echistatin and glycyl-arginyl-glycyl-aspartyl-serine

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.. 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.. 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.. 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.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Cattle; Cell Adhesion; Cells, Cultured; Crotalid Venoms; Dose-Response Relationship, Drug; Extracellular Matrix; Fluorescent Antibody Technique, Indirect; Integrins; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Oligopeptides; Peptides; Pigment Epithelium of Eye; Platelet Aggregation Inhibitors; Rabbits; Retinal Detachment; Viper Venoms; Vitreoretinopathy, Proliferative; Vitreous Body

In order to produce more potent and specific fibrinogen receptor (GpIIb/IIIa) antagonists, the Arg-Gly of a chemical series based upon Arg-Gly-Asp was replaced by alkyl chains of varying lengths. The most potent in this series, GR91669, inhibited aggregation of human gel-filtered platelets (GFP) in vitro induced by ADP or the thromboxane A2 mimetic, U46619, with IC50 values of 200nM and 500nM respectively and was selected for further studies. Its inhibitory effects on GFP were reversed by addition of excess fibrinogen. The compound also inhibited ADP- or U46619-induced platelet aggregation in human whole blood (IC50 values of 700nM in both cases). 125I-Fibrinogen binding to ADP-stimulated platelets was inhibited by GR91669 with an IC50 (65nM) similar to that against platelet aggregation. GR91669 (1mM) did not inhibit U46619-induced platelet shape change or 14C-5HT secretion from platelets stimulated by collagen, U46619 or thrombin. Therefore GR91669 inhibits aggregation but has no significant effect on stimulus-response events, a profile consistent with fibrinogen receptor blockade. In addition, GR91669 (1mM), unlike echistatin or Gly-Arg-Gly-Asp-Ser, did not disrupt vitronectin recptor-dependent attachment of cultured HUVECS in vitro and similarly did not inhibit Mac-1 dependent adhesion of human granulocytes. Thus, of the integrins tested, GR91669 appears to be specific for GpIIb/IIIa. Following intravenous administration to marmosets of 1 or 10 mg/kg GR91669, ADP (10 microM)-induced platelet aggregation ex vivo was abolished for 15 and 60 minutes respectively. Greater than 50% inhibition was maintained for 30 minutes and 2 hours respectively. GR91669, therefore appears to be a potent, specific fibrinogen receptor antagonist in vitro and which is also active in vivo.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Blood Platelets; Callithrix; Cell Adhesion; Dipeptides; Endothelium, Vascular; Granulocytes; Humans; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Oligopeptides; Peptides; Platelet Adhesiveness; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Serotonin; Structure-Activity Relationship; Thiourea; Viper Venoms

A range of cyclic RGD based peptides have been developed to mimic the conformation of RGD within fibrinogen. These peptides, as well as echistatin (IC50 = 0.05 microM) and GRGDS (IC50 = 25 microM) fully inhibited adenosine diphosphate (ADP) (10 microM)-induced platelet aggregation of human gel-filtered platelets (GFP). RGDF was the most potent linear peptide in inhibiting ADP-induced aggregation (IC50 = 8 microM) but cyclisation, using a 6,5 bicyclic coupling group to produce GR83895, led to an approximately 10-fold increase in potency (IC50 = 0.9 microM). In GFP, ADP-induced 125I-fibrinogen binding was inhibited (> 80%) by echistatin, GRGDS or GR83895 at concentrations (IC50 values 0.05 microM, 25 microM and 1.4 microM respectively) similar to those needed to inhibit aggregation. All three compounds also completely inhibited ADP- and U46619-induced aggregation in both platelet rich plasma (PRP) and whole blood. In contrast to platelet aggregation, U-46619-induced 14C-5HT secretion in PRP was not inhibited by GR83895 or echistatin, indicating that agonist-induced signal transduction is not affected by either agent, a profile consistent with that predicted for a specific fibrinogen receptor blocking drug. To test specificity of action, echistatin, GR83895 and GRGDS were also examined for their ability to detach cultured human umbilical vein endothelial cells attached to plastic through a vitronectin receptor dependent process. GR83895 only caused detachment at concentrations 100-fold greater than those required to inhibit platelet aggregation, in contrast to GRGDS and echistatin which caused cell detachment at concentrations similar to those inhibiting aggregation. In summary, cyclisation of RGD-containing peptides has led to both improved potency and specificity of action. Such specificity of action may prove to be an important consideration for the successful development of a fibrinogen receptor blocking drug as an anti-thrombotic drug.

Topics: Amino Acid Sequence; Humans; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Oligopeptides; Peptides; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Radioligand Assay; Viper Venoms