Chen H, Marjan J, Cox A D, Devine D V
Department of Pathology, University of British Columbia, Canada.
J Immunol. 1994 Feb 1;152(3):1332-8.
The breakdown products of the complement protein C3 function in receptor-mediated immune clearance. The catabolism of the C3 molecule, mediated by factors I and H, results in the generation of the fragments C3c and C3dg. C3dg binds to human platelets in a specific and saturable manner. The direct interaction of platelets with soluble C3dg may contribute to immune-mediated platelet destruction. More importantly, platelets may interact with opsonized pathogens or complement-activating immune complexes via C3dg. In this report, we investigated the interaction of C3dg with platelets and calculated the Ka to be 3.2 x 10(6) M-1 with 1100 to 2200 specific binding sites/platelet. In the presence of 5 mM calcium, both the Ka and the number of specific binding sites were modestly decreased to Ka = 2.8 x 10(6) M-1 with 1400 to 2400 specific binding sites/platelet. The Scatchard plots demonstrated a curvilinear character. On labeling C3dg with peroxidase and visualizing platelet-bound C3dg by electron microscopy, it was shown that binding sites for C3dg were restricted to the platelet plasma membrane. Using a cell attachment assay, platelet adhesion to C3dg was readily detectable; attachment to C3dg-coated plates was not blocked by fibrinogen or fibronectin. We have characterized the C3dg-binding protein of platelets using the chemical cross-linkers, SASD and DSS, to cross-link C3dg to thrombin-stimulated platelets. Gel filtration of the 125I-labeled C3dg-platelet complex revealed the presence of a large protein complex that was absent when 125I-labeled C3dg alone was analyzed. SDS-PAGE of the radiolabeled cross-linked protein, followed by autoradiography, identified a 95-kDa membrane protein. The relationship of this C3dg-binding protein to other platelet membrane proteins has yet to be determined.
补体蛋白C3的分解产物在受体介导的免疫清除中发挥作用。由因子I和H介导的C3分子分解代谢产生片段C3c和C3dg。C3dg以特异性和可饱和的方式与人血小板结合。血小板与可溶性C3dg的直接相互作用可能导致免疫介导的血小板破坏。更重要的是，血小板可能通过C3dg与调理过的病原体或补体激活的免疫复合物相互作用。在本报告中，我们研究了C3dg与血小板的相互作用，并计算出Ka为3.2×10⁶ M⁻¹，每个血小板有1100至2200个特异性结合位点。在5 mM钙存在的情况下，Ka和特异性结合位点的数量均适度降低，Ka = 2.8×10⁶ M⁻¹，每个血小板有1400至2400个特异性结合位点。Scatchard图显示出曲线特征。用过氧化物酶标记C3dg并通过电子显微镜观察血小板结合的C3dg，结果表明C3dg的结合位点仅限于血小板质膜。使用细胞附着试验，很容易检测到血小板对C3dg的粘附；对C3dg包被平板的附着不受纤维蛋白原或纤连蛋白的阻断。我们使用化学交联剂SASD和DSS将C3dg与凝血酶刺激的血小板交联，从而对血小板的C3dg结合蛋白进行了表征。对¹²⁵I标记的C3dg - 血小板复合物进行凝胶过滤，发现存在一种大的蛋白质复合物，而单独分析¹²⁵I标记的C3dg时不存在这种复合物。对放射性标记的交联蛋白进行SDS - PAGE，随后进行放射自显影，鉴定出一种95 kDa的膜蛋白。这种C3dg结合蛋白与其他血小板膜蛋白的关系尚待确定。
