Echistatin, α1 isoform

Biochemical characterization of the binding of echistatin to integrin alphavbeta3 receptor.[Pubmed:9353406]

J Pharmacol Exp Ther. 1997 Nov;283(2):843-53.

Echistatin is a 49-amino-acid peptide belonging to the family of disintegrins that are derived from snake venoms and are potent inhibitors of platelet aggregation and cell adhesion. Integrin alphavbeta3 receptor plays a critical role in several physiological processes such as tumor-induced angiogenesis, tumor cell metastasis, osteoporosis and wound repair. In this study, we have characterized the binding of echistatin to purified integrin alphavbeta3 receptor and the form expressed on human embryonic kidney 293 cells. We show that both purified and membrane-bound integrin alphavbeta3 binds to echistatin with a high affinity, which can be competed efficiently by linear and cyclic peptides containing the RGD sequence. Previous studies have shown that alphavbeta3 binds to vitronectin in a nondissociable manner, whereas an RGD-containing peptide derived from vitronectin binds in a dissociable manner with a Kd of 9.4 x 10(-7) M. Our studies indicate that radiolabeled echistatin binds to alphavbeta3 in a nondissociable manner, similar to native echistatin. However, echistatin does not support the adhesion of 293 cells expressing alphavbeta3 receptor because of poor binding to plastic dishes and is a potent antagonist of the adhesion of these cells to vitronectin. These studies demonstrate that echistatin binding to alphavbeta3 is of high affinity and irreversible similar to vitronectin and provides an alternate ligand for high-throughput screening for alphavbeta3 antagonists.

Inhibition of platelet adhesion to surfaces of extracorporeal circuits by disintegrins. RGD-containing peptides from viper venoms.[Pubmed:2364514]

Circulation. 1990 Jul;82(1):261-73.

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.

Echistatin is a potent inhibitor of bone resorption in culture.[Pubmed:2211834]

J Cell Biol. 1990 Oct;111(4):1713-23.

The venom protein, s-echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-echistatin. Antibody localization of echistatin on these cells showed much greater echistatin binding to rat osteoclasts than to chicken osteoclasts. Laser scanning confocal microscopy after immunohistochemical staining showed that s-echistatin binds to osteoclasts, that s-echistatin receptors are most abundant at the osteoclast/glass interface, and that s-echistatin colocalizes with vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-echistatin, demonstrated colocalization of s-echistatin with the outer edges of clusters of grey contacts at the tips of some lamellipodia. Identification of the echistatin receptor as an integrin was confirmed by colocalization of echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]proline to chicken osteoclasts confirmed that the mechanism of action of echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in bone resorption, that this RGD-binding integrin is at adhesion structures, and that it colocalizes with vinculin and has an alpha-like subunit.