Expression Of Ligand-induced Binding Sites Research Articles

Arg-Gly-Asp–Dependent Occupancy of GPIIb/IIIa by Applaggin: Evidence for Internalization and Cycling of a Platelet Integrin

Using indirect immunofluorescence microscopy we examined the distribution and cycling of GPIIb/IIIa after binding to applaggin, a high-affinity Arg-Gly-Asp (RGD)--containing ligand. Resting, unfixed platelets were incubated with applaggin for 30 minutes at 37 degrees C, and bound applaggin was detected by an affinity-purified rabbit anti-applaggin antibody. Examination of intact cells showed a rim pattern for applaggin, consistent with its binding to the platelet surface. Staining of Triton X-100--permeabilized cells showed an intracellular pool of applaggin. Competition of applaggin binding by either AP-2, an anti-GPIIb/IIIa monoclonal antibody (MoAb) that blocks fibrinogen binding, or the synthetic peptide RGDW eliminated both surface and intracellular staining, indicating that applaggin is binding to GPIIb/IIIa in an RGD-dependent manner. Inhibition of platelet activation by PGE1 and theophylline had no effect on the observed staining patterns, indicating that cellular activation is not required for surface binding and subsequent internalization. To evaluate whether occupancy of functional binding sites on GPIIb/IIIa is required for internalization, we used mAb15, an anti-GPIIIa antibody that neither blocks fibrinogen binding nor induces the expression of ligand-induced binding sites on GPIIb/IIIa. In these studies mAb15 was internalized in a manner analogous to both AP-2 and applaggin, showing that occupancy of the RGD binding site is not required to initiate receptor internalization. To estimate the size of the newly internalized pool of applaggin, 125I-applaggin--binding studies were performed. Displacement of bound 125I-applaggin by excess unlabeled applaggin or EDTA showed that at least 17% of bound applaggin was nondisplaceable when binding was performed under conditions permitting membrane flow and internalization. These data indicate that GPIIb/IIIa is internalized in unstimulated platelets independent of cellular activation or occupancy of the functional binding site(s) of GPIIb/IIIa by RGD-containing ligands. Thus, internalization of GPIIb/IIIa may represent a mechanism by which the surface expression of this adhesion receptor is regulated.

Loxosceles gaucho spider venom and its sphingomyelinase fraction trigger the main functions of human and rabbit platelets

Loxosceles venoms can promote severe local and systemic damages. We have previously reported that Loxosceles gaucho spider venom causes a severe early thrombocytopenia in rabbits. Herein, we investigated the in vitro effects of this venom and its sphingomyelinase fraction on the main functions of platelets. Whole venom and its fraction induced aggregation of both human and rabbit platelets. Aggregation was dependent of plasma component(s) but independent of venom-induced lysophosphatidic acid generation. There was no increase in the levels of lactate dehydrogenase during platelet aggregation, ruling out the possibility of platelet lysis. The increased expression of ligand-induced binding site 1 (LIBS1) induced by L. gaucho venom and its sphingomyelinase fraction, as well as of P-selectin by the whole venom, evidenced the activation state of both human and rabbit platelets. Adhesion assays showed an irregular response when platelets were exposed to the whole venom, whereas the sphingomyelinase fraction induced a dose-dependent increase in the platelet adhesion to collagen. These findings evidence that L. gaucho venom and its sphingomyelinase fraction trigger adhesion, activation, and aggregation of both human and rabbit platelets. Thus, this work justifies the use of rabbits to investigate Loxosceles venom-induced platelet disturbances, and it also supports research on the role of platelets in the pathogenesis of loxoscelism.

Combined defect in membrane expression and activation of platelet GPIIb–IIIa complex without primary sequence abnormalities in myeloproliferative disease

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.

Association Between Ligand-Induced Conformational Changes of Integrin αIIbβ3 and αIIbβ3-Mediated Intracellular Ca2+ Signaling

AbstractPlatelet αIIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of αIIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and αIIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various αIIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as αIIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both αIIb and β3 subunits. In group II, antagonists can induce LIBS on the αIIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on αIIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to αIIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable αIIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate αIIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and αIIbβ3-mediated intracellular Ca2+ signaling in platelets.

Association Between Ligand-Induced Conformational Changes of Integrin IIbβ3 and IIbβ3-Mediated Intracellular Ca2+ Signaling

Platelet IIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and β3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and IIbβ3-mediated intracellular Ca2+ signaling in platelets.

Expression of ligand-induced binding sites on glycoprotein IIb/IIIa complexes and the effect of various inhibitors

Glycoprotein (GP) IIb/IIIa is a therapeutic target for the blockade of platelet aggregation during acute coronary syndromes. Peptides, peptidomimetics, and antibodies are generated that competitively block the binding of fibrinogen or von Willebrand factor to the activated GPIIb/IIIa complex. Binding of these receptor blockades to GPIIb/IIIa effectively inhibits the formation of the platelet aggregate because ligand binding to the activated GPIIb/IIIa is the final common pathway to thrombus formation. In addition, bound antagonists induce a conformational change in the receptor. This conformational change, also called a ligand-induced binding site, can be used as a marker for receptor occupancy by GPIIb/IIIa receptor blockades. Using the binding properties of the D3 monoclonal antibody that binds with high affinity to the antagonist bound GPIIb/IIIa, we have developed a new method for monitoring the extent of receptor blockade by GPIIb/IIIa antagonists. This method has specific advantages over the interpatient variability of the aggregation assay and provides a method for the evaluation of appropriate target levels of GPIIb/IIIa blockade. (Am Heart J 1998;135:S179-S183.)

Effect of Glycoprotein IIb-IIIa Receptor Antagonism on Platelet Membrane Glycoproteins after Coronary Stent Placement

Platelet membrane glycoproteins play a crucial role in ischemic complications after coronary stenting. Glycoprotein IIb-IIIa blockade reduces adverse clinical events after angioplasty but is associated with rare but profound thrombocytopenia that might increase hemorrhagic complications. Changes in platelet membrane glycoproteins of patients with angina who underwent coronary stenting and were treated with the GPIIb-IIIa antagonist abciximab (n=20) or with heparin (n=23) were studied. GPIb-IIIa receptor blockade and membrane glycoproteins were evaluated with immunological markers in venous blood samples taken before. 10, 24, 48, 72, and 96 h after initial treatment with either abciximab or heparin. Patients receiving abciximab therapy showed a rapid inhibition of binding of fluorochrome-conjugated mAb CD41 and c7E3 concomitant with a reduction in platelet aggregation which was restored in part in the days after termination of abciximab infusion. Induction of ligand-induced binding sites on GPIIb-IIIa was increased in patients receiving abciximab. The expression of ligand-induced binding sites correlated inversely with platelet count. No significant change in platelet membrane markers were found in the heparin group. In vitro studies showed that abciximab induces ligand-induced binding sites on isolated platelets and on nuclear cells bearing recombinant GPIIb-IIIa. Abciximab rapidly achieves GPIIb-IIIa receptor blockade after coronary stent placement that might be beneficial in high-risk settings to bridge the delayed action of ticlopidine. Significant alterations of platelet membrane glycoproteins during GPIIb-IIIa antagonism might contribute to development of acute profound thrombocytopenia.

Significance of RGD Loop and C-Terminal Domain of Echistatin for Recognition of αIIbβ3 and αvβ3 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 αvβ3 and αIIbβ3 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 αIIbβ3 and αvβ3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to αIIbβ3 and decreased binding to αvβ3. This substitution impaired the ability of echistatin to induce LIBS in αvβ3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind αIIbβ3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with αIIbβ3 and αvβ3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized αvβ3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified αvβ3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of αIIbβ3 and αvβ3 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.

Significance of RGD Loop and C-Terminal Domain of Echistatin for Recognition of αIIbβ3 and αvβ3 Integrins and Expression of Ligand-Induced Binding Site

AbstractEchistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to αvβ3 and αIIbβ3 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 αIIbβ3 and αvβ3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to αIIbβ3 and decreased binding to αvβ3. This substitution impaired the ability of echistatin to induce LIBS in αvβ3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind αIIbβ3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with αIIbβ3 and αvβ3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized αvβ3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified αvβ3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of αIIbβ3 and αvβ3 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.

Stimulation of platelet glycoprotein IIb-IIIa (α IIbβ 3-integrin) functional activity by a monoclonal antibody to the N-terminal region of glycoprotein IIIa

Platelet glycoprotein (GP) IIb-IIIa complex (α IIbβ 3-integrin) changes its conformation upon platelet activation that results in binding of RGD-containing ligands and expression of ligand-induced binding site (LIBS) neoepitopes. Anti-GIIb-IIIa monoclonal antibody (monAB) CRC54 bound to ≤10% of GPIIb-IIIa on resting platelets but binding was enhanced by the occupation of GPIIb-IIIa with RGDS peptide and by platelet activation indicating that CRC54 is directed against LIBS epitope. The epitope was located within the first 100 N-terminal residues of GPIIIa and differed from other LIBS epitopes. CRC54 as well as its Fab fragments were able to induce platelet aggregation. CRC54 also stimulated interaction of GPIIb-IIIa with its ligands (fibrinogen and fibronectin) and conformation-dependent antibodies. The results indicated that changes of GPIIb-IIIa conformation, binding of ligands and platelet aggregation could be stimulated via interaction of anti-LIBS antibody with the N-terminal part of GPIIIa.

Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3.

Disintegrin Interaction with αvβ3Integrin on Human Umbilical Vein Endothelial Cells: Expression of Ligand-Induced Binding Site on β3Subunit

The effect of seven disintegrins (albolabrin, barbourin, bitistatin, echistatin, eristostatin, flavoridin, and kistrin) and the neurotoxin analogue, mambin, on the adhesion of human umbilical vein endothelial cells (HUVEC) to immobilized vitronectin and fibronectin has been studied. Adhesion to vitronectin was significantly inhibited by echistatin, kistrin, flavoridin, and mambin. Echistatin, flavoridin, and kistrin bound with high affinity to immobilized αvβ3in solid phase assay; other disintegrins bound at a much lower level. Echistatin and flavoridin had a modest inhibitory effect on HUVEC adhesion to fibronectin. HUVEC adhered to disintegrins with a high selectivity toward bitistatin, echistatin, flavoridin, kistrin, and mambin. Adhesion of HUVEC to fibronectin and vitronectin resulted in cell spreading, whereas cells adhering to immobilized echistatin remained globular and cells adhering to kistrin showed abnormal morphology. Echistatin and kistrin potently inhibited the binding of monoclonal antibody (Mab) 7E3, which recognizes the αvβ3complex, to HUVEC. Echistatin and kistrin also induced the binding to HUVEC of Mab 62, which recognizes the ligand-induced binding site (LIBS) epitope on the β3subunit, enhancing HUVEC binding to immobilized Mab 62. Similar results with both antibodies were obtained in Chinese hamster ovary cells transfected with αvβ3genes. In conclusion, disintegrin interaction with HUVEC appears to be selectively mediated by αvβ3receptors, and it results in an expression of LIBS epitope that may play a role in the regulation of ligand-binding affinity and intracellular signaling.