Immunological characterization of eristostatin and echistatin binding sites on alpha(IIb)beta(3) and alpha(v)beta(3) integrins

被引:47
作者
Marcinkiewicz, C
Rosenthal, LA
Mosser, DM
Kunicki, TJ
Niewiarowski, S
机构
[1] TEMPLE UNIV, SCH MED, SOL SHERRY THROMBOSIS RES CTR, DEPT PHYSIOL, PHILADELPHIA, PA 19140 USA
[2] TEMPLE UNIV, SCH MED, DEPT MICROBIOL & IMMUNOL, PHILADELPHIA, PA 19140 USA
[3] SCRIPPS RES INST, LA JOLLA, CA 92037 USA
关键词
D O I
10.1042/bj3170817
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (K-d 28 nM) and to 200000 sites (K-d 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (K-d 53 nM) per A5 cell and to 443000 sites (K-d 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 (LIES) 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).
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页码:817 / 825
页数:9
相关论文
共 48 条
[1]   INHIBITION OF FIBRINOGEN BINDING TO STIMULATED HUMAN-PLATELETS BY A MONOCLONAL-ANTIBODY [J].
BENNETT, JS ;
HOXIE, JA ;
LEITMAN, SF ;
VILAIRE, G ;
CINES, DB .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1983, 80 (09) :2417-2421
[2]   LABELING OF PROTEINS TO HIGH SPECIFIC RADIOACTIVITIES BY CONJUGATION TO A I-125-CONTAINING ACYLATING AGENT - APPLICATION TO RADIOIMMUNOASSAY [J].
BOLTON, AE ;
HUNTER, WM .
BIOCHEMICAL JOURNAL, 1973, 133 (03) :529-538
[3]   THE DISULFIDE BRIDGE PATTERN OF SNAKE-VENOM DISINTEGRINS, FLAVORIDIN AND ECHISTATIN [J].
CALVETE, JJ ;
WANG, YQ ;
MANN, K ;
SCHAFER, W ;
NIEWIAROWSKI, S ;
STEWART, GJ .
FEBS LETTERS, 1992, 309 (03) :316-320
[4]   IDENTIFICATION OF THE DISULFIDE BOND PATTERN IN ALBOLABRIN, AN RGD-CONTAINING PEPTIDE FROM THE VENOM OF TRIMERESURUS-ALBOLABRIS - SIGNIFICANCE FOR THE EXPRESSION OF PLATELET-AGGREGATION INHIBITORY ACTIVITY [J].
CALVETE, JJ ;
SCHAFER, W ;
SOSZKA, T ;
LU, WQ ;
COOK, JJ ;
JAMESON, BA ;
NIEWIAROWSKI, S .
BIOCHEMISTRY, 1991, 30 (21) :5225-5229
[5]  
CALVETE JJ, 1994, THROMB HAEMOSTASIS, V72, P1
[6]   PLATELET GLYCOPROTEIN-IIB AND GLYCOPROTEIN-IIIA - EVIDENCE FOR A FAMILY OF IMMUNOLOGICALLY AND STRUCTURALLY RELATED GLYCOPROTEINS IN MAMMALIAN-CELLS [J].
CHARO, IF ;
FITZGERALD, LA ;
STEINER, B ;
RALL, SC ;
BEKEART, LS ;
PHILLIPS, DR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1986, 83 (21) :8351-8355
[7]  
CHEN YQ, 1992, J BIOL CHEM, V267, P17314
[8]  
CHERESH DA, 1987, J BIOL CHEM, V262, P1434
[9]  
COLLER BS, 1991, BLOOD, V77, P75
[10]   A NEW MURINE MONOCLONAL-ANTIBODY REPORTS AN ACTIVATION-DEPENDENT CHANGE IN THE CONFORMATION AND OR MICROENVIRONMENT OF THE PLATELET GLYCOPROTEIN IIB/IIIA COMPLEX [J].
COLLER, BS .
JOURNAL OF CLINICAL INVESTIGATION, 1985, 76 (01) :101-108