Strong Hemolytic Activity Research Articles

Structure-Activity Relationship of Model Peptides Based on Pleurocidin, an Antibacterial Peptide

Abstract Pleurocidin (Ple) isolated from the skin of the winter flounder is a cationic amphiphilic peptide with antibacterial activity. To search for a simple peptide with strong antibacterial activity but no hemolytic activity, we designed three model peptides based on Ple, Ple I, Ple II, and Ple III. In the model peptides, Leu or Ala, and Lys were used instead of some hydrophobic amino acids and hydrophilic ones in Ple, respectively, to simplify the amino acid composition. The order of their α-helicity and hydrophobicity is Ple I > Ple II > Ple III. Ple I and Ple II showed antibacterial activity similar to Ple amide (Ple-NH2). However, Ple I had very strong hemolytic activity. Ple III exhibited negligible biological activity. Ple II was found to be a desirable candidate to promote the study of the structure-activity relationship.

Characterization of functional domains of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata.

CEL-III is a Ca(2+)-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectin isolated from the marine invertebrate Cucumaria echinata. This lectin exhibits strong hemolytic activity and cytotoxicity through pore formation in target cell membranes. The amino acid sequence of CEL-III revealed the N-terminal two-thirds to have homology to the B-chains of ricin and abrin, which are galactose-specific plant toxic lectins; the C-terminal one-third shows no homology to any known proteins. To examine the carbohydrate-binding ability of the N-terminal region of CEL-III, the protein comprising Pyr1-Phe283 was expressed in Escherichia coli cells. The expressed protein showed both the ability to bind to a GalNAc-immobilized column as well as hemagglutinating activity for rabbit erythrocytes, confirming that the N-terminal region has binding activity for specific carbohydrates. Since the C-terminal region could not be expressed in E. coli cells, a fragment containing this region was produced by limited proteolysis of the native protein by trypsin. The resulting C-terminal 15 kDa fragment of CEL-III exhibited a tendency to self-associate, forming an oligomer. When mixed with erythrocytes, the oligomer of the C-terminal fragment caused hemagglutination, probably due to hydrophobic interaction with cell membranes, while the monomeric fragment did not. Chymotryptic digestion of the preformed CEL-III oligomer induced upon lactose binding also yielded an oligomer of the C-terminal fragment comprising six molecules of the 16 kDa fragment. These results suggest that after binding to cell surface carbohydrate chains, CEL-III oligomerizes through C-terminal domains, leading to the formation of ion-permeable pores by hydrophobic interaction with the cell membrane.

Hemolytic activity of crystalline silica – Separated erythrocytes versus whole blood

Whole blood and 1% erythrocyte suspensions were treated with crystalline silica (quartz DQ12, Min-U-Sil5) at concentrations of 0.5, 1, 2, and 5 mg/ml. Quartz DQ12 and Min-U-Sil5 revealed a strong dose-dependent hemolytic activity in the 1% erythrocyte suspension reaching nearly total hemolysis (>80%) at the highest tested concentration of 5 mg/ml. This effect may be ascribed to surface reactivity by silanol groups. In contrast, using whole blood cultures the tested silica dusts caused no or only minor hemolytic activity (<4%). The mechanism by which the hemolytic activity is prevented in whole blood cultures can be attributed to a number of factors such as the presence of metal binding proteins and free radical scavenger, antioxidant mechanisms and to coating of the silica surface by proteins, antibodies and complement. In contrast to separated erythrocytes whole blood represents an independent physiological compartment with functions of host defence and regulatory functions against cell damaging effects produced by oxidative stress.

Oligomerization process of the hemolytic lectin CEL-III purified from a sea cucumber, Cucumaria echinata.

CEL-III is a Ca(2+)-dependent lectin purified from a sea cucumber, Cucumaria echinata. This protein exhibits strong hemolytic activity as well as cytotoxicity toward some cultured cell lines. Hemolysis is caused by CEL-III oligomers formed in the cell membrane after binding to specific carbohydrate chains on the cell surface. We have found that the oligomerization of CEL-III is also induced by the binding of simple carbohydrates, such as lactose, in aqueous solution under high pH and high ionic strength conditions. From gel filtration analysis of the oligomerization of CEL-III, it was found that the formation of the CEL-III oligomer is effectively induced by the binding of lactose and lactulose, disaccharides containing a beta-galactoside structure. Electron micrographs of the resulting oligomers revealed them to exist as particles with a size of approximately 20-30 nm. The oligomerization process required more than 1 h, which is consistent with the increase in surface hydrophobicity as measured using a fluorescent probe, 8-anilinonaphthalene-1-sulfonate. However, a change in the far-UV CD spectra as well as small-angle X-ray scattering occurred within a few minutes, suggesting that a structural change in the protein takes place rapidly, but the following growth of the oligomer is a much slower process.

Structure-Activity Relationships of Haemolytic Saponins

The haemolytic activity of 59 samples, natural or partially synthetic pure saponins and mixtures thereof, was determined. The tested saponins possessed triterpenic genins and six structural skeleton types were studied. Structure-activity relationships were established by comparison of the functional groups of each saponin and of the branched sugar chains attached to aglycone. The haemolytic activity is shown to depend on the number of sugar units in the chains, as well as on the presence of an osidic chain in position 3 or of functional groups on the genin such as carboxylic or 16a-OH. Comparison of activities of monodesmosidic and bidesmosidic saponins showed that monodesmosidic saponins were generally more active and suggested a polar balance between the two sugar chains at positions 3 and 28. Twelve saponins showed strong haemolytic activity and five possessed a Haemolytic Index (HI) greater than 100,000, such as the a- and ß-aescines (HI: 200,000).

Characterization of unique amphipathic antimicrobial peptides from venom of the scorpion Pandinus imperator

Two novel antimicrobial peptides have been identified and characterized from venom of the African scorpion Pandinus imperator. The peptides, designated pandinin 1 and 2, are α-helical polycationic peptides, with pandinin 1 belonging to the group of antibacterial peptides previously described from scorpions, frogs and insects, and pandinin 2 to the group of short magainin-type helical peptides from frogs. Both peptides demonstrated high antimicrobial activity against a range of Gram-positive bacteria (2.4–5.2μM), but were less active against Gram-negative bacteria (2.4–38.2μM), and only pandinin 2 affected the yeast Candida albicans. Pandinin 2 also demonstrated strong haemolytic activity (11.1–44.5μM) against sheep erythrocytes, in contrast with pandinin 1, which was not haemolytic. CD studies and a high-resolution structure of pandinin 2 determined by NMR, showed that the two peptides are both essentially helical, but differ in their overall structure. Pandinin 2 is composed of a single α-helix with a predominantly hydrophobic N-terminal sequence, whereas pandinin 1 consists of two distinct α-helices separated by a coil region of higher flexibility. This is the first report of magainin-type polycationic antimicrobial peptides in scorpion venom. Their presence brings new insights into the mode of action of scorpion venom and also opens new avenues for the discovery of novel antibiotic molecules from arthropod venoms.

Mechanisms and Surface Chemical Prediction of Imipramine ‐Induced Hemolysis Suppressed by Modified Cyclodextrins

The suppression of imipramine hydrochloride (IMP)‐ induced hemolysis by native cyclodextrins (α‐, β‐, and γ‐CDs) and β‐CD derivatives is measured as a function of CD concentration and is quantitatively correlated with the surface tension of the solution determined at 37.0 °C. The modified β‐CDs are more or less adsorbed onto the air–water interface and occupy larger areas than the wider rim of β‐CD. The surface tension data at low CD concentrations in the presence of 3 mM IMP allow us to estimate the 1:1 binding constants of IMP with CDs. Both the capabilities of hemolysis suppression and surface tension elevation for 3 mM IMP are strong in the order carboxymethyl‐β‐CD (CM) > β‐CD ≈ 6‐O‐glucosyl‐β‐CD (G1) > γ‐CD > 2‐hydroxypropyl‐β‐CD (HP) > α‐CD ≥ 2,6‐di‐O‐methyl‐β‐CD (DM). The suppression of IMP‐induced hemolysis is ascribed to the decrease in the concentration of free IMP molecules. This concentration can be quantitatively estimated from the surface tension data determined at 37 °C. Therefore, the suppression of IMP‐induced hemolysis by most of the CDs can be quantitatively predicted from these surface tension data, regardless of the kind and concentration of CD. However, α‐CD, HP, and DM are outliers of this prediction. This failure for α‐CD and HP is ascribed to their weaker competitive binding to IMP than to membrane phospholipid. Because DM has a strong hemolytic activity, it does not almost suppress the IMP‐induced hemolysis. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1056–165, 2001

Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins. Four monoclonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin. Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two antibodies (mAbs C and G) was weaker. Subsequently, nine truncated PLOs were derived from recombinant Escherichia coli by various deletions from the N-terminus. Strong hemolytic activity was recognized in truncates of PLO following the deletion of 30 or 55 amino acids, but not in the truncate with deletion of 74 residues. Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs. The epitope for mAbs C and G lies within the undecapeptide region (amino acids 487–505) of the C-terminus of PLO, which seems to be the binding site to erythrocytes. In contrast, the epitopes for mAbs S and H, which showed strong neutralizing activity, were found to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. From these results, it seems that the N-terminal region of PLO, in particular, the region of amino acids 55–74 is important for hemolytic activity.

Calcium ions stabilize a protein structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata.

CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc)-specific lectin purified from a marine invertebrate, Cucumaria echinata, has a strong hemolytic activity, especially toward human and rabbit erythrocytes in the presence of Ca2+. We evaluated the role of Ca2+ in hemagglutinating and hemolytic activities of CEL-III. We found that Ca2+ is closely associated with both activities of CEL-III. The fluorescence spectra of CEL-III upon binding to Ca2+ were measured. The result showed a structural change of CEL-III in the presence of Ca2+. The structural change of CEL-III upon Ca2+ binding was further demonstrated by stabilization against urea denaturation and by insusceptibility to protease digestions. CEL-III was completely unfolded at a low concentration of 2 M urea, while CEL-III complexed with Ca2+ was stable in 6 M urea. As for protease digestions, CEL-III monomer and oligomer were readily digested by trypsin, chymotrypsin, and papain in the absence of Ca2+, while they were insusceptible to the three proteases in the presence of Ca2+. The papain digestion of the decalcified oligomer produced a large C-terminal peptide, suggestting that the C-terminal region of CEL-III may participate in oligomerization of CEL-III as a core domain.

Characterization, crystallization and preliminary X--ray diffraction analysis of acutohaemolysin, a haemolytic toxin from Agkistrodon acutus venom.

Acutohaemolysin, a phospholipase A(2) (PLA(2)) from the venom of the snake Agkistrodon acutus, has been isolated and purified to homogeneity by anion-exchange chromatography on a DEAE-Sepharose column followed by cation-exchange chromatography on a CM-Sepharose column. It is an alkaline protein with an isoelectric point of 10.5 and is comprised of a single polypeptide chain of 13 938 Da. Its N-terminal amino-acid sequence shows very high similarity to Lys49-type PLA(2) proteins from other snake venoms. Although its PLA(2) enzymatic activity is very low, acutohaemolysin has a strong indirect haemolytic activity and anticoagulant activity. Acutohaemolysin crystals with a diffraction limit of 1.60 A were obtained by the hanging-drop vapour-diffusion method. The crystals belong to the space group C2, with unit-cell parameters a = 45.30, b = 59.55, c = 46.13 A, beta = 117.69 degrees. The asymmetric unit contains one molecule.

Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants.

Adjuvant and haemolytic activities of 47 saponins purified from medicinal and food plants were examined. The compounds showed various levels of both adjuvant and haemolytic activities. Soyasaponins and lablabosides showed strong adjuvant activity but little haemolytic activity. Jujubosides showed strong adjuvant and haemolytic activities. Escins showed weaker adjuvant activity than the adjuvant-control, but strong haemolytic activity. Comparison of the functional groups of each saponin revealed that the acyl residue in saponin, the aldehyde group at carbon 4 in aglycone, and branched sugar chains attached to aglycone, were not essential for adjuvant activity. Furthermore, saponins with an acyl residue or oxide-ring moiety tended to show haemolytic activity. These results suggest that the adjuvant activity of saponins does not relate with haemolytic activity. It is considered that not only the functional groups themselves, but the overall conformation harmoniously consisting of such functional groups, affects adjuvant activity of saponins.

Primary structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata and its cDNA: structural similarity to the B-chain from plant lectin, ricin

CEL-III, a galactose/ N-acetylgalactosamine (Gal/GalNAc) specific lectin purified from a marine invertebrate Cucumaria echinata has a strong hemolytic activity especially toward human and rabbit erythrocytes. We determined the primary structure of the CEL-III by examining the amino acid sequences of the protein and the nucleotide sequence of the cDNA. The cDNA encoding CEL-III has 1823 nucleotides and an open reading frame of 1296 nucleotides. CEL-III is composed of 432 amino acid residues with a M r of 47 457 and has six internal tandem repeats, each with of 40–50 amino acids, comprising the N-terminal two-thirds of the molecule. Similar repeats are found in the B-chains of cytotoxic plant lectins, such as ricin and abrin, where six repetitive sequences extend throughout the molecules. A hydropathy plot predicts hydrophobic segments in the C-terminal region of CEL-III. These findings suggest that the N-terminal region of CEL-III plays an important role in binding to carbohydrate receptors on the target cell membranes, an event which triggers an intermolecular hydrophobic interaction of the C-terminal region, the result being oligomerization of CEL-III to lead to pore-formation in erythrocyte membrane.

Influence of antigenic forms and adjuvants on the IgG subclass antibody response to Aujeszky's disease virus in mice

The influence of antigenic forms of Aujeszky's disease virus (ADV) and adjuvant types on the production of IgG subclass antibodies in mice was investigated. Particulate antigen, inactivated ADV, alone induced IgG1 and lower IgG2a antibody production, while the antigen adsorbed onto aluminum phosphate gel (alum) enhanced IgG1 antibody production but suppressed IgG2a antibody production as well as solubilized ADV antigen adsorbed onto alum. QS21 saponin purified from Quillaja saponaria promoted the production of IgG1 and IgG2a antibodies in a large extent against the both particulate and soluble antigens, while this saponin has strong hemolytic activity. Lablaboside F saponin isolated from Dolichos lablab without hemolytic activity, also induced the production of large IgG1 and little IgG2a antibody against both antigens. Oil-based adjuvant, ISA70 of water-in-oil type and ISA25 of oil-in-water type, increased IgG1 and IgG2a antibodies against the both soluble and particulate antigens, whereas a combination of ISA25 and soluble antigen reduced IgG2a antibody response. These results indicate that IgG1 antibody production was not suppressed by a combination of antigenic form and adjuvant type, however, IgG2a antibody production was influenced.

Chemical modification of the hemolytic lectin CEL-III by succinic anhydride: involvement of amino groups in the oligomerization process.

CEL-III is a Ca(2+)-dependent lectin from a marine invertebrate, Cucumaria echinata, which shows strong hemolytic activity toward human and rabbit erythrocytes. After binding to carbohydrate receptors, CEL-III oligomerizes in the erythrocyte membrane to form ion-permeable pores, leading to the colloid osmotic rupture of the cells. Since hemolysis was greatly increased in the alkaline pH, especially above pH 9, involvement of amino groups of CEL-III in its hemolytic activity was evaluated using chemical modification by succinic anhydride. After modification of 7 amino groups per protein molecule, the hemolytic activity of CEL-III was reduced to 23% of the native protein, but hemagglutinating and carbohydrate-binding activities were only slightly affected even after modification of 14 amino groups. A circular dichroism spectrum of modified CEL-III showed almost no change in the secondary structure from that of the native protein, indicating that the decrease of hemolytic activity was not caused by partial unfolding of the protein. Immunoblotting analysis of the erythrocyte membrane treated with modified CEL-III showed a decrease in the formation of CEL-III oligomer in the membrane in parallel with the decrease in hemolytic activity. These results suggest that amino groups of CEL-III are involved in its oligomerization in the cell membrane, and their modification leads to inactivation of the protein without much influence on the carbohydrate-binding activity.

α-Helix to β-sheet transition within the Leu i Lys j (i=2j) series of lytic amphipathic peptides by decreasing their size

De novo designed extremely simplified amphipathic basic LeuiLysj (i=2j) peptides of 8, 9 and 15 residues were synthesized to clarify the mechanism of action of natural cytotoxic and hemolytic small proteins or peptides. They proved to have strong hemolytic activity towards human erythrocytes which increases with peptide length. These peptides are highly surface active and form stable peptidic films at the air/water interface. The sensitive and efficient FTIR modulated polarization technique (PMIRRAS) allows one to obtain in situ structural and orientational information about the peptides at the interface. A transition of secondary structure is observed: the shorter peptides (8 and 9 residues) adopt β-sheet structures while the longer one (15 residues) is folded into an α-helix. In both cases, the peptides lie with the axis parallel to the interface. Their insertion into a dimyristoylphosphatidylcholine monolayer can be followed from the increase in the surface and/or pressure of the films. In the mixed films, the peptides adopt the same structure and orientation as observed at the air/water interface. Therefore, among the same series of peptides, a transition from β-sheet to α-helix occurs when the length increases (roughly>10 aa), but despite this drastic change both types of structures result in strongly hemolytic peptides.

Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs.

We examined the taxonomic position of seven Aeromonas isolates, recovered from Flemish and Scottish drinking water production plants and reservoirs, which were previously recognized by numerical analysis of genomic AFLP fingerprints as members of an unknown Aeromonas taxon that most closely resembled the species Aeromonas bestiarum (DNA hybridization group [HG] 2). The new phenotypic and DNA-DNA hybridization data obtained in this study show that the A. bestiarum-like strains constitute a separate Aeromonas species, for which the name Aeromonas popoffii sp. nov. is being proposed. The new species exhibited an internal DNA relatedness ranging from 79 to 100% and was 22 to 63% related to the type or reference strains of other Aeromonas spp. The highest DNA binding values were determined with A. bestiarum (51 to 63%), followed by Aeromonas hydrophila sensu stricto (HG1; 50 to 60%) and Aeromonas salmonicida (HG3; 39 to 55%). Although fingerprints generated by ribotyping and cellular fatty acid analysis often were highly similar, minor differences between the respective fingerprints were of significance for the differentiation of A. popoffii from its closest taxonomic neighbors, HG1, HG2, and HG3. Phenotypically, all seven strains of A. popoffii were positive for acid and gas production from D-glucose and glycerol, growth in KCN broth, arginine dihydrolase, DNase, Voges-Proskauer reaction, and resistance to vibriostatic agent O/129 and ampicillin but displayed negative reactions for production of urease, tryptophan deaminase, ornithine decarboxylase, and lysine decarboxylase (LDC). None of the strains displayed strong hemolytic activity. The lack of D-sucrose fermentation and LDC production and the ability to utilize DL-lactate as the sole energy and carbon source were useful characteristics for the biochemical separation of A. popoffii from A. bestiarum. Other Aeromonas spp. could be differentiated phenotypically from the new species by at least two features. The chromosomal G+C content of A. popoffii ranges from 57.7 to 59.6 mol%. Strain LMG 17541 is proposed as the type strain.

Cell membrane permeability- and mitochondrial dysfunction-inducing activities in cell free supernatants from Serpulina hyodysenteriae serotypes 1 and 2.

Serpulina hyodysenteriae is a Gram-negative, anaerobic, oxygen-tolerant spirochete recognized as the etiologic agent of swine dysentery. Currently 9 serotypes/serogroups of S. hyodysenteriae are recognized on the basis of antigenicity of cell wall lipooligosaccharide antigens. Characteristics that distinguish S. hyodysenteriae from other intestinal spirochetes of swine include strong hemolytic activity and enteropathogenicity for swine, mice, guinea pigs and chicks.KeywordsToxic ActivityCell Free SupernatantCell Membrane PermeabilityAmerican Veterinary Medical AssociationPeripheral Blood LymphoThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

CHARACTERIZATION OF AEROMONAS SPECIES ISOLATED FROM FOOD

ABSTRACT Characterization of ten Aeromonas spp. isolates from local foods and A. hydrophila ATCC 7965, revealed that all strains of A. hydrophila, A. sobria and one strain of A. caviae showed strong hemolytic activity. All of them were protease producers except one A. caviae strain. Some A. hydrophila and A. caviae strains fermented lactose, coagulated milk and reduced litmus. The majority of strains were resistant to at least 5 of 14 antibiotics tested. All the strains grew over the range of 5–42C except those of A. caviae which failed to grow at 42C. Frozen storage at ‐16C greatly decreased the viability of the tested strains. Radiation D10values of strains ranged from 0.109–0.253 kGy in nutrient broth and increased when some strains were irradiated in ground meat at room temperature. Both strains of A. hydrophila (ATCC andFI), A. sobria FI and A. caviae FI grew normally in the presence of 1 and 3% salt. Only A. hydrophila FI tolerated 6.5% salt. These four strains were able to grow in the presence of 0.05–0.3% potassium sorbate at pH 7.0, while at pH 5.0 the presence of sorbate prevented their growth.

Coelenterolysin: a hemolytic polypeptide associated with the coelenteric fluid of sea anemones

The gastrovascular fluids of the sea anemone Phymactis clematis display strong hemolytic activity, which has basic pH optimum, is thermolabile and is sensitive to proteases. The hemolytic agent from the gastrovascular fluid was partially purified by ammonium sulfate precipitation, chromatography on Dowex-50W cation exchanger and gel filtration on Sephadex G-50. A single peak elutes from the latter with an estimated mol. wt of 18,000. This elution pattern is unaffected if the chromatography is carried out in the presence of 5 M urea. These results indicate that the hemolytic activity is due to a single peptide or a group of peptides of similar size, which we here designate as “coelenterolysin”. Coelenterolysin is also present in sea anemone tissue homogenates, is different from the nematocyst toxin and is not associated with phospholipase activities. It is inhibited by sphingomyelin. This is the first report of hemolytic polypeptides associated with the coelenteric fluid of sea anemones. Coelenterolysin may have a role in extracellular digestion, defense against predators and invasion of the coelenteron by foreign organisms.

Synthetic dipeptide, N-stearoyl-D-Ser-L-Pro-OEt, induces release of tissue-type plasminogen activator in cultured cells and in experimental animals.

The tissue-type plasminogen activator (t-PA)-releasing action of synthetic dipeptides containing Gly, Ser or Pro was investigated. Among 10 dipeptides, Boc-L-Ser-L-Pro-OH and H-L-Ser-L-Pro-OH induced t-PA release in vitro, but the others were inactive. Since Boc-L-Ser-L-Pro-OH was more effective than H-L-Ser-L-Pro-OH, 7 related dipeptides with N-acylation were synthesized. Five of them enhanced the release of t-PA; N-stearoyl-L-Ser-L-Pro-OH (FK-5) had the greatest effect. Four compounds were further examined for activity to enhance the release of t-PA in rats. FK-5 produced a two-fold increase in fibrinolytic activity, and N-palmitoyl-L-Ser-L-Pro-OH (FK-4) also markedly enhanced the release of t-PA. Since FK-5 caused severe hemolysis, 7 analogues of FK-5 were synthesized. All of them enhanced the release of t-PA from melanoma (Bowes) cells. In rats, FK-5, N-stearoyl-D-Ser-L-Pro-OH (FK-8) and N-stearoyl-D-Ser-L-Pro-OEt (FK-10) enhanced the fibrinolytic activity two-fold. FK-5 and FK-8 also exhibited strong hemolytic activity, but FK-10 did not induce hemolysis. Therefore, FK-10 was examined in rabbits. After the injection of this compound, the fibrinolytic activity in the euglobulin fraction was markedly enhanced without accompanying hemolysis. Thus, FK-10 potently enhances fibrinolytic activity both in vitro and in vivo.