Klegerman Melvin E, Zou Yuejiao, McPherson David D
Division of Cardiology, Department of Internal Medicine, University of Texas Health Science Center-Houston, Houston, TX 77030, USA.
J Liposome Res. 2008;18(2):95-112. doi: 10.1080/08982100802118482.
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 K(D) approximately 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.
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理想的，但纤维蛋白特异性抗体难以产生且存在免疫原性问题。组织纤溶酶原激活剂（tPA）是一种内源性蛋白质，已证明其能以高亲和力结合纤维蛋白，且可能避免抗体相关的难题。然而，使用tPA衍生的蛋白质或肽时，需要中和或去除纤溶酶原激活的蛋白水解活性。作为确定该靶向策略可行性的第一步，人重组tPA（阿替普酶）用D-苯丙氨酸-L-脯氨酸-L-精氨酸-氯甲基酮（PPACK）进行不可逆抑制，并通过硫醚偶联方案与固有回声脂质体（ELIP）偶联。用一种新型的两阶段纤维蛋白垫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tPA与纤维蛋白的结合活性，解离常数（K(D)）分别约为2 nM和33 nM。对PPACK失活的tPA（tPA(P)）的热力学分析显示出高度放热的相互作用，表明存在离子缔合，尤其是对于较高亲和力的情况。tPA(P)与ELIP的偶联效率在先前针对IgG所达到的范围内，并表现出令人满意的纤维蛋白靶向性，其特征是焓和熵增量显著增加。在先前的IgG抗体偶联中观察到的非共价缔合能量学与磷脂酰乙醇胺主要相变偶联的证据，在这种情况下也很明显，但转导机制的性质不同。这些结果表明，缺乏蛋白水解活性的tPA衍生成分可作为纤维蛋白靶向剂用于递送治疗和诊断制剂。
