New insights on disintegrin-receptor interactions: eristostatin and melanoma cells.

Viper venom disintegrins have been used frequently to study the cellular receptors which characterize various types of cells, including platelets, endothelial cells and cancer cells. While the majority of such analyses have pointed to involvement of integrin receptors alphavbeta3, alpha5beta1 or alphaIIbbeta3, this may not always be so. Eristostatin, from Eristocophis macmahoni, is a potent inhibitor of ADP-induced platelet aggregation as well as of human and murine melanoma metastases in mouse model systems. This disintegrin requires an RGDW motif, as well as an intact C-terminus, in order to interact with both platelets and four different types of melanoma cells. Eristostatin causes nonmetastatic SBc12 melanoma cells to show higher susceptibility to specific killing by NK-like TALL-104 cells. While it is known that eristostatin binds to alphaIIbbeta3 on platelets, the receptor with which eristostatin binds to the melanoma cells has not yet been identified.

EC3, a heterodimeric disintegrin from Echis carinatus, inhibits human and murine alpha4 integrin and attenuates lymphocyte infiltration of Langerhans islets in pancreas and salivary glands in nonobese diabetic mice.

The venom of Echis carinatus suchoreki contains a monomeric disintegrin echistatin (Mr 5,500 Da) that strongly inhibits alphaIIbbeta3, alphavbeta3, and alpha5beta1 integrins and a heterodimeric disintegrin called EC3 (M(r) 14,762 Da). At nanomolar concentration, EC3 inhibits adhesion of human cell lines expressing alpha4beta1 and alpha4beta7 to immobilized VCAM-1; it has a lower inhibitory effect on alpha5beta1-mediated cell adhesion. In this study, we demonstrated that EC3, in contrast to echistatin, inhibited binding of monoclonal anti-alpha4 and anti-alpha5 antibodies to cells expressing alpha4beta7. In a dose-dependent manner and to the same extent, EC3 inhibited adhesion of Jurkat cells and murine splenic lymphocytes to immobilized VCAM-1, whereas echistatin was not active. EC3 injected intraperitoneally into nonobese diabetic (NOD mice) suppressed development of insulitis and sialoadenitis, whereas echistatin had no significant effect. We propose that the effect of EC3 is mediated, at least, in part, by blocking alpha4beta1 and alpha4beta7 on murine lymphocytes.

Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins.

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.

The disulphide bond pattern of bitistatin, a disintegrin isolated from the venom of the viper Bitis arietans.

The disulphide bond pattern of the long disintegrin bitistatin (83 amino acids, 14 cysteines) was established using structural information gathered by amino acid analysis, N-terminal sequencing, and molecular mass determination of fragments isolated by reversed-phase HPLC after polypeptide degradation with trypsin and oxalic acid. A computer program was used to calculate all possible combinations of disulphide-bonded peptides matching the mass spectrometric data, and the output was filtered using compositional and sequence data. Disulphide bonds between cysteines 16-34, 18-29, 28-51, 42-48, 47-72, and 60-79 are conserved in medium-long disintegrins flavoridin and kistrin (70 amino acids, 12 cysteines), and the two cysteine residues at positions 5 and 24 found in bitistatin but not in other disintegrin molecules are disulphide-bridged. This linkage creates an extra, large loop, which, depending on whether the NMR structure of flavoridin or kistrin is used for modelling the structure of bitistatin, lies opposite or nearly parallel, respectively, to the biologically active RGD-containing loop.

Significance of RGD loop and C-terminal domain of echistatin for recognition of alphaIIb beta3 and alpha(v) beta3 integrins and expression of ligand-induced binding site.

Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to alpha(v) beta3 and alphaIIb beta3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with alphaIIb beta3 and alpha(v) beta3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to alphaIIb beta3 and decreased binding to alpha(v) beta3. This substitution impaired the ability of echistatin to induce LIBS in alpha(v) beta3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind alphaIIb beta3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with alphaIIb beta3 and alpha(v) beta3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized alpha(v) beta3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified alpha(v) beta3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of alphaIIb beta3 and alpha(v) beta3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.

Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of alpha IIb beta 3 and alpha v beta 3 integrins.

Echistatin and eristostatin are structurally homologous distintegrins which exhibit significant functional differences in interaction with various integrins. We hypothesized that this may reflect differences in the sequences of their RGD loops: 20CKRARGDDMDDYC32 AND 23CRVARGDWNDDYC35, respectively. Mapping of eristostatin peptides obtained by proteolytic digestion suggested that it has the same alignment of S-S bridges as echistatin. Synthetic echistatin D27W resembled eristostatin since it had increased platelet aggregation inhibitory activity, increased potency to block fibrinogen binding to alpha IIb beta 3, and decreased potency to block vitronectin binding to alpha v beta 3 as compared to wild-type echistatin. Since eristostatin and echistatin have a similar pattern of disulfide bridges, we constructed molecular models of eristostatin based on echistatin NMR coordinates. The RGD loops of eristostatin and echistatin D27W were wider than echistatin's due to the placement of tryptophan (rather than aspartic acid) immediately after the RGD sequence. We propose a hypothesis that the width and shape of the RGD loop are important ligand structural features that affect fitting of ligand to the binding pocket of alpha IIb beta 3 and alpha v beta 3.

Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation.

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year-old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction-independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.

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.

Effects of the disintegrin eristostatin on individual steps of hematogenous metastasis.

Adhesion molecules, including integrins, are important for interactions of cancer cells with vessel walls, a step leading to cancer metastasis. Disintegrins block the action of integrins by binding to them. We tested the hypothesis that the disintegrin eristostatin would block metastasis by interfering with cancer cell adhesion to vessel walls, thus preventing extravasation. Experimental metastasis assays, in which B16F1 melanoma cells (controls vs eristostatin-treated, 25 micrograms/ml) were injected via mesenteric veins of anesthetized C57BL/6 mice, showed that eristostatin reduced (P < 0.05) the mean number of liver metastases from 14.4 to 0.6 at 11 days postinjection (p.i.). We examined three different steps in metastasis at which eristostatin could have exerted its effect, namely, cell arrest, extravasation, and migration. Control and eristostatin-treated B16F1 cells were fluorescently labeled and examined by videomicroscopy in liver microcirculation in vivo at various times up to 14 days p.i. Measurements of vessel size in which cell arrest occurred and length/width ratio of arrested cells showed only small differences between control and eristostatin-treated cells. Eristostatin treatment did not prevent extravasation, and the timing and process of extravasation were similar for both treated and control cells; by 3-4 days p.i. more than 90% of the cells had extravasated or were in the process. Eristostatin also did not affect the ability of extravasated cells to migrate through the extracellular matrix to the subcapsular region where tumors later form. Therefore, we conclude that eristostatin exerted its primary effect by regulating the number of individual cancer cells that grow after extravasation.

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.

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.

Interaction of disintegrins with the alpha IIb beta 3 receptor on resting and activated human platelets.

Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3.

Characterization of the cross-linking site of disintegrins albolabrin, bitistatin, echistatin, and eristostatin on isolated human platelet integrin GPIIb/IIIa.

Disintegrins, a family of low molecular weight, RGD-containing peptides found in snake venoms prevent the binding of adhesive ligands to a number of integrin receptors. Albolabrin, bitistatin, echistatin, and eristostatin bind to the platelet fibrinogen receptor (GPIIb/IIIa) acting thus as potent inhibitors of platelet aggregation. Here, we have determined the cross-linking of these disintegrins on isolated GPIIb/IIIa. The cross-linking site of all of them was within GPIIIa 217-302, a domain that has been implicated in a number of receptor functions including heterodimer association, activation-dependent conformational changes, and fibrinogen binding.

Glycoprotein IIb peptide 656-667 mimics the fibrinogen gamma chain 402-411 binding site on platelet integrin GPIIb/IIIa.

The human integrin glycoprotein IIb/IIIa complex plays a central role in haemostasis as an inducible receptor for fibrinogen and other adhesive proteins at the platelet plasma membrane. Current evidence indicates that the ligand-binding domain of GPIIb/IIIa is discontinuous and placed at the subunit interface. Here we show that a synthetic peptide containing the polypeptide stretch GPIIb 656-667, which is hidden within the resting platelet GPIIb/IIIa heterodimer but becomes exposed following platelet activation with thrombin, binds to soluble fibrinogen (n = 2.3 +/- 1.3; Kd = 2 +/- 0.8 x 10(-5) M). This interaction is Ca(2+)-independent and can be partially inhibited with synthetic fibrinogen gamma-chain peptide 400-411 but not with GRGDS. In addition, peptide GPIIb 656-667 inhibits in a dose-dependent manner the aggregation of activated platelets (IC50 = 170 microM). Altogether, our results indicate that the GPIIb 656-667 region may form part of the inducible fibrinogen binding site and may not overlap with the integrin RGD-recognition domain.

Mouse antithrombotic assay. Inhibition of platelet thromboembolism by disintegrins.

The mouse antithrombotic assay represents a model of fatal pulmonary thromboembolism induced by intravenous injection of collagen and epinephrine. Mice were protected by low doses of two disintegrins, albolabrin (10 micrograms/mouse) and eristostatin (0.6 micrograms/mouse), whereas high doses of a thrombin inhibitor and an inhibitor of von Willebrand Factor binding to glycoprotein Ib were not effective. Injection of collagen and epinephrine resulted in the drop of platelet count and accumulation of platelet aggregates in the lung that appears to be the immediate cause of death. Albolabrin or eristostatin administration did not prevent the decrease of platelet count. Injection of albolabrin resulted in the formation of smaller and reversible platelet aggregates in the lungs and decreased accumulation of 51Cr-labeled platelets in the lung suggesting that this disintegrin decreases formation of platelet aggregates in vivo. We compared the effects of albolabrin and erisostatin on platelet aggregation, tail bleeding time, and survival of challenged animals. Eristostatin was about 5 times more potent in inhibiting platelet aggregation in vitro than albolabrin and 38 times more potent than albolabrin in protecting animals from sudden death. Both disintegrins, at the same doses (0.6-5 micrograms/mouse), caused similar dose-dependent prolongation of the bleeding time; however, only eristostatin exerted a protective effect. In conclusion, a) the mouse antithrombotic assay is a suitable model to screen and to evaluate the potency of platelet fibrinogen receptor antagonists in vivo; b) the results of the antithrombotic assay correlate better with the inhibition of platelet aggregation in vitro than with the prolongation of bleeding time.

Exposure of platelet fibrinogen receptors by zinc ions: role of protein kinase C.

Previous studies demonstrated that Zn2+ at a concentration of 50 microM increases the number of fibrinogen receptors exposed on ADP-stimulated platelets and that higher concentrations of Zn2+ induce platelet aggregation that appears to be mediated by receptors associated with the glycoprotein IIb/IIIa complex. The purpose of this study was to identify the mechanism by which Zn2+ modulates exposure of fibrinogen receptors on the surface of human washed platelets. We determined that Zn2+ (300-800 microM)-induced platelet aggregation that was not accompanied by the release of [14C]serotonin was not blocked by ADP scavenging enzymes and 5'-p-fluorosulfonylbenzoyl-adenosine, an affinity label for ADP binding sites, but it was inhibited by disintegrins, staurosporine, and EDTA. Zn2+ (50-200 microM) showed a synergistic effect on platelet aggregation and platelet release caused by ADP and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, a Zn2+ chelator, and inhibited ADP-induced platelet aggregation that was reversed by Zn2+ (50 microM). Zn2+ (200 microM) increased the number of fibrinogen binding sites and the affinity of albolabrin (a disintegrin isolated from Trimeresurus albolabris snake venom that has been shown to bind to the fibrinogen receptor) on ADP-activated platelets. On the other hand, Zn2+ (100-800 microM) did not increase fibrinogen binding to the purified receptor. Incubation of platelets with Zn2+ (200 microM) resulted in the phosphorylation of a 47-kDa protein that was blocked by staurosporine, an inhibitor of protein kinase C. In conclusion, Zn2+ ions activate protein kinase C and enhance fibrinogen receptor exposure on the surface of platelets stimulated by ADP.

Beta 3 integrin derived peptide 217-230 inhibits fibrinogen binding and platelet aggregation: significance of RGD sequences and fibrinogen A alpha-chain.

beta 3 Integrin derived peptides 217-230 (DAPEGGFDAIMQAT) and 217-231 (Y) (DAPEGGFDAIMQATVY) at 100 microM inhibited 125I-fibrinogen binding to ADP-stimulated platelets and platelet aggregation. Peptide 217-231 (Y) (100 microM) significantly inhibited the binding of 125I-albolabrin (a disintegrin with a single RGD sequence) to ADP- and thrombin-activated platelets while it had only a slight effect on albolabrin binding to resting platelets. The 125I-beta 3 217-231 (Y) cross-linked selectively to the fibrinogen A alpha-chain. The interaction of the RGD sequence in the A alpha-chain of fibrinogen with beta 3 217-231 sequence appears to play a significant role in the events leading to platelet aggregation.