaria: Actiniidae) / Tamaños de cnidae en dos morfotipos de la anémona gigante del Caribe Condylactis gigantea (Actiniaria: Actiniidae)

Abstract The sea anemone Condylactis gigantea is an ecologically important member of the benthic community in coral reefs of the tropical Atlantic, and displays two morphotypes with respect to the color in their tentacular tips: the green tip morphotype and the pink/purple tip morphotype. Although some molecular and ecological differences have been found between these morphotypes, no other morphological distinctions have been reported, and currently both are still considered a single taxonomic species. In the present study, we perform an exploration on the variability in the size of cnidae between these two morphotypes and performed statistical analyses to compare the 10 categories of cnidae from specimens hosted in the Cnidarian Collection of Gulf of Mexico and Mexican Caribbean, of the Universidad Nacional Autónoma de México, which were previously collected in several coral reefs localities of the Yucatán Peninsula. Results reveal no significant variation in cnidae size between the two morphotypes, but significant variations were found within each morphotype. In addition, we update the composition of the cnidom of C. gigantea, and the utility of the size of cnidae to distinguish between morphotypes or closely related species is discussed. Rev. Biol. Trop. 66(3): 1055-1064. Epub 2018 September 01.

Resumen La anémona Condylactis gigantea es un miembro ecológicamente importante de la comunidad bentónica en arrecifes de coral del Atlántico tropical, y exhibe dos morfotipos con respecto al color de las puntas de sus tentáculos: el morfotipo de puntas verdes y el morfotipo de puntas rosadas/púrpuras. Aunque se han encontrado algunas diferencias moleculares y ecológicas entre estos morfotipos, no se han reportado otras distinciones morfológicas, y actualmente ambos siguen siendo considerados una sola especie taxonómica. En el presente estudio, realizamos una exploración sobre la variabilidad en el tamaño de los cnidocistos entre estos dos morfotipos y realizamos un análisis estadístico de 10 categorías de cnidocistos a partir de especímenes albergados en la Colección de cnidarios del Golfo de México y Caribe Mexicano, de la Universidad Nacional Autónoma de México, los cuales fueron previamente recolectados en varias localidades arrecifales de la Península de Yucatán. Los resultados no revelan variación significativa en el tamaño de los cnidocistos entre los dos morfotipos, aunque fueron encontradas variaciones significativas dentro de cada morfotipo. Adicionalmente, actualizamos la composición del cnidoma de C. gigantea, y discutimos sobre la utilidad de la talla de los cnidocistos para distinguir entre morfotipos o entre especies estrechamente relacionadas.

Peces arrecifales someros en el islote rocoso La Viuda, Golfo Dulce, Costa Rica / Shallow reef fishes around "La Viuda" rocky islet, Golfo Dulce, Costa Rica

Resumen Los arrecifes rocosos proveen una alta diversidad de organismos. Se presentaron cambios en la estructura de la comunidad íctica a lo largo de la estación climática y la acción del oleaje. Este estudio evaluó la diversidad y la abundancia de los peces del arrecife rocoso La Viuda, Golfo Dulce, Costa Rica (8°37'33" N - 83°14'08" W). Los peces fueron estudiados con el método de Bohnsack y Bannerot comparando las estaciones seca y lluviosa, las zonas protegidas y expuestas al oleaje usando índices ecológicos. El total de especies fue 28, las familias más abundantes fueron Pomacentridae y Labridae. La diversidad de Shannon-Wiener resultó entre 1,1 y 1,73 nits/ ind. El índice de Pielou en La Viuda fue 0.43. Se encontró mayor diversidad de especies en la época seca y en la zona protegida al oleaje.

Abstract Rocky reefs maintain a high diversity of organisms. Differences in fish community could be expected by seasonal events and wave action on rocky reefs. I studied "La Viuda" rocky islet, Golfo Dulce, CostA Rica (8°37'33" N - 83°14'08" W) with Bohnsack and Bannerot visual census methods four times between February and December 2002. I identified 28 species and the most abundant families were Pomacentridae and Labridae. Shannon-Wiener diversity was between 1.1 and 1.73 nits/ind., and Pielou 0.43. There is a higher diversity in the dry season and in areas protected from wave action. Rev. Biol. Trop. 63 (Suppl. 1): 329-338. Epub 2015 April 01.

Seabather's eruption in two triathletes at Samal Island, Davao, Philippines

Seabather's eruption (SBE) is characterized by pruritic erythematous papules on the covered areas of the body that appear within 24 hours after exposure to seawater. SBE is known to be caused by the planula of a thimble jellyfish (Linuche unguiculata) or a sea anemone (Edward siellalineata). We report cases of two adult male triathletes who developed pruritic erythematous papules on the chest and back after a swim training along the coastal waters of Samal island, Davao City. Examination of samples of the seawater revealed multiple planulae or larval forms of cnidarians. The histopathologic examination revealed moderately dense superficial and deep perivascular and periadnexal inflammatory infiltrates consisting predominantly of lymphocytes, few eosinophils and neutrophils. Treatment with a short course of systemic corticosteroids proved beneficial in both patients. A review of published literature regarding this interesting aquatic sports dermatosis was also conducted.

Ocurrence of the sea anemone Telmatactis panamensis (Verrill, 1869) (Cnidaria: Anthozoa: Actiniaria) at Isla del Coco National Park, Costa Rica / Ocurrencia de la anémona de mar Telmatactis panamensis (Verrill, 1869) (Cnidaria: Anthozoa: Actiniaria) en el Parque Nacional Isla del Coco, Costa Rica

The sea anemone fauna of Isla del Coco National Park (also known as Cocos Island Nacional Park), Pacific Costa Rica is poorly known. In the present work we report the first occurrence of the species Telmatactis panamensis. Individuals of this sea anemone (n=24) were collected at Chatham Bay intertidal and at 15m depth in Punta Ulloa, in both cases attached to rocks; during the expedition UCR-UNA-COCO-I in April 2010. We provide photographs of live individuals, external anatomy and an inventory of cnidae of the studied specimens. Possibly this species is extended to greater depth as observed by other authors in the Galápagos Islands.

La fauna de anémonas de mar es prácticamente desconocida para el Parque Nacional Isla del Coco (Costa Rica). En el presente trabajo se reporta por primera vez la presencia de la especie Telmatactis panamensis. Individuos de esta anémona de mar fueron colectados en el intermareal de Bahía Chatham y a 15m de profundidad en Punta Ulloa, en ambos casos adheridas a rocas; durante la expedición UCR-UNA-COCO-I en Abril de 2010. Se proveen fotografías de ejemplares vivos, datos de su anatomía externa y un inventario del cnidae de los especímenes estudiados. Posiblemente esta especie se extienda a mayor profundidad, tal como fue observado por otros autores para ejemplares de las Islas Galápagos.

Assessment of biomedical and pharmacological activities of sea anemones Stichodactyla mertensii and Stichodactyla gigantea from Gulf of Mannar Biosphere Reserve, southeast coast of India

Cnidarians comprise an old and diverse animal phylum, and possess a wide variety of biologically active substances. Sea anemones contain a diversity of interesting biologically active compounds including some potent toxins. In the present work, the sea anemones Stichodactyla mertensii and Stichodactyla gigantea, collected from the Mandapam coast, are characterized biomedically and pharmacologically. The crude protein was obtained by using methanol and aqueous extracts. The respective protein contents of S. mertensii and S. gigantea were found to be 2.10 µg/mL and 1.87 µg/mL. The methanol and aqueous extracts of S. mertensii and S. gigantea yielded six and nine bands by SDS-PAGE on 12 percent gel. In the hemolytic assay, both extracts exhibited hemolytic effect on chicken, goat, cow and human erythrocytes ('A', 'B' and 'O'). The neurotoxic effects of these crude extracts were determined in vivo using the sea shore crab Ocypode macrocera and mortality was observed. The mouse bioassay for lethality was performed on male albino mice. The crude extract of S. mertensii showed higher lethality (58 seconds at 1 mL-dose) than that of S. gigantea (2 minutes and 10 seconds at 0.75 mL-dose). The analgesic activity test was also carried out on albino mice by Eddy's hot plate and tail-flick methods. The extracts showed moderate analgesic effect by both hot-plate and tail-flick methods. These characteristics emphasize the need for the isolation and molecular characterization of new active toxins in S. mertensii and S. gigantea.(AU)

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.(AU)

Pharmacological and biomedical properties of sea anemones Paracondactylis indicus, Paracondactylis sinensis, Heteractis magnifica and Stichodactyla haddoni from East coast of India

OBJECTIVE@#To explore the biomedical and pharmacological activity of Paracondactylis indicus (P. indicus), Paracondactylis sinensis (P. sinensis), Heteractis magnifica (H. magnifica) and Stichodactyla haddoni (S. haddoni).@*METHODS@#The live sea anemones were kept inside the glass bowl along with some amount of distilled water in an ice container for 15 min. During stress condition, nematocysts released from the tentacles were collected and centrifuged at 5,000 rpm for 15 min. The supernatant were collected in separate cleaned beakers for lyophilisation.@*RESULTS@#The protein content of crude extracts was 15.2, 28.7, 18.2 and 35.4 μg/mL. In hemolytic assay, the P. indicus was sensitive (16.842 HT/mg) on chicken blood but P. sinensis was less sensitive (1.114 HT/mg) on chicken and goat blood. Whereas H. magnifica and S. haddoni showed hemolysis (0.879, 0.903 HT/mg and 56.263, 0.451 HT/mg) in chicken and goat blood. In antimicrobial assay, the methanol extract of P. indicus showed maximum inhibition zone of 9.7 mm against S. typhii and P. sinensis showed 9.8 mm against K. pneumonia in methanol and ethanol extracts. Whereas the H. magnifica and S. haddoni showed maximum of 10 mm against S. typhii, K. pneumonia in methanol and ethanol extracts.@*CONCLUSIONS@#The high toxic sea anemones may also contain some biologically active agents which has haemolytic, analgesic and anti-infilamatory activity.

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAE-Sepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE.(AU)

Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

It is well established that sea anemones comprise a rich source of cytolytic toxins. The present study reports the isolation and characterization of a cytolysin obtained from the sea anemone Heteractis magnifica collected in the Andaman Islands of the Indian Ocean. The crude extract was screened for hemolytic activity by a blood agar plate method and a 6-mm zone of clearance was observed after incubation. The hemolytic property of the crude extract, tested by the microtiter plate method, revealed positive results at concentrations as low as 120 ng/mL. Furthermore, it was favored by alkaline pH and was stable up to 60°C. On the other hand, the hemolytic effect was abolished by the addition of human serum. Purification steps involved ammonium sulfate precipitation and subsequent desalting by dialysis, followed by anion- and cation-exchange chromatographies. The purified fractions displayed the presence of a 19-kDa cytolysin when analyzed by SDS-PAGE. The conserved region of the cytolysin (with 303 bp) was amplified by RT-PCR and was sequenced. The sequence showed maximum homology (97 percent) with the already reported cytolysins from other sea anemone species.(AU)

Construction of an immunotoxin by linking a monoclonal antibody against the human epidermal growth factor receptor and a hemolytic toxin

Hybrid molecules obtained through conjugation of monoclonal antibodies and toxins constitute an approach under exploration to generate potential agents for the treatment of cancer and other diseases. A frequently employed toxic component in the construction of such immunotoxins is ricin, a plant toxin which inhibits protein synthesis at ribosomal level and so requires to be internalized by the cell. A hemolytic toxin isolated from the sea anemone Stichodactyla helianthus, which is active at the cell membrane level, was linked through a disulfide bond to the anti-epidermal growth factor receptor monoclonal antibody ior egf/r3. The resulting immunotoxin did not exhibit hemolytic activity except under reducing conditions. It was toxic for H125 cells that express the human epidermal growth factor receptor, but non-toxic for U1906 cells that do not express this receptor.

A Case of Sea Anemone Dermatitis / 대한피부과학회지

Sea anemone dermatitis, caused by the toxin of sea anemones (member of phylum Cnidaria), is characterized by erythematous papules, wheals, or vesicles, accompanied by pain and itching. A 25-year old woman presented with multiple erythematous papules on her right forearm, after contact with a sea anemone while scuba diving. She took both courses of an acute toxic reaction and a delayed allergic reaction.

Asexual reproduction and molecular systematics of the sea anemone Anthopleura krebsi (Actiniaria: Actiniidae)

In this paper we use allozyme analyses to demonstrate that individuals in Anthopleura krebsi aggregates are monoclonal. Additionally, sympatric samples of the red and the green colour-morphs of A. krebsi from Pernambuco, Brazil were genetically compared and no significant differences were observed between them (gene identity = 0.992), indicating that they do not belong to different biological species. All individuals within aggregates of the green colour-morph were found to be identical over the five polymorphic loci analysed. Such results would be extremely unlikely (P < 10(-11)) if the individuals analysed had been generated through sexual reproduction, thus confirming the presence of asexual reproduction in this species.

Identification of four new ceramides from Anthopleura pacifica / 药学学报

<p><b>AIM</b>To study the bioactive constituents from Anthopleura pacifica.</p><p><b>METHODS</b>Compounds were separated by Pyricularia oryzae bioassay-guided fractionation method with a combination of multi-chromatography. Their chemical structures were determined on the basis of spectral analysis and chemical evidence.</p><p><b>RESULTS</b>A portion showing activity against P. oryzae was obtained and from the portion four compounds were identified as N-hydroxyethyl-N-tetradecanoyl-(2S,3R)-octadecasphinga-4(E), 8(E)-dienine (a), N-hydroxyethyl-N-(9Z-hexadecenoyl)-(2S,3R)-octadecasphinga-4 (E), 8 (E)-dienine (b), N-hydroxyethyl-N-hexadecanoyl-(2S,3R)-octadecasphinga-4(E), 8 (E)-dienine (c) and N-hydroxyethyl-N-(13Z-docosenoyl-(2S,3R)-octadecasphinga-4(E), 8(E)-dienine(d).</p><p><b>CONCLUSION</b>All the four compounds are new ceramides.</p>

The construction of cDNA expression library from the tentacles of Sagartia rosea / 生物工程学报

A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.

Chemical constituents from marine fungus Penicillium thomii / 药学学报

<p><b>AIM</b>To investigate the bioactive constituents from the mycelium of Penicillium thomii. Which isolated from Anemone collected in Qingdao beach.</p><p><b>METHODS</b>The constituents were separated by using various chromatography and the structures were identified on the basis of extensive spectral analysis.</p><p><b>RESULTS</b>Five compounds, namely penicillixanthone A (I), p-methylbenzolic acid (II), 1-O-hexadecanoyl-2-O-(9-octadecenoyl)-3-O-(9, 12-octadecadienoyl) glycerol (III), 5 alpha, 8 alpha-epidioxy-24 zeta-methylcholesta-6, 22-dien-3 beta-ol (IV) and 1, 6, 8-trihydroxyl-3-methyl-9, 10-anthracenedione (V), were isolated from the mycelium of Penicillium thomii.</p><p><b>CONCLUSION</b>Penicillixanthone A is a new compound, while the others are isolated from Penicillium thomii for the first time.</p>

Identification of hemolytic and neuroactive fractions in the venom of the sea anemone Bunodosoma cangicum

Sea anemones are a rich source of biologically active substances. In crayfish muscle fibers, Bunodosoma cangicum whole venom selectively blocks the I K(Ca) currents. In the present study, we report for the first time powerful hemolytic and neuroactive effects present in two different fractions obtained by gel-filtration chromatography from whole venom of B. cangicum. A cytolytic fraction (Bcg-2) with components of molecular mass ranging from 8 to 18 kDa elicited hemolysis of mouse erythrocytes with an EC50 = 14 æg/ml and a maximum dose of 22 æg/ml. The effects of the neuroactive fraction, Bcg-3 (2 to 5 kDa), were studied on isolated crab nerves. This fraction prolonged the compound action potentials by increasing their duration and rise time in a dose-dependent manner. This effect was evident after the washout of the preparation, suggesting the existence of a reversible substance that was initially masking the effects of an irreversible one. In order to elucidate the target of Bcg-3 action, the fraction was applied to a tetraethylammonium-pretreated preparation. An additional increase in action potential duration was observed, suggesting a blockade of a different population of K+ channels or of tetraethylammonium-insensitive channels. Also, tetrodotoxin could not block the action potentials in a Bcg-3-pretreated preparation, suggesting a possible interaction of Bcg-3 with Na+ channels. The present data suggest that B. cangicum venom contains at least two bioactive fractions whose activity on cell membranes seems to differ from the I K(Ca) blockade described previously

Behavioral and electroencephalographic analysis of seizures induced by intrahippocampal injection of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

In this study, the behavioral and electroencephalographic (EEG) analysis of seizures induced by the intrahippocampal injection in rats of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera, was determined. The first alterations occurred during microinjection of granulitoxin (8 µg) into the dorsal hippocampus and consisted of seizure activity that began in the hippocampus and spread rapidly to the occipital cortex. This activity lasted 20-30 s, and during this period the rats presented immobility. During the first 40-50 min after its administration, three to four other similar short EEG seizure periods occurred and the rats presented the following behavioral alterations: akinesia, facial automatisms, head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements. Within 40-50 min, the status epilepticus was established and lasted 8-12 h. These results are similar to those observed in the acute phase of the pilocarpine model of temporal lobe epilepsy and suggest that granulitoxin may be a useful tool not only to study the sodium channels, but also to develop a new experimental model of status epilepticus.

Efecto agregante plaquetario de 2 citolisinas: sti y stii purificadas de la anémona marina Stichodactyla helianthus / Platelet aggregation effect of 2 cytolysins: sti and stii purified from the sea anemone Stichodactyla helianthus

Las citolisinas Sticholysina I (St I) y Sticholysina II (St II) inducen la agregación plaquetaria en el plasma rico en plaquetas en el rango de concentraciones ensayadas (0,5 a 10 µg/mL). Para ambas citolisinas se obtienen porcentajes de agregación plaquetaria superiores al 90 porciento con menos del 50 porciento de lisis celular. La agregación plaquetaria se mantiene elevada aún cuando la lisis celular disminuye a menos del 20 porciento. El EDTA 2 mM/L y el verapamilo 100 mM/L inhiben significativamente la agregación inducida por StI, lo que evidencia que el calcio extracelular tiene una función importante en este proceso y probablemente esta citolisina tiene una función similar a la de un ionóforo de calcio. Con StII no se obtuvo inhibición significativa de la agregación en presencia de EDTA y verapamilo. La agregación inducida por ambas citolisinas no está influida por el aumento del AMPc intracelular y es independiente de la formación de tromboxano A2 en la plaqueta