Byun Y, Yang V C
Department of Materials Science and Engineering, Institute of Science and Technology, Kwangju, Korea.
ASAIO J. 1998 Sep-Oct;44(5):M638-41. doi: 10.1097/00002480-199809000-00068.
Cardiovascular diseases that result from thrombosis of critically situated blood vessels remain the leading cause of death in industrialized countries. One primary clinical treatment is dissolution of the thrombus with thrombolytic agents, plasminogen activators (PA). Activation of plasminogen by a PA agent produces plasmin that degrades fibrin. However, plasmin also degrades other circulating clotting factors. Therefore, thrombolytic therapy, which introduces systemic generation of excess plasmin, carries the risk of hemorrhage. We propose a novel approach that could lead to targeted thrombolysis without bleeding risk. The system is comprised of a protein conjugate made of two parts: a fibrin-targeting antibody (Ab) linked with anionic heparin; and a PA derivatized with cationic species. These two parts are linked via an electrostatic interaction. Because the cationic species are relatively small, the derivatized PA would retain its thrombolytic activity, but this activity would be inhibited after binding with the Ab-heparin counterpart because of the blockage of the PA's active site by these appended macromolecules. Because protamine is a clinical heparin antagonist with a much stronger affinity for heparin than the incorporated cations, it can be used safely to dissociate the modified PA from the Ab-heparin counterpart. Therefore, this approach would permit administration of a fibrin-targeting but inactive thrombolytic, and subsequently, a triggered release of the active modified PA drug in close proximity to the fibrin deposit. These features would enhance the potency and the specificity of the thrombolytic agent and alleviate the bleeding risk by avoiding systemic generation of excess plasmin. In this report, we present preliminary results demonstrating the feasibility of the approach. A cationic octapeptide, (Arg)7-Cys, was linked to urokinase (UK) using the N-succinimidyl-3-[2-pyridylidithio]propionate activation method. This UK peptide retained a significant amount of its catalytic activity, as measured by the S-2251 chromogenic assay. However, this activity was almost completely inhibited (approximately 99%) after the addition of heparin, but was fully reversed (100%) after the addition of protamine.
由关键部位血管血栓形成导致的心血管疾病仍然是工业化国家的主要死因。一种主要的临床治疗方法是用溶栓剂（纤溶酶原激活剂，PA）溶解血栓。PA 剂激活纤溶酶原会产生降解纤维蛋白的纤溶酶。然而，纤溶酶也会降解其他循环凝血因子。因此，引入全身过量纤溶酶生成的溶栓治疗存在出血风险。我们提出了一种新方法，可实现靶向溶栓且无出血风险。该系统由一种蛋白质偶联物组成，它由两部分构成：与阴离子肝素相连的纤维蛋白靶向抗体（Ab）；以及用阳离子物质衍生化的 PA。这两部分通过静电相互作用相连。由于阳离子物质相对较小，衍生化的 PA 会保留其溶栓活性，但与 Ab - 肝素对应物结合后，由于这些附加大分子对 PA 活性位点的阻断，该活性会受到抑制。因为鱼精蛋白是一种临床肝素拮抗剂，对肝素的亲和力比掺入的阳离子强得多，所以它可以安全地用于使修饰后的 PA 与 Ab - 肝素对应物解离。因此，这种方法允许给予靶向纤维蛋白但无活性的溶栓剂，随后在纤维蛋白沉积物附近触发释放活性修饰 PA 药物。这些特性将增强溶栓剂的效力和特异性，并通过避免全身过量纤溶酶的生成来减轻出血风险。在本报告中，我们展示了证明该方法可行性的初步结果。使用 N - 琥珀酰亚胺基 - 3 - [2 - 吡啶二硫基]丙酸酯活化方法将阳离子八肽（Arg）7 - Cys 与尿激酶（UK）相连。通过 S - 2251 显色测定法测量，这种 UK 肽保留了大量催化活性。然而，加入肝素后该活性几乎完全被抑制（约 99%），但加入鱼精蛋白后完全恢复（100%）。
