Makowski Gregory S, Ramsby Melinda L, Ramsby Gale R
Department of Laboratory Medicine, School of Medicine, University of Connecticut Health Center, Farmington, CT, USA.
J Biomed Sci. 2005;12(2):421-9. doi: 10.1007/s11373-004-0226-6.
The ability of indium to target fibrin in vitro was evaluated. The radionuclide (114m)Indium (114mIn) was prepared as a soluble and colloidal (In:In) form, as well as, a mixed indium:calcium phosphate (In:CaP) colloid. Soluble 114mIn was prepared by maintaining acid pH (50 mM HCl). Colloidal 114mIn (In:In) was prepared under slightly basic conditions (50 mM Tris-Cl, pH 7.6). The mixed In:CaP colloid was prepared by incubation of 114mIn with calcium (10 mM) and phosphate (250 microM) under slightly basic conditions (50 mM Tris-Cl, pH 7.6). To assess fibrin binding, the three 114mIn preparations were mixed with diluted human plasma (source of fibrinogen). Fibrin polymerization was initiated by addition of calcium (5 mM) and thrombin (0.5 U/ml). Following incubation (15 min, 37 degrees C), the fibrin matrix was condensed, removed from the reaction mixture, and washed briefly. Fibrin uptake of 114mIn (soluble, colloidal, or In:CaP) was determined by gamma counting. Results demonstrated that soluble 114mIn exclusively bound a plasma protein electrophoretically and immunologically identified as transferrin. Although both colloidal 114mIn and 114mIn:CaP bound fibrin, the mixed 114mIn:CaP colloid demonstrated substantially higher fibrin binding activity (about 2-fold). The target of indium binding was confirmed as fibrin due to the presence of characteristic cross-linked gamma-gamma dimers (100 kDa) and beta-monomers (58 kDa) by SDS-PAGE. 114mIn colloid and the mixed 114mIn:CaP colloid demonstrated no ability to bind fibrin's precursor, fibrinogen. 114mIn:CaP fibrin binding was associated with formation of CaP, as evidenced by its dependence on phosphate concentration. The biocompatibility of CaP including its ability to bind 114mIn and specifically target fibrin may be of potential value for diagnostic imaging studies to identify regions of occult vascular stenosis (i.e., atherosclerotic plaques, deep vein thrombosis, pulmonary embolus).
评估了铟在体外靶向纤维蛋白的能力。放射性核素（114m）铟（114mIn）制备成可溶胶体形式（In:In）以及铟与磷酸钙的混合胶体（In:CaP）。通过维持酸性pH值（50 mM HCl）制备可溶性114mIn。在略碱性条件下（50 mM Tris-Cl，pH 7.6）制备胶体114mIn（In:In）。通过在略碱性条件下（50 mM Tris-Cl，pH 7.6）将114mIn与钙（10 mM）和磷酸盐（250 microM）孵育来制备混合In:CaP胶体。为了评估纤维蛋白结合情况，将三种114mIn制剂与稀释的人血浆（纤维蛋白原来源）混合。通过添加钙（5 mM）和凝血酶（0.5 U/ml）引发纤维蛋白聚合。孵育（15分钟，37℃）后，纤维蛋白基质凝聚，从反应混合物中取出并短暂洗涤。通过γ计数测定114mIn（可溶性、胶体或In:CaP）对纤维蛋白的摄取。结果表明，可溶性114mIn仅与一种经电泳和免疫鉴定为转铁蛋白的血浆蛋白结合。虽然胶体114mIn和114mIn:CaP都能结合纤维蛋白，但混合的114mIn:CaP胶体表现出显著更高的纤维蛋白结合活性（约2倍）。通过SDS-PAGE检测到特征性的交联γ-γ二聚体（100 kDa）和β-单体（），证实铟结合的靶点为纤维蛋白。114mIn胶体和混合的114mIn:CaP胶体均无结合纤维蛋白前体纤维蛋白原）的能力。114mIn:CaP与纤维蛋白的结合与磷酸钙的形成有关，这通过其对磷酸盐浓度的依赖性得以证明。磷酸钙的生物相容性，包括其结合114mIn并特异性靶向纤维蛋白的能力，对于识别隐匿性血管狭窄区域（即动脉粥样硬化斑块、深静脉血栓形成、肺栓塞）的诊断成像研究可能具有潜在价值。 58 kDa58 kDa
