Tan L, Kowalska M A, Romo G M, Lopez J A, Darzynkiewicz Z, Niewiarowski S
Department of Physiology, and Sol Sherry Thrombosis Research Center, Philadelphia, PA, USA.
Blood. 1999 Apr 15;93(8):2605-16.
The expression and function of a glycoprotein Ib (GPIb) complex on human umbilical vein endothelial cells (HUVECs) is still a matter of controversy. We characterized HUVEC GPIb using viper venom proteins: alboaggregins A and B, echicetin, botrocetin, and echistatin. Echicetin is an antagonist, and alboaggregins act as agonists of the platelet GPIb complex. Botrocetin is a venom protein that alters von Willebrand factor (vWF) conformation and increases its binding affinity for the GPIb complex. Echistatin is a disintegrin that blocks alphavbeta3. Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese hamster ovary (CHO) cells transfected with alphavbeta3. We found the following: (1) Binding of monoclonal antibodies against GPIbalpha to HUVECs was moderately increased after stimulation with cytokines and phorbol ester. Echicetin demonstrated an inhibitory effect. (2) Both echicetin and echistatin, an alphavbeta3 antagonist, inhibited the adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The inhibitory effect was additive when both proteins were used together. (3) Botrocetin potentiated the adhesion of HUVECs to vWF, and this effect was completely abolished by echicetin, but not by echistatin. (4) CHO cells expressing GPIbalphabeta/IX adhered to vWF (in the presence of botrocetin) and to alboaggregins; GPIbalpha was required for this reaction. Echicetin, but not echistatin, inhibited the adhesion of cells transfected with GPIbalphabeta/IX to immobilized vWF. (5) HUVECs adhered strongly to immobilized vWF and alboaggregins with extensive spreading, which was inhibited by LJ1b1, a monoclonal antibody against GPIb. The purified alphavbeta3 receptor did not interact with the alboaggregins, thereby excluding the contribution of alphavbeta3 in inducing HUVEC spreading on alboaggregins. In conclusion, our data confirm the presence of a functional GPIb complex expressed on HUVECs in low density. This complex may mediate HUVEC adhesion and spreading on immobilized vWF and alboaggregins.
糖蛋白Ib（GPIb）复合物在人脐静脉内皮细胞（HUVECs）上的表达及功能仍存在争议。我们使用蛇毒蛋白对HUVEC的GPIb进行了特性分析：白蝰凝集素A和B、echicetin、巴曲酶和echistatin。Echicetin是一种拮抗剂，白蝰凝集素作为血小板GPIb复合物的激动剂。巴曲酶是一种能改变血管性血友病因子（vWF）构象并增加其与GPIb复合物结合亲和力的蛇毒蛋白。Echistatin是一种阻断αvβ3的整合素。Echistatin而非echicetin抑制了转染αvβ3的中国仓鼠卵巢（CHO）细胞与vWF的黏附。我们发现如下：（1）用细胞因子和佛波酯刺激后，抗GPIbα单克隆抗体与HUVECs的结合适度增加。Echicetin表现出抑制作用。（2）Echicetin和αvβ3拮抗剂echistatin均以剂量依赖方式抑制HUVECs与固定化vWF的黏附。两种蛋白一起使用时，抑制作用具有相加性。（3）巴曲酶增强了HUVECs与vWF的黏附，且这种作用被echicetin完全消除，但未被echistatin消除。（4）表达GPIbαβ/IX的CHO细胞黏附于vWF（在巴曲酶存在下）和白蝰凝集素；此反应需要GPIbα。Echicetin而非echistatin抑制了转染GPIbαβ/IX的细胞与固定化vWF的黏附。（5）HUVECs强烈黏附于固定化vWF和白蝰凝集素并广泛铺展，这被抗GPIb单克隆抗体LJ1b1抑制。纯化的αvβ3受体不与白蝰凝集素相互作用，从而排除了αvβ3在诱导HUVECs在白蝰凝集素上铺展中的作用。总之，我们的数据证实低密度表达于HUVECs上存在功能性GPIb复合物。该复合物可能介导HUVECs与固定化vWF和白蝰凝集素的黏附及铺展。
