Crystal structure of flavocetin-A, a platelet glycoprotein Ib-binding protein, reveals a novel cyclic tetramer of C-type lectin-like heterodimers

Snake venom contains a number of the hemostatically active C-type lectin-like proteins, which affect the interaction between von Willebrand factor (vWF) and the platelet glycoprotein (GP) Ib or platelet receptor to inhibit/induce platelet activation. Flavocetin-A (FL-A) is a high-molecular mass C-type lectin-like protein (149 kDa) isolated from the habu snake venom. FL-A binds with high affinity to the platelet GP Ibalpha-subunit and functions as a strong inhibitor of vWF-dependent platelet aggregation. We have determined the X-ray crystal structure of FL-A and refined to 2.5 A resolution. This is a first elucidation of a three-dimensional structure of the platelet GP Ib-binding protein. The overall structure reveals that the molecule is a novel cyclic tetramer (alphabeta)(4) made up of four alphabeta-heterodimers related by a crystallographic 4-fold symmetry. The tetramerization is mediated by an interchain disulfide bridge between cysteine residues at the C-terminus of the alpha-subunit and at the N-terminus of the beta-subunit in the neighboring alphabeta-heterodimer. The high affinity of FL-A for the platelet GP Ib alpha-subunit could be explained by a cooperative-binding action through the multiple binding sites of the tetramer.     

Chelation affects the conformation, lability and aggregation of channel catfish (Ictalurus punctatus) phosphorylcholine-reactive protein (PRP).

1. Phosphorylcholine-reactive protein (PRP) affinity-purified from channel catfish (Ictalurus punctatus) serum on phosphorylcholine-Sepharose, eluted from Bio-Gel A-5M as a 94.6 +/- 2.4 kDa protein when the gel filtration column buffer (Tris-saline) contained 25mM ethylenediaminetetraacetic acid (EDTA). 2. PRP chelated with EDTA immediately after affinity purification and gel-filtered in Tris-saline-EDTA, eluted as a 75.5 +/- 2.67 kDa protein referred to as fast-PRP (F-PRP). 3. PRP and F-PRP were identical on SDS-PAGE. Both resolved as a broad band of protein (ca 86-100 kDa) on non-reducing gels or as a ca 100 kDa protein after reduction with 2-mercaptoethanol (2-ME). 4. After gel-filtration in Tris-saline-EDTA, nearly complete reduction of 100 kDa PRP was achieved on SDS-PAGE. However, the protein regained its resistance to reduction upon storage at -60 degrees C. 5. SDS-PAGE and native PAGE also revealed that during storage, PRP and F-PRP combined to form 3 different aggregates referred to as aggregated-PRP (aggPRP). These aggregates are readily dissociated in the presence of 2-ME, suggesting a covalent interaction between adjacent pentamers comprising decameric aggPRPs. 6. PRP, F-PRP, and aggPRP have similar amino acid compositions.     

Cloning and Nucleotide Sequence of the Bacillus subtilis K trp B Gene Encoding Tryptophan Synthase β-Subunit

When defatted soy milk was ultracentrifuged, 34kDa allergenic soybean protein Gly m Bd 30 К was more abundant in the precipitate than in the supernatant by an SDS-PAGE analysis. The addition of more than 10 тм of 2-mercaptoethanol (2-ME) to the soy milk resulted not only in further removal of the 34 kDa allergenic protein to the precipitate, but also in better recovery of conglycinin in the supernatant.

After a two-dimensional SDS-PAGE analysis (the first dimension, minus 2-ME; the second, plus 2-ME) of the precipitates, superimposition between the CBB-stained gel and the electroblotted membrane stained with a monoclonal antibody specific to Gly m Bd 30 К indicated that part of Gly m Bd 30 К was preferentially bound to the a’- and a-subunits of conglycinin, and that part of them had formed the dimer through a disulfide bond.     

Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom

A novel C-type lectin protein (CLP), lebecetin, was purified to homogeneity from the venom of Macrovipera lebetina by gel filtration on a Sephadex G75 column and ion exchange chromatography on Mono S column. Lebecetin is a basic protein with a pHi=9.9 and migrates in SDS-PAGE as a single band or two distinct bands under nonreducing and reducing conditions, respectively. These results are further confirmed by MALDI-TOF mass spectrometry that indicates a molecular mass of 29779 Da for native lebecetin and molecular masses of 15015 and 16296 Da for alpha and beta subunits, respectively. The N-terminal amino acid sequences of lebecetin subunits show a high degree of similarity with those of C-type lectin-like proteins. In addition, functional studies showed that lebecetin has a potent inhibitory effect on platelet aggregation induced by thrombin in a concentration-dependent manner. In contrast, no inhibitory effect is observed when platelets are exposed to thromboxane A2 (TxA2) mimetic (U46619) or arachidonic acid. Moreover, there was no effect either on blood coagulation or A, B and O washed human erythrocytes agglutination. Furthermore, flow cytometric analysis revealed that fluoro-isothiocyanate (FITC)-labelled lebecetin bound to human formalin fixed platelets in a saturable and concentration manner and this binding was specifically prevented by anti-glycoprotein Ib (GPIb) mAb. These observations suggest that lebecetin is a C-type lectin-like protein that selectively binds to platelet GPIb.     

Structure of rhodocetin reveals noncovalently bound heterodimer interface

Rhodocetin is a unique heterodimer consisting of alpha- and beta-subunits of 133 and 129 residues, respectively. The molecule, purified from the crude venom of the Malayan pit viper, Calloselasma rhodostoma, functions as an inhibitor of collagen-induced aggregation. Rhodocetin has been shown to have activity only when present as a dimer. The dimer is formed without an intersubunit disulfide bridge, unlike all the other Ca(2+)-dependent lectin-like proteins. We report here the 1.9 A resolution structure of rhodocetin, which reveals the compensatory interactions that occur in the absence of the disulfide bridge to preserve activity.     

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus . Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol (2-ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. When FSG was first carboxymethylated with non-radioactive iodoacetic acid and then reduced with 2-ME and finally carboxymethylated with ¹⁴C-iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted-FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.     

Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases

Antioxidant defenses include a group of ubiquitous, non-heme peroxidases, designated the peroxiredoxins, which rely on an activated cysteine residue at their active site to catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and peroxynitrite. In the typical 2-Cys peroxiredoxins, a second cysteinyl residue, termed the resolving cysteine, is also involved in intersubunit disulfide bond formation during the course of catalysis by these enzymes. Many bacteria also express a flavoprotein, AhpF, which acts as a dedicated disulfide reductase to recycle the bacterial peroxiredoxin, AhpC, during catalysis. Mechanistic and structural studies of these bacterial proteins have shed light on the linkage between redox state, oligomeric state, and peroxidase activity for the peroxiredoxins, and on the conformational changes accompanying catalysis by both proteins. In addition, these studies have highlighted the dual roles that the oxidized cysteinyl species, cysteine sulfenic acid, can play in eukaryotic peroxiredoxins, acting as a catalytic intermediate in the peroxidase activity, and as a redox sensor in regulating hydrogen peroxide-mediated cell signaling.     

Native and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformis

Many branchiopod crustaceans are endowed with extracellular, high-molecular-weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically 'oldest' extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35,775+/-4 and 36,055+/-4 Da, respectively, determined by ESI-MS. Nonreducing conditions showed only two disulfide-bridged dimers, a homodimer of TcHbA, designated D1 (71,548+/-5 Da), and the heterodimer D2 (71,828+/-5 Da). Carbamidomethylation of free SH groups revealed the presence of three cysteines per subunit and indicated one intrasubunit and one intersubunit disulfide bridge. Ultracentrifugation and light-scattering experiments under nondenaturating conditions yielded mass estimates that suggested an uneven number of 17 subunits forming the native hemoglobin. This unrealistic number resulted from the presence of two size classes (16-mer and 18-mer), which were recognized by native PAGE and Ferguson plot analysis. ESI-MS revealed three hemoglobin isoforms with masses of 588.1 kDa, 662.0 kDa, and 665.0 kDa. The 16-mer and the smaller 18-mer species are supposed to be composed of TcHbA only, given the dominance of this subunit type in SDS/PAGE. Transmission electron microscopy of negatively stained specimens showed a population of compact molecules with geometrical extensions of 14, 16 and 9 nm. The proposed stoichiometric model of quarternary structure provides the missing link to achieve a mechanistic understanding of the structure-function relationships among the multimeric arthropodan hemoglobins.     

Identification of cyst and trophozoite antigens from Colombian Giardia duodenalis isolates recognized by IgA

Little is known about the role of IgA in the immune response against Giardia duodenalis infection. The current study identified the antigens of Colombian G. duodenalis isolates which stimulate the production of IgA anti-G. dudoenalis. Cyst and trophozoite stage proteins were separated by SDS-PAGE and their antigenicity was determined by Western blot. Without 2-mercapto ethanol (2-ME), the protein profile of the cyst stage showed 24 proteins within a molecular weight range of 23-270 kDa; with 2-ME, 35 polypeptides ranging from 22 to 241 kDa were distinguished. The trophozoite stage protein profile without 2-ME was formed by 16 proteins within the range of 24-270 kDa; with 2-ME, 45 proteins were present between 18 and 241 kDa. The identification of 20 and 29 antigens from the cyst and trophozoite stage, respectively, suggested that G. duodenalis stimulates a specific humoral immune response in the human host. The antigens of 31, 57, 110, 133, and 170 kDa recognized by anti-G duodenalis IgA in both cysts and trophozoites corresponded with G. duodenalis isolates from other geographic regions, whereas those of 35, 38, 43, 45, 49, 52, 60, 62, 65, 72, 82, 99, 145, 155, and 185 kDa seemed specific to Colombian isolates. This indicated that antigens of 57, 65, 145, and 170 kDa, recognized by anti-G. duodenalis IgA antibodies in cysts (with frequencies between 82% and 96%) and trophozoites (with frequencies between 86% and 97%) can be considered identification markers for G. duodenalis infections.     

Rapid maturation of glycoprotein hormone free alpha-subunit (GPHalpha) and GPHalpha alpha homodimers

The dynamics of glycoprotein hormone alpha-subunit (GPHalpha) maturation and GPHalpha alpha homodimer formation were studied in presence (JEG-3 choriocarcinoma cells) and absence (HeLa cells) of hCGbeta. In both cases, the major initially occurring GPHalpha variant in [35S]Met/Cys-labeled cells carried two N-glycans (M(r app) = 22 kDa). Moreover, a mono-N-glycosylated in vivo association-incompetent GPHalpha variant (M(r app) = 18 kDa) was observed. In JEG-3 cells the early 22-kDa GPHalpha either associated with hCGbeta, or showed self-association to yield GPHalpha alpha homodimers, or was later converted into heavily glycosylated large free GPHalpha (M(r app) = 24 kDa). Micro-preparative isolation of intracellular GPHalpha alpha homodimers of JEG-3 cells and their conversion by reduction revealed that they consisted of 22-kDa GPHalpha monomers and not of large free GPHalpha. In HeLa cells, the large free GPHalpha variant was not observed, whereas GPHalpha alpha homodimers were present. Intracellularly, early GPHalpha alpha homodimers (35 kDa) and late variants (JEG-3: 44 kDa, HeLa: 39 kDa) were found. Both cell types secreted 45 kDa GPHalpha alpha homodimers. Large free GPHalpha and GPHalpha alpha homodimers were more rapidly sialylated than hCG alphabeta-heterodimers indicating a sequestration mechanism in the secretory pathway. In GPHalpha alpha homo- as well as hCG alphabeta-heterodimers the subunit interaction site, located on loop 2 of GPHalpha (amino acids 33-42), became immunologically inaccessible indicating similar spatial orientation of GPHalpha in both types of dimers. The studies demonstrate the formation, in vivo dynamics of GPHalpha alpha homodimers, and the pathways of the cellular metabolism of variants of GPHalpha, monoglycosylated GPHalpha and large free GPHalpha.     

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.     

Crystal structure of rhodocytin, a ligand for the platelet-activating receptor CLEC-2

Binding of the snake venom protein rhodocytin to CLEC-2, a receptor on the surface of human platelets, initiates a signaling cascade leading to platelet activation and aggregation. We have previously solved the structure of CLEC-2. The 2.4 A resolution crystal structure of rhodocytin presented here demonstrates that it is the first snake venom or other C-type lectin-like protein to assemble as a non-disulfide linked (alphabeta)(2) tetramer. Rhodocytin is highly adapted for interaction with CLEC-2 and displays a concave binding surface, which is highly complementary to the experimentally determined binding interface on CLEC-2. Using computational dynamic methods, surface electrostatic charge and hydrophobicity analyses, and protein-protein docking predictions, we propose that the (alphabeta)(2) rhodocytin tetramer induces clustering of CLEC-2 receptors on the platelet surface, which will trigger major signaling events resulting in platelet activation and aggregation.     

Rhodocetin, a novel platelet aggregation inhibitor from the venom of Calloselasma rhodostoma (Malayan pit viper): synergistic and noncovalent interaction between its subunits.

A novel platelet aggregation inhibitor, rhodocetin, was purified from the crude venom of Calloselasma rhodostoma. It inhibited collagen-induced platelet aggregation in a dose-dependent manner, with an IC50 of 41 nM. Rhodocetin has a heterodimeric structure with alpha and beta subunits, which could be separated on a nonreducing denaturing gel or reverse-phase HPLC column. Individually neither subunit inhibited platelet aggregation even at 2.0 microM concentration. Titration and reconstitution experiments showed that, when these subunits are mixed to give a 1:1 complex, most of its biological activity was recovered. The reconstituted complex inhibited platelet aggregation with an IC50 of 112 nM, about 3-fold less effective than the native molecule. Circular dichroism analysis revealed that the reconstituted complex had a spectrum similar to that of the native protein. By using surface plasmon resonance studies, we established that the stoichiometry of binding between the two subunits is 1:1 and the subunits interact with a Kd of 0.14 +/- 0.04 microM. The complete amino acid sequences of the alpha (15956.16 Da, 133 residues) and beta (15185.10 Da, 129 residues) subunits show a high degree of homology with each other (49%) and with the Ca2+-dependent lectin-related proteins (CLPs) (typically 29-48%) isolated from other snake venoms. Unlike the other members of the family in which the subunits are held together by an interchain disulfide bond, rhodocetin subunits are held together only through noncovalent interactions. The cysteinyl residues forming the intersubunit disulfide bridge in all other known CLPs are replaced by Ser-79 and Arg-75 in the alpha and beta subunits of rhodocetin, respectively. These studies support the noncovalent and synergistic interactions between the two subunits of rhodocetin. This is the first reported CLP dimer with such a novel heterodimeric structure.     

Involvement of tryptophan in sodium dodecyl sulfate-induced conformation of K99 pilin

K99 pili from bovine strain B44 ofEscherichia coli were incubated in a solution containing 1% sodium dodecyl sulfate (SDS), 4M urea, 1% 2-mercaptoethanol (2-ME), and 10% glycerol at room temperature. After 2 weeks, there was a partial conversion of the pilin from its apparent molecular weight of 17, 000 to 21, 000 due to change in conformation as studied by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After 10–12 weeks, this change in conformation was complete. The presence of 2-ME in the incubation mixture was essential for this change; this suggested the involvement of a disulfide bond. The role of the disulfide bond could also be shown by reduction and carboxamidation of the pilin. Other modifications such as performic acid oxidation, treatment with iodoacetic acid at acid pH, and 2-hydroxy-5-nitrobenzyl bromide showed that only performic acid oxidation and HNBB modification prevented change in conformation. From these data, it was concluded that the disulfide bond and tryptophan residues of the pilin are essential for the change in conformation in SDS.     

Characteristics of seminal plasma proteins and their correlation with canine semen analysis

The objectives were to separate canine seminal plasma proteins (with SDS-PAGE) and to determine the correlation between specific proteins and semen characteristics. Three ejaculates from 20 mixed-breed dogs, of unknown fertility, were collected by digital manipulation. Ejaculate volume and color, sperm motility, sperm vigor, percentage of morphologically normal spermatozoa, and membrane integrity (hypoosmotic swelling test and fluorescent staining) were assessed. For each dog, seminal plasma was pooled from all three ejaculates and proteins were separated with SDS-PAGE, using polyacrylamide concentrations of 13% and 22% in the separation gels. After staining, gel images were digitized to estimate molecular weights (MW) and integrated optical density (IOD) of each lane and of individual bands. Total seminal plasma protein concentration was 2.19+/-1.56 g/dL (mean+/-SD; range 1.12-5.19 g/dL). A total of 37 protein bands were identified (although no dog had all 37 bands). In the 13% gel, molecular weights ranged from 100.6 to 17.1 kDa, with four bands (49.7, 33.2, 26.4, and 19.5 kDa) present in samples from all dogs. In the 22% gel, molecular weights ranged from 15.6 to 3.6 kDa, with nine bands (15.6, 13.5, 12.7, 11.7, 10.5, 8.7, 7.8, 5.6, and 4.9 kDa) present in samples from all dogs. Combined for both gels, the majority of bands (85%) had molecular weights <17 kDa, with B20 (15.6 kDa) in high concentrations in samples from all dogs. There were positive correlations (P < or = 0.01) between two bands, B4 (67 kDa) and B5 (58.6 kDa), and sperm motility (r=0.66 and r=0.46), sperm vigor (r=0.56 and r=0.66), percentage of morphologically normal spermatozoa (r=0.55 and r=0.59), the hypoosmotic swelling test (r=0.76 and r=0.68), and fluorescent staining (r=0.56 and r=0.59), respectively. In conclusion, 37 proteins were identified in seminal plasma; two were significantly correlated with semen characteristics.     

Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node

A new C-type lectin-like gene encodes 293 amino acids and maps to chromosome 19p13.3 adjacent to the previously described C-type lectin genes, CD23, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and DC-SIGN-related protein (DC-SIGNR). The four genes form a tight cluster in an insert size of 105 kb and have analogous genomic structures. The new C-type lectin-like molecule, designated liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), is a type II integral membrane protein of approximately 40 kDa in size with a single C-type lectin-like domain at the COOH terminus, closest in homology to DC-SIGNR, DC-SIGN, and CD23. LSECtin mRNA was only expressed in liver and lymph node among 15 human tissues tested, intriguingly neither expressed on hematopoietic cell lines nor on monocyte-derived dendritic cells (DCs). Moreover, LSECtin is expressed predominantly by sinusoidal endothelial cells of human liver and lymph node and co-expressed with DC-SIGNR. LSECtin binds to mannose, GlcNAc, and fucose in a Ca(2+)-dependent manner but not to galactose. Our results indicate that LSECtin is a novel member of a family of proteins comprising CD23, DC-SIGN, and DC-SIGNR and might function in vivo as a lectin receptor.     

Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).     

Purpureotin: a novel di-dimeric C-type lectin-like protein from Trimeresurus purpureomaculatus venom is stabilized by noncovalent interactions

Purpureotin, a novel di-dimeric C-type lectin-like protein (CLP) from Trimeresurus purpureomaculatus, was purified and sequenced. While its native molecular mass was determined to be 63kDa, purpureotin showed a single band of 30kDa on nonreducing SDS-PAGE and two polypeptide chains (16.0 and 14.5kDa) under reducing condition. These results were subsequently confirmed by mass spectrometric analyses. Based on these results, we postulate that purpureotin is a dimer of the alpha,beta-heterodimer which is held together by noncovalent interactions. Molecular modeling studies indicate that a dimer of alpha,beta-heterodimers can be formed where the alpha chains are held together by electrostatic charges and beta chains via hydrophobic interactions. Functionally, purpureotin induced platelet aggregation without any cofactor in a dose-dependent manner. However, the platelet aggregation effect was blocked by echicetin. Therefore, purpureotin is assumed to be a GPIb-binding protein which binds to the same or a closely related GPIb site on platelets as echicetin.