Bombeli T, Schwartz B R, Harlan J M
Division of Hematology, University of Washington, Seattle, Washington 98195-7710, USA.
J Exp Med. 1998 Feb 2;187(3):329-39. doi: 10.1084/jem.187.3.329.
Although it has been reported that activated platelets can adhere to intact endothelium, the receptors involved have not been fully characterized. Also, it is not clear whether activated platelets bind primarily to matrix proteins at sites of endothelial cell denudation or directly to endothelial cells. Thus, this study was designed to further clarify the mechanisms of activated platelet adhesion to endothelium. Unstimulated human umbilical vein endothelial cell (HUVEC) monolayers were incubated with washed, stained, and thrombin-activated human platelets. To exclude matrix involvement, HUVEC were harvested mechanically and platelet binding was measured by flow cytometry. Before the adhesion assay, platelets or HUVEC were treated with different receptor antagonists. Whereas blockade of platelet beta1 integrins, GPIbalpha, GPIV, P-selectin, and platelet-endothelial cell adhesion molecule (PECAM)-1 did not reduce platelet adhesion to HUVEC, blockade of platelet GPIIbIIIa by antibodies or Arg-Gly-Asp (RGD) peptides markedly decreased adhesion. Moreover, when platelets were treated with blocking antibodies to GPIIbIIIa-binding adhesive proteins, including fibrinogen and fibronectin, and von Willebrand factor (vWF), platelet binding was also reduced markedly. Addition of fibrinogen, fibronectin, or vWF further increased platelet adhesion, indicating that both endogenous platelet-exposed and exogenous adhesive proteins can participate in the binding process. Evaluation of the HUVEC receptors revealed predominant involvement of intercellular adhesion molecule (ICAM)-1 and alphavbeta3 integrin. Blockade of these two receptors by antibodies decreased platelet binding significantly. Also, there was evidence that a component of platelet adhesion was mediated by endothelial GPIbalpha. Blockade of beta1 integrins, E-selectin, P-selectin, PECAM-1, vascular cell adhesion molecule (VCAM)-1 and different matrix proteins on HUVEC did not affect platelet adhesion. In conclusion, we show that activated platelet binding to HUVEC monolayers is mediated by a GPIIbIIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1, alphavbeta3 integrin, and, to a lesser extent, GPIbalpha.
尽管已有报道称活化血小板可黏附于完整的内皮细胞，但所涉及的受体尚未完全明确。此外，尚不清楚活化血小板主要是在内皮细胞剥脱部位与基质蛋白结合，还是直接与内皮细胞结合。因此，本研究旨在进一步阐明活化血小板黏附于内皮的机制。将未刺激的人脐静脉内皮细胞（HUVEC）单层与洗涤、染色并经凝血酶活化的人血小板共同孵育。为排除基质的影响，机械收获HUVEC，并通过流式细胞术检测血小板结合情况。在黏附试验前，用不同的受体拮抗剂处理血小板或HUVEC。虽然阻断血小板β1整合素、糖蛋白Ibα（GPIbalpha）、糖蛋白IV（GPIV）、P-选择素和血小板内皮细胞黏附分子（PECAM）-1并不会降低血小板对HUVEC的黏附，但用抗体或精氨酸-甘氨酸-天冬氨酸（RGD）肽阻断血小板糖蛋白IIbIIIa可显著降低黏附。此外，当用针对与糖蛋白IIbIIIa结合的黏附蛋白（包括纤维蛋白原、纤连蛋白和血管性血友病因子（vWF））的阻断抗体处理血小板时，血小板结合也显著减少。添加纤维蛋白原、纤连蛋白或vWF可进一步增加血小板黏附，表明内源性血小板暴露的和外源性黏附蛋白均可参与结合过程。对HUVEC受体的评估显示细胞间黏附分子（ICAM）-1和αvβ3整合素起主要作用。用抗体阻断这两种受体可显著降低血小板结合。此外，有证据表明血小板黏附的一个成分是由内皮GPIbalpha介导的。阻断HUVEC上的β1整合素、E-选择素、P-选择素、PECAM-1、血管细胞黏附分子（VCAM）-1和不同的基质蛋白并不影响血小板黏附。总之，我们表明活化血小板与HUVEC单层的结合是由一种依赖糖蛋白IIbIIIa的桥接机制介导的，该机制涉及血小板结合的黏附蛋白以及内皮细胞受体ICAM-1、αvβ3整合素，在较小程度上还涉及GPIbalpha。
