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PubMed1996

Interferon therapy, but not busulfan restores normal-sized megakaryopoiesis in CML--a comparative histo- and immunomorphometric study.

To assess possible alterations of megakaryocytes associated with interferon (IFN) and busulfan (BU) therapy of Ph(1+)-CML, an immunohistochemical and morphometric study was performed on trephine biopsies of the bone marrow taken before and at varying intervals during treatment. For the identification of megakaryopoiesis and its endoreduplicative activity the monoclonal antibodies CD61 (anti-platelet glycoprotein IIIa) and PC10 raised against proliferating cell nuclear antigen (PCNA) were used. We compared 60 specimens from 20 patients following IFN alpha-2b administration (in combination with IFN gamma in seven patients) with 57 specimens from 22 patients after monotherapy with BU. A close correlation with clinical follow-up studies revealed that in the IFN-treated group the prevalence of atypical micro-megakaryocytes, usually characterizing CML, was conspicuously reduced in repeatedly taken bone marrow samples. Initially, even an increase in size which was levelled to normal values during maintenance therapy was observed. These features were most prominently expressed in the 13 patients with a complete hematologic and/or partial cytogenetic response. Associated with this phenomenon was a significant enhancement of the PCNA-labelling index which indicated a stimulation of endoreduplicative (endomitotic) activity necessary for achieving normal size and ploidy. In the second group of patients treated by BU these changes were absent. For this reason, our findings are in keeping with the assumption that during IFN treatment, there is at least partial recovery and expansion of a putative normal (Ph1-) megakaryopoiesis. In conclusion, megakaryocyte morphology, i.e. normalization in size, is thought to be a useful indicator to evaluate the response to IFN in CML patients.

Adult↗
PubMed1997

Relationship between the cellular distribution of the alpha(v)beta3/5 integrins and adenoviral infection in salivary glands.

This study compared the localization of the alpha(v)beta3/5 integrins in the different cell types of the rat submandibular gland with the susceptibility of these cells to infection by an intraductally administered replication-deficient recombinant type 5 adenovirus coding for rat aquaporin 5 (termed AdrAQP5). We used a panel of antibodies and immunohistochemical procedures to determine the luminal membrane distribution of the integrins. The alpha(v)beta3/5 integrin subunits were present in luminal membranes of all ductal cell types and acini; however, the alpha(v) subunit was found to a lesser extent in the acini. After AdrAQP5 infection, the expression of AQP5 exhibited a similar, though not identical, cellular localization to that seen for alpha(v)beta3/5 integrins. AdrAQP5 infected all cell types in these glands, except excretory ducts, after intraductal administration, directing the transient expression of AQP5. In addition, the localization of alpha(v)beta3/5 integrins was examined in rabbit, monkey, and human submandibular and parotid glands. Although there were some interspecies differences, glands generally displayed the presence of these integrin subunits on luminal membranes of ductal cells and most acinar cells. These findings show that the presence of alpha(v)beta3/5 integrins on the luminal membranes of rat salivary epithelial cells is associated with the susceptibility of these cells to act as targets for adenoviral-mediated gene transfer. However, the abundance of alpha(v)beta3/5 integrin expression was not necessarily predictive of the extent of transgene expression in a particular cell type. Furthermore, because the salivary glands of rabbits, monkeys, and humans show a similar luminal membrane distribution of these integrins, it is likely that recombinant type 5 adenoviruses may be able to mediate in vivo gene transfer to salivary glands in these species.

Adenoviridae↗
PubMed1997

Integrin expression on normal and neoplastic human breast epithelium.

Integrin adhesion receptor expression of different benign and malignant breast tumours was examined by means of immunohistochemical techniques. A panel of seven different anti-alpha and two different anti-beta subunit antibodies was used. Normal breast epithelium displayed a well characterized and constant pattern of integrin expression consisting of strong alpha 1,2,3,6 and alpha v, and a relatively weaker beta 1 and beta 3 staining. No staining for alpha 4 or alpha 5 could be detected on the epithelial cells. Benign fibroadenomas did not show changes in their receptor expression compared to normal tissues. In the cases of different types of breast cancer, there was a significant downregulation of all subunits. The staining pattern was distinct if there could a basement membrane like structure be detected around the invading tumour nodules. When laminin and collagen type IV surrounded the tumour cells, those cells in contact with the extracellular matrix components still displayed strong positivity for the integrin subunits. Other cells inside the tumour cell nests or not surrounded by basement membrane did not express integrins. The positively staining cells might be more differentiated owing to the effect the basement membrane. Myoepithelial labeling of the integrin expressing cells gave negative results. The observed integrin expression heterogeneity renders the histologic picture difficult to interpret with regard to clinical behavior of the tumour.

Antibodies↗
PubMed1998

In vitro platelet binding compared with in vivo thrombus imaging using alpha(IIb)beta3-targeted radioligands.

Radioligands for the alpha(IIb)beta3 integrin on platelets are being studied for their ability to image venous thrombi and pulmonary emboli. One such radioligand, 123I-bitistatin, was previously shown to have higher thrombotic uptake in an animal model than other disintegrins, but the reason for this difference was not clear. The purpose of this study was to evaluate three labeled disintegrins, bitistatin, kistrin and barbourin, to look for in vitro differences in platelet binding which could explain the in vivo behavior. Disintegrins labeled with 121I were compared in vitro for extent of binding to platelets and rates of binding and dissociation. These findings were related to organ distribution and image quality for imaging thrombotic lesions, following administration of 123I-disintegrins in an animal model. Fibrinogen at 8.8 micromol/l was able to displace 125I-barbourin and 125I-kistrin more rapidly from ADP-stimulated platelets, with half-times of 3.5 and 10.7 min, compared with 125I-bitistatin (31.6 min). At equivalent concentrations in whole blood, a higher percentage of bitistatin bound to platelets compared with the other two. In vivo, kistrin and barbourin had significantly lower thrombus:muscle and pulmonary embolus:lung ratios in images compared with bitistatin. There was evidence of more metabolic deiodination of labeled kistrin and barbourin in vivo compared with bitistatin. A surprising finding was that conventional in vitro platelet binding studies did not predict the relative in vivo behavior of labeled disintegrins. The results suggest that labeled bitistatin has improved targeting of thrombi because it is less easily displaced from stimulated platelets, permitting longer lesion retention. It also appears to have a greater association with resting platelets in the blood, which may increase bioavailability and delay metabolic breakdown.

Animal Testing Alternatives↗
PubMed2005

Tricyclic pharmacophore-based molecules as novel integrin alpha(v)beta3 antagonists. Part III: synthesis of potent antagonists with alpha(v)beta3/alpha(IIb)beta3 dual activity and improved water solubility.

In order to optimize our novel integrin alpha(v)beta3/alpha(IIb)beta3 dual antagonists, spatial screening at the N-terminus was performed. The alpha(v)beta3 antagonistic activity varied depending on the space that was occupied by the N-terminus, but high potency against alpha(IIb)beta3 was well maintained. The (3S)-aminopiperidine analogue had the strongest activity against alpha(v)beta3, and the S isomer at piperidine was more potent than the R isomer. Compounds selected on the basis of SAR analysis of a novel lead compound showed acceptable early absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles and sufficient water solubility for use as infusion drugs. Docking studies with the alpha(v)beta3 receptor were performed to confirm the SAR findings.

Animals↗
PubMed1999

Clustering of integrin alphaIIb-beta3 differently regulates tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK in concanavalin A-stimulated platelets.

Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72syk and pp125FAK and of the gamma2 isoform of phospholipase C (PLCgamma2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72syk and PLCgamma2 with a similar kinetics, while tyrosine phosphorylation of pp125FAK occurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCgamma2 and pp125FAK, while tyrosine phosphorylation of pp72syk induced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK was not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin alphaIIb-beta3 was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK required the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAK and pp72syk induced by Concanavalin A required the expression of integrin alphaIIb-beta3 on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCalpha2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin alphaIIb-beta3 induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72syk and pp125FAK and provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.

Blood Platelets↗
PubMed1998

Structural analysis of the sixth immunoglobulin-like domain of mouse neural cell adhesion molecule L1 and its interactions with alpha(v)beta3, alpha(IIb)beta3, and alpha5beta1 integrins.

Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to alpha(v)beta3, alpha(IIb)beta3, and alpha5beta1 integrins. The domain was found to have the expected beta-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to alpha(v)beta3, alpha(IIb)beta3, and alpha5beta1 integrins, suggesting specific interactions with these ligands. However, only the interaction to alpha(v)beta3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC50 was determined to be approximately 20 nM. Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562-564 is involved in the interaction to alpha(v)beta3. Our findings indicate an RGD-independent mechanism for the interactions to alpha(IIb)beta3 and alpha5beta1, as no involvement of any RGD motif could be demonstrated.

Amino Acid Sequence↗
PubMed2005

Direct observation of ligand binding to membrane proteins in living cells by a saturation transfer double difference (STDD) NMR spectroscopy method shows a significantly higher affinity of integrin alpha(IIb)beta3 in native platelets than in liposomes.

About 30% of the proteins in mammalian systems are membrane bound or integrated (e.g., GPCRs). It is inherently difficult to investigate receptor-ligand interactions on a molecular level in their natural membrane environment. Here, we present a new method based on saturation transfer difference (STD) NMR to characterize at an atomic level binding interactions of cell surface proteins in living cells. Implemented as a double difference technique, STD NMR allows the direct observation of binding events and the definition of the binding epitopes of ligands. The binding of the pentapeptide cyclo(RGDfV) to the surface glycoprotein integrin alpha(IIb)beta3 of intact human blood platelets can be detected by saturation transfer double difference (STDD) NMR in less than an hour. A 5-fold higher STD response reflects a significantly higher affinity of integrin alpha(IIb)beta3 in native platelets than in liposomes, which demonstrates the importance of studying membrane proteins in their natural environment. Also, the binding mode of cyclo(RGDfV) in the arginine glycine region is slightly different when interacting with native integrin in platelets compared to integrin reintegrated into liposomes.

Blood Platelets↗
PubMedDate not supplied

[Limited proteolysis of integrin alpha v beta3 from human placenta].

Purification of alpha v beta 3 integrin from human placenta with successive usage of two affinity sorbents--immobilized monoclonal antibodies to alpha v beta 3 integrin and immobilized RGD-containing decapeptide allowed to purify this integrin's partially degraded fraction, that was nevertheless able to interact with its ligand. During the incubation of partially degraded alpha v beta 3 integrin at 37 degrees C its further degradation went on. Addition of serine proteinase inhibitors: (phenylmethilsulfonyl fluoride, leupeptin and aprotinin) completely suppressed integrin further degradation of alpha v beta 3. In preparations of intact and partially degraded alpha v beta 3 integrin specific activity of two serine proteinases--urokinase and dipeptidilpeptidase IV--was discovered. alpha v beta 3 integrin, undergoing limited proteolysis, had lesser affinity towards RGD peptide, that intact integrin. The results show, that alpha v beta 3 integrin from human placenta co-purifies with serine proteinases. It is suggested that a definite part of functionally active alpha v beta 3 integrin, extracted from human placenta by triton X-100, forms a stable complex with serine proteinases.

Female↗
PubMed1997

Differential adhesion of metastatic RAW117 large-cell lymphoma cells under static or hydrodynamic conditions: role of integrin alpha(v) beta3.

RGD-containing substrates were used to study static and hydrodynamic adhesion of murine RAW117 large-cell lymphoma sublines with differential liver-metastatic potentials. Highly liver-metastatic RAW117-H10 cells had higher rates of static adhesion to vitronectin, fibronectin and (GRGDS)4 than poorly metastatic RAW117-P and moderately liver-metastatic RAW117-L17 cells. Under hydrodynamic conditions, adhesion stabilization was more rapid for H10 cells compared to P or L17 cells. Among the RGD peptides, only the polymeric RGD peptide (GRGDS)4 mediated strong static adhesion of H10 cells. Interestingly, all the RGD peptides mediated adhesion stabilization for H10 cells but still not for L17 or P cells under hydrodynamic conditions. Integrin alpha(v) beta3 was involved in stabilizing hydrodynamic adhesion to (GRGDS)4, monomeric RGD peptide R1, but was less important in static adhesion to monomeric RGD peptides. Differential adhesion to liver sinusoidal endothelial cell-derived extracellular matrix (H10 >> L17 > P) was observed under hydrodynamic but not static conditions. Integrin alpha(v) beta3 was also important in hydrodynamic adhesion to liver sinusoidal endothelial cell-derived extracellular matrix. We believe that strong static and hydrodynamic adhesion of H10 cells and their capability of altering adhesive behavior in response to fluid shear may contribute to liver metastasis.

Amino Acid Sequence↗
PubMed2000

Expression of alpha(v)beta6 integrin in oral leukoplakia.

The distribution of alpha(v)beta6 integrin was examined in oral leukoplakia, lichen planus and squamous cell carcinomas using immunohistochemistry. Controls included oral mucosal wounds, chronically inflamed and normal oral mucosa. Integrins beta1, beta3, beta4, beta5, fibronectin and tenascin were also studied. The integrin alpha(v)beta6 was highly expressed throughout the whole lesion of 90% of the squamous cell carcinomas but was not present in any of the normal specimens. alpha(v)beta6 integrin was also expressed in 41% of the leukoplakia specimens, and 85% of the lichen planus samples, but in none of the tissues with inflammatory hyperplasia or chronic inflammation. The expression of beta1 integrins was localized in the basal layer, and that of the beta4 at the cell surface facing the basement membrane of all specimens. The integrins beta3 and beta5 were absent from all normal and leukoplakia specimens. Fibronectin and tenascin were present in the connective tissue underneath the epithelium of all the sections, and their expression was similar in both alpha(v)beta6-positive and alpha(v)beta6-negative tissues. A group of 28 leukoplakia patients were followed 1-4 years after first diagnosis. In this group, initially alpha(v)beta6 integrin-positive leukoplakia specimens had high tendency for disease progression while alpha(v)beta6-negative specimens did not progress. These results suggest that the expression of alpha(v)beta6 integrin could be associated in the malignant transformation of oral leukoplakias.

Antibodies, Monoclonal↗
PubMed1999

Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3.

This paper reports a detailed conformational characterization in solution by 1H-NMR in H2O and DMSO-d6 and molecular modeling simulations of cyclic peptides containing the RGDDV pharmacophore and the RGDY(Me)R pharmacophore. These two pentapeptide sequences when properly constrained in cyclic peptides are low to sub-nanomolar inhibitors of integrin alpha(v)beta3. The peptides containing the RGDDY(Me)R sequence bind potently to integrin alphaIIb3 as well. The conformations found in H2O and in DMSO-d6 solutions are valuable for the design of peptidomimetics of these two pharmacophores. The structure-activity relationships of the RGDDV and RGDY(Me)R pharmacophores within cyclic peptides are discussed. Specifically, the orientation of surface-accessible chemical features on the ligand, such as hydrophobic, positive and negative ionizable groups, which are considered to be responsible for the desired biological activity, is focused on.

Magnetic Resonance Spectroscopy↗
PubMed1999

Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumor cell survival.

Growth and dissemination of malignant melanoma has a profound impact on our population, and little is known concerning the mechanisms controlling this disease in humans. Evidence is provided that integrin alpha(v)beta3 plays a critical role in M21 melanoma tumor survival within human skin by a mechanism independent of its known role in angiogenesis. Antagonists of alpha(v)beta3 blocked melanoma growth by inducing tumor apoptosis. Moreover, M21 melanoma cell interactions with denatured collagen, a known ligand for alpha(v)beta3, caused a 5-fold increase in the relative Bcl-2:Bax ratio, an event thought to promote cell survival. Importantly, denatured collagen colocalized with alpha(v)beta3-expressing melanoma cells in human tumor biopsies, suggesting that alpha(v)beta3 interaction with denatured collagen may play a critical role in melanoma tumor survival in vivo.

Animals↗
PubMed1998

Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and alpha5 beta1 integrin ligands.

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.

Animals↗
PubMed2000

Coexpression of integrin alpha(v)beta3 and matrix metalloproteinase-2 (MMP-2) coincides with MMP-2 activation: correlation with melanoma progression.

Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin alphavbeta3 correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to alpha(v)beta3 was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, alpha(v)beta3-negative melanoma cell lines MV3 and BLM, their beta3-transfected alpha(v)beta3 expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription-polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both alpha(v)beta3-negative and alpha(v)beta3-positive cell lines Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of alpha(v)beta3-negative cell lines and undetectable in the alpha(v)beta3-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta3 expressing transfectants. Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in beta3 transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the alpha(v)beta3 expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin alpha(v)beta3 is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alphavbeta3-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and alpha(v)beta3 increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and alphavbeta3-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of alpha(v)beta3 in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation.

Disease Progression↗
PubMed1997

Human fibroblasts bind directly to fibrinogen at RGD sites through integrin alpha(v)beta3.

Fibroblast migration into the blood clot initially filling a wound requires close interaction between fibroblasts and the matrix of the fibrin clot. However, very little is known about the specific receptor-ligand interactions that mediate fibroblast attachment to fibrin. Using an attachment assay developed to measure even relatively weak interactions, we demonstrate here that normal human dermal fibroblasts can attach to substrates coated with fibrinogen, fibrin, or the fibrinogen breakdown product I-9D. Fibroblast attachment to these ligands did not require the presence of fibronectin on the cell surface or as a component of the substrate. Cells treated with cycloheximide and monensin, to limit the synthesis and secretion of endogenous fibronectin, attached as well as untreated cells. The synthetic peptide GRGDS inhibited adhesion to fibrinogen, fibrin, and fibrinogen I-9D by about 60%, while the control peptide GRGES had no substantial effect. We conclude that attachment to these ligands is mediated at least partially by direct interactions between the substrates and one specific receptor, the integrin alpha(v)beta3. Affinity chromatography demonstrated that alpha(v)beta3 from detergent lysates of fibroblasts bound to a fibrinogen matrix and was eluted with EDTA. Furthermore, antibodies against the alpha(v)beta3 complex or against the alpha(v) subunit inhibited fibroblast attachment to fibrinogen and fibrin by 50-70%. An inhibitory antibody against the integrin beta1 subunit had no effect. The observation that integrin antagonists could not produce complete inhibition suggests that there may be other fibroblast cell surface proteins that can bind directly to fibrinogen.

Cell Adhesion↗
PubMed2005

A novel anti-platelet monoclonal antibody (3C7) specific for the complex of integrin alpha IIb beta3 inhibits platelet aggregation and adhesion.

Activation or ligand binding induces conformational changes in alpha IIb beta3, resulting in exposure of neoepitopes named ligand-induced binding sites. We reported here a novel monoclonal antibody developed by using Chinese hamster ovary (CHO) cells expressing an activated alpha IIb beta3 mutant (CHO alpha IIb beta3Delta717) as the immunogen. This IgG 2b kappa named 3C7 was specific for the complex of alpha IIb beta3 as demonstrated by flow cytometry, immunoprecipitation, and EDTA chelating. The binding of 3C7 to platelets increased significantly when platelets were activated by ADP/thrombin or occupied by RGDS peptides, fibrinogen, or PAC-1, suggesting that 3C7 was an anti-ligand-induced binding site antibody. The antibody failed to bind to the CHO cells expressing another alpha IIb beta3 mutant (beta3Y178A) suggesting that the Cys177-Cys184 loop of beta3 was likely the epitope for 3C7. 3C7 inhibited platelet aggregation, which was initiated by ADP or thrombin in a dose-dependent manner (IC50s of 5.6 and 0.05 microg/ml, respectively). The antibody also inhibited platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. These findings suggested that 3C7 was a potent antagonist of integrin alpha IIb beta3 and a potential anti-thrombotic agent.

Adenosine Diphosphate↗