Fibrin-specific molecular targeting strategies are desirable for site-specific imaging and treatment of late stage atheroma, but fibrin-specific antibodies are difficult to produce and present immunogenicity problems. Tissue plasminogen activator (tPA) is an endogenous protein that has been shown to bind fibrin with high affinity and may circumvent antibody difficulties. Use of tPA-derived proteins or peptides, however, requires that the plasminogen-activating proteolytic activity be neutralized or removed. As an initial step in determining the feasibility of this targeting strategy, human recombinant tPA (Activase®) was irreversibly inhibited with D-phe-L-pro-L-arg-chloromethyl ketone (PPACK) and conjugated to intrinsically echogenic liposomes (ELIP) by a thioether coupling protocol. Fibrin-binding affinities were assessed with a novel two‐stage fibrin pad ELISA. We achieved 95–99% inactivation, while retaining both tPA fibrin-binding activities of KD ∼ 2 nM and 33 nM. Thermodynamic analysis of the PPACK-inactivated tPA (tPA(P)) revealed highly exothermic interactions, indicative of ionic associations, especially for the higher affinity. The conjugation efficiency of tPA(P) to ELIP was within the range of that previously achieved for IgG and exhibited satisfactory fibrin targeting, characterized by striking increases of enthalpy and entropy increments. Evidence for coupling of noncovalent association energetics with the phosphatidylethanolamine major phase transition, observed in previous IgG antibody conjugations, was also evident in this case, but the nature of the transduction mechanism was different. These results demonstrate that tPA-derived components lacking proteolytic activity can be employed as fibrin-targeting agents for delivery of therapeutic and diagnostic formulations.

中文翻译：

---

具有灭活组织纤溶酶原激活剂的回声脂质体的纤维蛋白靶向

纤维蛋白特异性分子靶向策略对于晚期动脉粥样硬化的位点特异性成像和治疗是可取的，但纤维蛋白特异性抗体难以产生并存在免疫原性问题。组织纤溶酶原激活剂 (tPA) 是一种内源性蛋白质，已被证明能以高亲和力结合纤维蛋白，并可避免抗体困难。然而，使用 tPA 衍生的蛋白质或肽需要中和或去除纤溶酶原激活蛋白水解活性。作为确定这种靶向策略可行性的第一步，人类重组 tPA (Activase®) 被 D-phe-L-pro-L-arg-氯甲基酮 (PPACK) 不可逆地抑制并与内在回声脂质体 (ELIP) 结合通过硫醚偶联方案。使用新型两阶段纤维蛋白垫 ELISA 评估纤维蛋白结合亲和力。我们实现了 95-99% 的失活，同时保留了 KD ~ 2 nM 和 33 nM 的 tPA 纤维蛋白结合活性。PPACK 灭活的 tPA (tPA(P)) 的热力学分析揭示了高度放热的相互作用，表明离子协会，特别是对于更高的亲和力。tPA(P) 与 ELIP 的结合效率在之前 IgG 的结合效率范围内，并表现出令人满意的纤维蛋白靶向，其特征是焓和熵增量的显着增加。在之前的 IgG 抗体偶联中观察到的非共价缔合能量与磷脂酰乙醇胺主要相变耦合的证据在这种情况下也很明显，但转导机制的性质不同。