Marcinkiewicz C, Rosenthal L A, Mosser D M, Kunicki T J, Niewiarowski S
Department of Physiology, Sol Sherry Thrombosis Research Center, Philadelphia, PA 19140, USA.
Biochem J. 1996 Aug 1;317 ( Pt 3)(Pt 3):817-25. doi: 10.1042/bj3170817.
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.
两种氨基酸序列相似度很高的去整合素——echistatin和eristostatin，与中国仓鼠卵巢（CHO）细胞的相互作用水平较低，但它们能以可逆且饱和的方式与转染了αIIbβ3基因的CHO细胞（A5细胞）以及转染了αvβ3基因的CHO细胞（VNRC3细胞）结合。Scatchard分析表明，eristostatin分别与每个A5细胞上的816000个位点（解离常数Kd为28 nM）以及每个VNRC3细胞上的200000个位点（Kd为14 nM）结合。然而，VNRC3细胞不与固定化的eristostatin结合。Echistatin与每个A5细胞上的495000个位点（Kd为53 nM）以及每个VNRC3细胞上的443000个位点（Kd为20 nM）结合。通过流式细胞术、放射结合测定和黏附研究确定，这两种去整合素与A5细胞和静息血小板的结合以及echistatin与VNRC3细胞的结合均导致β3亚基上配体诱导结合位点（LIBS）的表达。Eristostatin比echistatin更强烈地抑制三种单克隆抗体与A5细胞、静息和活化血小板以及纯化的αIIbβ3的结合，这三种单克隆抗体分别是：OPG2（依赖RGD基序）、A2A9（依赖αIIbβ3复合物）和7E3（依赖αIIbβ3和αvβ3复合物）。单独或联合使用echistatin和eristostatin抑制7E3和OPG2抗体与静息血小板结合的实验表明，这两种去整合素与αIIbβ3整合素上不同但重叠的位点结合。单克隆抗体LM 609和echistatin似乎与αvβ3整合素上不同的位点结合。然而，echistatin抑制7E3抗体与VNRC3细胞和纯化的αvβ3的结合，这表明αvβ3和αIIbβ3可能共享echistatin和7E3都能结合的相同表位。Eristostatin在这些系统中没有作用，这进一步证明它与αvβ3上不同的表位结合。
