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PubMed1996

Inhibition of retinal pigment epithelial cell-induced tractional retinal detachment by disintegrins, a group of Arg-Gly-Asp-containing peptides from viper venom.

PURPOSE: Integrin-mediated extracellular matrix (ECM) attachment plays an important role in vitreous contraction of retinal pigment epithelial (RPE) cells. Disintegrins, a group of Arg-Gly-Asp (RGD)-containing peptides from viper venom, are potential anti-adhesion agents that interfere with integrin-ECM binding. This study was performed to determine whether disintegrins were effective in inhibiting RPE cell-induced matrix attachment in vitro and tractional retinal detachment in a rabbit model in vivo. METHODS: Two disintegrins, echistatin from viper Echis carinatus and flavoridin from Trimeresurus flavoviridis, were used. The expression of integrins on the surface of bovine and rabbit RPE cells was examined by indirect immunofluorescent stain with specific anti-integrin monoclonal antibodies. The inhibitory effect of disintegrins on RPE cell-mediated ECM attachment and vitreous contraction was evaluated with cell adhesion and vitreous contraction assays. In the in vivo model, rabbit eyes were injected intravitreously with either homologous rabbit RPE cells alone or together with disintegrins to induce tractional retinal detachment. The cytotoxic effect of disintegrins was examined with a cell proliferation assay using the alamar blue method. Retinal toxicity of disintegrins was evaluated with electroretinograms and histologic examination of the rabbit eyes. RESULTS: Bovine and rabbit RPE cells showed the positive staining for the integrins alpha 2 beta 1 and alpha 5 beta 1 on cell surface. Disintegrins, echistatin, and flavoridin inhibited RPE cell attachment to the ECM. The potency of disintegrins was 150 to 300 times higher than that of Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. The disintegrins also inhibited RPE cell-induced vitreous contraction in a dose-dependent manner, whereas the GRGDS peptide had no effect. In the in vivo experiment, echistatin (50 microgram/ml) or flavoridin (80 microgram/ml) significantly inhibited RPE cell-induced tractional retinal detachment compared with the control group at week 2 (P< 0.05) and week 4 (P< 0.01) after surgery. Disintegrins were nontoxic to RPE cells and rabbit retina as evaluated by cytotoxicity tests, electroretinograms, and histologic examinations. CONCLUSIONS: The disintegrins were effective in inhibiting RPE cell attachment to the ECM and vitreous contraction in vitro. They also were effective in suppressing RPE cell-induced tractional retinal detachment in the rabbit eyes. They were nontoxic. Disintegrins and their analogs might be potential anti-adhesion therapeutic agents in the treatment of proliferative vitreoretinopathy.

Amino Acid Sequence↗

PubMed1997

Function of disintegrin-like/cysteine-rich domains of atrolysin A. Inhibition of platelet aggregation by recombinant protein and peptide antagonists.

Snake venom hemorrhagic metalloproteinase toxins that have metalloproteinase, disintegrin-like and cysteine-rich domains are significantly more potent than toxins with only a metalloproteinase domain. The disintegrin-like domains of these toxins differ from the disintegrin peptides found in crotalid and viperid venoms by the nature of their different disulfide bond structure and, in lieu of the disintegrins' signature Arg-Gly-Asp (RGD) integrin binding sequence, there is an XXCD disulfide-bonded cysteinyl sequence in that region. Due to these apparent differences, the contribution to the overall function of the hemorrhagic metalloproteinases by the disintegrin-like domain has been unknown. In this investigation we have expressed in insect cells the disintegrin-like/cysteine-rich (DC) domains of the Crotalus atrox hemorrhagic metalloproteinase atrolysin A and demonstrated that the recombinant protein (A/DC) can inhibit collagen- and ADP-stimulated platelet aggregation. Using synthetic peptides, we have evidence that the region of the disintegrin-like domain that is positionally analogous to the RGD loop of the disintegrins is the site responsible for inhibition of platelet aggregation. For these synthetic peptides to have significant inhibitory activity, the -RSECD- cysteinyl residue must be constrained by participation in a disulfide bond with another cysteinyl residue. The two acidic amino acids adjacent to the middle cysteinyl residue in these peptides are also important for biological activity. These studies emphasize a functional role for the disintegrin-like domain in toxins and suggest structural possibilities for the design of antagonists of platelet aggregation.

Amino Acid Sequence↗

PubMed1994

Disintegrins and other naturally occurring antagonists of platelet fibrinogen receptors.

Disintegrins represent a family of low-molecular weight, cysteine-rich, RGD-containing peptides that inhibit fibrinogen binding to glycoprotein IIb/IIIa complex as well as binding of other ligands to RGD-dependent integrins on the surface of other cells. Disintegrins occur in the venom of various vipers. Disintegrin domains have been identified in viper venom hemorrhagins and in sperm proteins involved in the sperm-egg fusion. Amino acid sequences of 25 disintegrins, alignment of S-S bridges in four disintegrins, and stereo models of five disintegrins are presented. Primary structures of disintegrins differ significantly from other fibrinogen receptor antagonists occurring in the venoms of Elapidae, leeches, and ticks. Several aspects of structure-function relationship of disintegrins, their biological activities, and possible clinical applications are discussed.

Amino Acid Sequence↗

PubMed1996

Metargidin, a membrane-anchored metalloprotease-disintegrin protein with an RGD integrin binding sequence.

Cellular disintegrins are a family of membrane-anchored proteins with structural homology to snake venom metalloproteases and disintegrins. We report here the cDNA cloning and initial biochemical characterization of the first cellular disintegrin protein with an RGD sequence in its disintegrin domain, which we propose to name metargidin (for metalloprotease-RGD-disintegrin protein). The domain organization of metargidin is identical with that of previously reported members of the cellular disintegrin family, comprising (i) a pro- and a metalloprotease domain including a zinc-binding consensus motif, (ii) a disintegrin domain containing the RGD motif, (iii) a cysteine-rich domain, (iv) an epidermal growth factor-like domain, (v) a hydrophobic transmembrane domain, and (vi) a cytoplasmic tail with proline-rich sequences that could act as potential SH3 ligands. Antibodies raised against the cytoplasmic tail of metargidin recognize a glycoprotein of 110 kDa in MDA-MB-468 mammary epithelial carcinoma cells, which can be cell surface-biotinylated, indicating its localization in the plasma membrane. A second protein of 56 kDa co-immunoprecipitates with metargidin, suggesting that it is part of a protein complex. These features are consistent with a model in which metargidin is an integrin ligand which, as a transmembrane protein, might function in cell-cell adhesion and/or signaling.

ADAM Proteins↗

PubMed1995

[Disintegrins: potent inhibitors of platelet aggregation].

Disintegrins are a family of highly homologous polypeptides purified from snake venoms, which contain the arginine-glycine-aspartic acid (RGD) sequence. The RGD tripeptide acts as an integrin recognition sequence; it is also present on several proteins involved in cell adhesion such as fibrinogen, fibronectin, von Willebrand factor, and collagen. Disintegrins can therefore competitively inhibit integrin-ligand interactions: they block fibrinogen binding to its platelet receptor, alpha IIb beta 3: hence they are potent platelet aggregation inhibitors. Disintegrins are up to 2000 times more potent than short synthetic linear RGD-containing peptides in blocking fibrinogen-dependent platelet aggregation. Likely, the amino acids surrounding the RGD sequence and intrachain disulphide bridges force the RGD sequence in an appropriate conformation which accounts for the high, but variable, platelet inhibitory activity exhibited by disintegrin molecules. Disintegrins block the adhesive functions of the RGD-dependent integrins present on different cell types in different tissues: for this reason they are not alpha IIb beta 3-specific. A single disintegrin polypeptide, barbourin, was found to be a specific alpha IIb beta 3 antagonist: unlike all other disintegrins it contains the lysine-glycine-aspartic acid (KGD) sequence instead of the RGD one, which solely imparts alpha IIb beta 3 specificity to the molecule. This finding led to the synthesis of small, conformationally constrained KGD-containing peptides, which proven to be specific and potent inhibitors of alpha IIb beta 3 function; these compounds are presently undergoing evaluation in clinical trials as antithrombotic agents.

Cell Adhesion↗

PubMed1993

Characterization of the integrin specificities of disintegrins isolated from American pit viper venoms.

A new series of homologous disintegrins was isolated from the venoms of new world pit viper genus Bothrops, Crotalus, and Lachesis. The relative activities of each disintegrin in blocking adhesive protein binding activities of GPIIb-IIIa, alpha v beta 3, and alpha 5 beta 1 were determined and correlated with their primary amino acid sequences. Four disintegrins contained the RGDW sequence and were found to be approximately twice as effective in blocking the binding of fibrinogen to GPIIb-IIIa than inhibiting the binding of vitronectin to alpha v beta 3 in solid-phase ligand binding assays (IC50 = 7.3 and 17.2 nM, respectively). A second group of seven disintegrins contained the RGDNP sequence and were found to be more potent inhibitors of vitronectin binding to alpha v beta 3 than fibrinogen binding to GPIIb-IIIa (IC50 = 4.3 and 19 nM, respectively). The RGDNP containing disintegrins were also greater than 10-fold more potent than RGDW containing disintegrins in blocking the adhesion of cells mediated by alpha 5 beta 1. These data illustrate that amino acid sequences immediately adjacent to the RGD site of disintegrins can create an extended RGD locus which coupled with conformational display of the RGD sequence may be involved in determining integrin selectivity and affinity. This information has been used in separate studies to design conformationally constrained integrin antagonists with high affinity for platelet GPIIb-IIIa.

Amino Acid Sequence↗

PubMed1996

Comparison of iodine-123-disintegrins for imaging thrombi and emboli in a canine model.

UNLABELLED: Disintegrins are peptides found in viper venoms which bind to platelets through the glycoprotein IIb-IIIa receptor. The purpose of this work was to evaluate the ability of disintegrins to image thrombi and emboli in vivo. METHODS: Eight disintegrins (bitistatin, albolabrin, echistatin, eristostatin, kistrin, mambin, halysin and barbourin) were purified from snake venom. After radiolabeling with 123I, disintegrins were tested for their ability to image 24-hr-old experimental deep vein thrombi (DVT) and pulmonary emboli in a canine model. Labeled fibrinogen and platelets were used as controls. Gamma camera imaging was performed during the first 4 hr, after which tissue samples were collected for counting. RESULTS: Of the disintegrins tested, 123I-bitistatin had higher uptake in DVT (0.21 +/- .06% ID/g) than any other disintegrin (0.009-0.036%/g, p < 0.05). Bitistatin had higher DVT-to-blood ratios (9.8 +/- 2.5) than all other disintegrins, 125I-fibrinogen or 99mTc-HMPAO-platelets (p < 0.05). Images of DVT obtained with 123I-bitistatin were focally positive within 1 hr and improved by 4 hr. In pulmonary emboli, the absolute uptake of 123I-bitistatin (0.64 +/- 0.17% ID/g) was higher than all other compounds (p < 0.05), although barbourin had moderate uptake (0.23 +/- 0.11% ID/g) and may also be useful for imaging pulmonary embolism (PE). The uptake of bitistatin in PE was superior to both 125I-fibrinogen (0.18 +/- 0.02% ID/g) (p < 0.05) and 99mTc-HMPAO-platelets (0.14 +/- 0.02% ID/g, p < 0.05). Iodine-123-bitistatin had embolus-to-blood ratios averaging 27 +/- 7, which was higher than platelets, fibrinogen, echistatin, mambin or halysin (p < 0.05). Iodine-123-bitistatin background in lungs, liver and heart were low, which permitted visualization of all pulmonary emboli by 2-4 hr after injection. CONCLUSION: Labeled bitistatin should be investigated further as an agent which may permit rapid imaging of both thrombi and emboli.

Amino Acid Sequence↗

PubMed1992

Structural domains in venom proteins: evidence that metalloproteinases and nonenzymatic platelet aggregation inhibitors (disintegrins) from snake venoms are derived by proteolysis from a common precursor.

A comparison of the structures of a precursor of trigramin (a disintegrin), metalloproteinases, disintegrins and related proteins, suggests the existence of common precursors for metalloproteinases and disintegrins. The proposed common precursor and related proteins have four distinct domains (A-D). Domain B contains the metal binding site and the catalytic Glu residue, which comprise the active site of metalloproteinases. Domain C contains the Arg-Gly-Asp sequence and hence the ability to inhibit the activity of integrins. Domains A and D are unique and their biochemical or biological activity is unknown. The proposed precursor can be proteolytically cleaved at several interdomain sites, releasing the disintegrins and metalloproteinases. A survey of more than 100 venom metalloproteinases and disintegrins strongly supports the existence of precursor proteins and their structural domains. This is also upheld by the co-occurrence occurrence of metalloproteinases and disintegrins in the venoms of several genera of crotalid and viperid snakes. The likelihood of intradomain disulfide bridges, and accessibility of all interdomain cleavage sites also supports our contention. The susceptibility of the cleavage sites appears to be determined by nearby disulfide bridges and glycosylation. Recognition of the proposed structural domains of venom proteinases should help clarify the structure-function relationships of several related proteins, and influence the synthesis of recombinant disintegrins, metalloproteinases and related polypeptides.

Amino Acid Sequence↗

PubMed1995

A comparison of the effect of decorsin and two disintegrins, albolabrin and eristostatin, on platelet function.

Naturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP-induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the beta 3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 micrograms/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of MADM: a catalytically active mammalian disintegrin-metalloprotease expressed in various cell types.

A peptide sequence of a metalloprotease purified from bovine brain [Chantry, Gregson and Glynn (1989) J. Biol. Chem. 264, 21603-21607] was used to design an oligonucleotide probe for screening a bovine brain cDNA library. A contig of the two overlapping cDNA clones that were isolated encoded a 748-amino-acid polypeptide with similarity to the disintegrin-metalloprotease precursor proteins of haemorrhagic snake venom. The bovine protein has been named MADM, for mammalian disintegrin-metalloprotease. The predicted mature protein has 534 amino acids arrayed as extracellular metallo-protease and disintegrin (potential integrin-binding) domains, a transmembrane helix and a basic/proline-rich cytoplasmic C-terminus. Highly conserved homologues of bovine MADM were found in cDNA libraries of rat brain and a human U937 histiocytic lymphoma cell line. A wide variety of mammalian cell lines expressed low levels of MADM mRNA (4.5 and 3.2 kb transcripts) and mature polypeptide (M(r) 62000), as assessed by Northern analysis and Western blotting with an antiserum raised to a peptide within the disintegrin domain. MADM appears to be a rather distantly related member of the reprolysin protein family, which includes both the snake venom disintegrin-metalloproteases and a number of predicted cell-surface disintegrin-containing mammalian proteins.

Amino Acid Sequence↗

PubMed1994

Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition.

The inhibitory capacities of six different disintegrins and one related neurotoxin analogue for the binding of RGD-dependent integrins to either fibrinogen, vitronectin or fibronectin were compared in solid phase assays. Echistatin and flavoridin were the most active inhibitors for alpha V beta 3 and alpha 5 beta 1 integrins and moderately exceeded the activity of the natural protein ligands. The same disintegrins together with eristostatin, bitistatin and barbourin were also very potent inhibitors of fibrinogen binding to alpha IIb beta 3 integrin. For all three integrins, albolabrin showed the lowest affinity, but it still clearly exceeded that of synthetic GRGDS. However, assay conditions may determine these relative affinities, as shown for the alpha IIb beta 3 and alpha V beta 3 integrins when used either in immobilized or soluble form. For alpha IIb beta 3, however, a close correlation was found between KD values determined in platelet binding assays and the concentrations required for half maximal inhibition of three disintegrins. The inhibiting capacity of disintegrins in assays with purified integrins also correlated reasonably well with their inhibition of cell attachment to RGD-dependent protein substrates. However, sequence differences in the RGD loops of the various disintegrins may not fully account for the 20-100-fold difference in their binding capacities. This was particularly evident for echistatin and albolabrin, which differ in this region only by two conservative substitutions but have considerably different inhibitory activities. More remote regions of the disintegrins and alignment of disulfide bridges are therefore likely to contribute to their affinity and selectivity.

Amino Acid Sequence↗

PubMed1992

Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin gene family.

A large hemorrhagin, jararhagin, has been cloned from a Bothrops jararaca venom gland cDNA expression library. The cDNA sequence predicts a 421-amino acid residue molecule with strong amino acid sequence homology and similar domain structure to HR1B, a high molecular weight hemorrhagic metalloprotease isolated from Trimeresurus flavoviridis (Habu) venom. Like HR1B, jararhagin contains enzyme, disintegrin, and cysteine-rich carboxyl-terminal regions. In the disintegrin region, the Arg-Gly-Asp sequence is replaced by Glu-Cys-Asp, as found in non-Arg-Gly-Asp disintegrin regions of HR1B and a guinea pig sperm fusion protein PH-30 beta. The cDNA sequence of jararhagin predicts a precursor protein (proprotein) with striking similarity to cryptic regions in precursors of the disintegrin peptides trigramin and rhodostomin. Comparison of jararhagin with disintegrin precursors highlights the modular arrangement of proprotein, metalloprotease, and disintegrin domains in the metalloprotease/disintegrin family and provides an insight into their biosynthesis and evolution.

Amino Acid Sequence↗

PubMed1992

Structure, function and evolutionary relationship of proteins containing a disintegrin domain.

Disintegrins are soluble integrin ligands from snake venoms that disrupt cell-matrix interactions. Recently, the nuclear magnetic resonance structures of two disintegrins were determined, providing provocative molecular insight into how a disintegrin may engage an integrin. In addition, it has recently been realized that disintegrins are derived from larger multifunctional proteins, and that there is a family of membrane-anchored, disintegrin domain-containing proteins that may promote important cell-cell interactions.

Amino Acid Sequence↗

PubMed1993

Primary structures of platelet aggregation inhibitors (disintegrins) autoproteolytically released from snake venom hemorrhagic metalloproteinases and new fluorogenic peptide substrates for these enzymes.

A hemorrhagic protein (60 kDa), HR1B, present in the venom of Trimeresurus flavoviridis is a mosaic protein consisting of an NH2-terminal metalloproteinase-domain, a disintegrin (platelet aggregation inhibitor)-like domain, and a unique COOH-terminal Cys-rich domain. Since the gross structures of HR1B and protein precursors of disintegrins, trigramin, and rhodostomin, all of which contain the metalloproteinase domain, are similar, many disintegrins so far detected in snake venoms are assumed to be autoproteolytic fragments released from precursors. In ongoing related experiments, the newly purified hemorrhagic metalloproteinases, HR1A from T. flavoviridis venom and HT-1 from Crotalus ruber ruber venom, in addition to HR1B, were autoproteolyzed, in the absence of Ca2+, at 37 degrees C for 3-12 h. Under these conditions, HR1A, HR1B, and HT-1 each released a single major fragment of 32, 34, and 31 kDa, respectively. The entire amino acid sequences of the isolated fragments indicated the presence of disintegrin-like and Cys-rich domains in the COOH-terminal regions of HR1A, HR1B, and HT-1, respectively. It seems likely that so-called disintegrins probably originate from various metalloproteinases present in venom. On the bases of peptide sequences close to the autoproteolytic cleavage sites of these metalloproteinases and the sites of fibrinogen cleaved by these enzymes, we synthesized new intramolecularly quenched fluorogenic peptide substrates. Among the 10 peptides tested, 2-aminobenzoyl (Abz)-Ser-Pro-Met-Leu-2,4-dinitroanilinoethylamide (Dna) proved to be the best substrate for venom metalloproteinase, as deduced from kinetic analyses.

Amino Acid Sequence↗

PubMed1994

Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry.

Disintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein IIb-IIIa (GPIIb-IIIa) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-IIIa complex on platelet membrane with a very high affinity (Kd, 10(-7)-10(-8) M). In this study, we analyzed GPIIb-IIIa complex on platelet membrane by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were. The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy) and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-IIIa. ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used, whereas it had little effect on that of FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case of FITC-Rn as the probe. The platelets from three patients with Glanzmann's thrombasthenia were probed with FITC-disintegrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1997

A role for the disintegrin domain of cyritestin, a sperm surface protein belonging to the ADAM family, in mouse sperm-egg plasma membrane adhesion and fusion.

Sperm-egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell-cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm-egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (alpha and beta subunits). Fertilin alpha and beta are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin beta functions in sperm-egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin beta, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm-egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80-90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin beta active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin beta also strongly inhibited (80-90%) both sperm-egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin beta showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin beta in sperm-egg plasma membrane adhesion leading to fusion.

ADAM Proteins↗

PubMed1990

Inhibition of platelet adhesion to surfaces of extracorporeal circuits by disintegrins. RGD-containing peptides from viper venoms.

Previous studies indicate that exposure of fibrinogen receptors associated with glycoprotein IIb/IIIa complex contributes to platelet loss during cardiopulmonary bypass. Recently, we isolated a number of RGD (Arg-Gly-Asp)-containing, low molecular weight, cysteine-rich peptides from viper venoms. These peptides, which we propose to call "disintegrins," block platelet-fibrinogen interaction and platelet aggregation. We compared the effect of RGDS (Arg-Gly-Asp-Ser) and four disintegrins (echistatin, flavoridin, albolabrin, and bitistatin) on platelet behavior in a membrane oxygenator. During simulated extracorporeal circulation for 2 hours, platelet count decreased to about 30% of initial values. Addition of echistatin (60-200 nM), albolabrin (60-200 nM), bitistatin (60 nM), and flavoridin (45 nM) significantly inhibited platelet loss in the circuit. RGDS (33 microM) did not show any significant inhibitory effect. ADP-induced platelet aggregation was inhibited in samples of platelet-rich plasma taken from the circuits containing disintegrins. However, echistatin appeared to be a more potent inhibitor of platelet aggregation, whereas albolabrin and flavoridin interfered more selectively with platelet loss from the circuit. Echistatin prevented the accumulation of glycoprotein IIIa on the surface of the circuit. Echistatin (60-200 nM), flavoridin (45 nM), bitistatin (60 nM), and albolabrin (200 nM) significantly inhibited the loss of beta-thromboglobulin from platelets into circulating plasma. Electron microscopy studies demonstrated shape change but not degranulation in platelets circulating in the presence of 200 nM echistatin. On the other hand, this peptide (up to 1,000 nM) did not prevent loss of alpha granules and beta-thromboglobulin from thrombin-stimulated platelets, although it prevented their aggregation. In conclusion, disintegrins protect platelets in the circuit by preventing their adhesion to surfaces and, therefore, preventing fragmentation of adhered platelets under the shear stress of flowing blood. This study indicates that disintegrins may be potential candidates for platelet protection during cardiopulmonary bypass.

Amino Acid Sequence↗

PubMed1995

ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain.

Fertilin alpha and beta, previously known as PH-30 alpha and beta, are two subunits of a guinea pig sperm integral membrane protein implicated in sperm-egg binding and fusion. They are derived from sequence-similar precursors which contain a metalloprotease-like and a disintegrin-like domain and which are related to a family of metalloprotease and disintegrin domain-containing snake venom proteins. We report here the cloning, sequencing, and characterization of mouse fertilin alpha and beta as well as five additional sequence-similar cDNAs from guinea pig and mouse testis. We name this gene family ADAM, for proteins containing A Disintegrin And Metalloprotease domain, and in honor of its dual origins in the fields of snakes and fertility. In situ hybridization demonstrated that, in testis, RNA encoding these ADAMs is expressed only in spermatogenic cells and that this expression is developmentally regulated. PCR analysis of mouse tissue cDNA showed that these ADAMs display different patterns of tissue distribution. Some ADAMs (e.g., fertilin alpha) have the consensus active-site sequence for a zinc-dependent metalloprotease in their metalloprotease-like domain. All have a disintegrin-like domain, which could bind integrins or other receptors. Some have sequences which may be active in membrane fusion. All encode potential membrane-spanning domains. Searches of sequence databases revealed that additional mammalian members of the ADAM gene family have been cloned from a variety of tissues. Thus, the ADAMs are a large, widely expressed, and developmentally regulated family of proteins with multiple potential functions in cell-cell and cell-matrix interactions.

ADAM Proteins↗