【作者】 董宁征；

【导师】 阮长耿；

【作者基本信息】 苏州大学 ， 内科血液学， 2002， 博士

【摘要】 血栓性疾病，尤其是急性心肌梗塞（AMI）和缺血性脑梗塞严重威胁着人类的健康，是人类致死、致残的首要原因。在发生AMI后尽早应用溶栓剂进行治疗，能显著降低AMI死亡率，改善预后。但是，现有的溶栓剂还存在着多种缺陷，如出血、缺血心肌没有明显的再灌注，以及溶栓治疗后再梗塞。因而对纤溶系统及其调节机制的研究，以及对高效、安全、方便的溶栓剂的研制具有重要作用，是该领域的研究重点。 本文围绕纤溶系统的调控机制和溶栓剂DSPA进行了一系列研究。首先利用大鼠肺纤维蛋白沉积模型，研究PAI-1和TAFIa对纤溶的调控作用；再从分子生物学及生物化学的角度探讨新型纤溶剂DSPA（Desmodus rotundus salivary plasminogen activator）的作用机制；最后研制P-selectin导向的DSPA，试图得到一个更好的溶栓剂。 PAI-1和TAFI是纤溶系统的两大重要调控因子。为研究PAI-1与TAFI的调控作用，我们首先建立以¹²⁵I示踪的batroxobin导致的大鼠肺纤维蛋白沉积模型，随后采用该模型研究PAI-1和TAFIa的作用。结果：1、Batroxobin能导致纤维蛋白在大鼠肺组织沉积，表现为5分钟肺组织放射性记数显著升高。而30分钟由於内源性纤溶系统的激活，肺组织放射性记数降低至与对照组类似水平；2、给予重组PAI-1和LPS都能升高血浆PAI-1的水平和活性，抑制30分钟肺组织放射性记数的降低；3、注射经凝血酶／TM活化的TAFIa能升高大鼠血浆TAFIa的水平，抑制30分钟肺组织放射性记数的降低。证实PAI-1和TAFIa都能抑制内源性纤溶，抑制纤维蛋白的降解。首次证实活化的TAFI（TAFIa）能直接抑制内源性纤溶；另外我们的结果还显示是TAFI的活性而不是TAFI的抗原量影响了内源性纤溶。 DSPA是一种从吸血蝙蝠（Desmodus rotundus）唾液中提取的纤溶酶原激活剂，结构上它包括Finger、EGF、Kringle和蛋白酶区。与tPA相比，DSPA对纤维蛋白具有高度的特异性。以往的研究结果提示Finger区可能与DSPA与纤维蛋白的结合有关，Finger区介导的纤维蛋白与DSPA的结合，可能导致了DSPA酶活性区与纤维蛋白表面特殊结构的结合，从而增强了DSPA的功能。为验证这纤溶系统的调控及溶栓剂的分子生物学研究中文摘要一推断，我们构建并表达了缺失Finger和EG厂区的OSPA的缺失突变体（OSPK），并对OSPA和OSpK进行了研究。结果:1、在无纤维蛋白存在下，OSPA和05户K在同等分子数时对小分子底物52765的催化活性没有显著区别，表现为它们的酶动力学常数K山和kca灯Km基本一致。但在纤维蛋白存在下，两者的kcat都明显升高，为11也增大。比较有无纤维蛋白存在时，它们的kcat/Km值，OSPA的kcat/.弋tn值增加了3.9倍，而OSPK仅增加了1.6倍。2、Miero Plate法进行的溶栓试验显示DSpK的溶栓能力降低了48倍。在1251标记的血浆凝块溶解实验，OSPK浓度升高至50 nM时也未见溶栓作用。3、尸Al一1对OSPK和OSPA具有同等的抑制作用。这些研究结果说明，OSPA的Finger和EGF区对OSPA的纤溶功能至关重要。DSPK的纤溶功能的降低，并非由于它对PAI一1的抑制作用更为敏感，而是由于纤维蛋白对OSPK的刺激作用明显降低所致。提示纤维蛋白对OSPA的刺激作用的机制是:纤维蛋白与OSPA结合，诱导OSPA蛋白酶活性中心的构型发生改变，从而增强了OSRA的活性。 新鲜动脉血栓主要由活化血小板和纤维蛋白组成，而陈旧性止血血栓主要由纤维蛋白组成。P一selectin是活化血小板的标志物，是新鲜动脉血.栓导向的良好靶抗原。我室于1989年研制了抗活化血小板表面队selectin的单克隆抗体5251，研究显示它具有很好的血栓显像效果。另外，5251导向的单链u队（SZ51scuPA）在体内外溶栓实验中也显示出比uPA更好的溶栓作用。为研制P一selectin导向的DSPA，我们构建了全长5251一DSRA、5251 Fab’一DSPA和SZ51seFv一DSPA的表达载体，并根据它们所表达的融合蛋白的抗原结合特性和溶栓能力，从中选出525，一OSPA作进一步研究;建立了4株高效表达5251一DS以（>3 ug/m1）的稳定表达细胞株;经proteinA亲和柱和凝胶柱分离后得到了纯化的融合蛋白:纯化的5251一OSPA与5251抗体具有相同的抗原结合特异性，两者的kd值类似。体外溶栓实验显示5251一OSPA与OSPA具有同等的溶栓作用。这些结果显示，我们成功地研制了P一selectin导向的OSPA，它保留了5251的抗原结合特性和OSPA溶栓功能。下一步我们将采用狗的股动脉血栓模型进行溶栓试验，以明确525，一OSPA在动物体内是否优于OSPA本身。之更多还原

【Abstract】 Thrombolytic diseases, such as acute myocardial infarction and stroke, are life-threatening diseases that affect millions of people each year. Therapeutic approaches to treat arterial thrombosis use plaminogen activators in combination with antiplatelet drugs and anticoagulants. Despite its tremendous clinical success, several major limitations of the therapy still need to be addressed. These include major bleeding, inadequate reperfusion, and thrombotic reocclusion. Thus, identification of new therapeutic targets and/or development of new generations of thrombolytic agents are the focal points in the field of fibrinolysis.The theme of my graduate study is to focus on regulation of fibrinolysis and molecular biology of a highly fibrin-selective plasminogen activator, DSPA, using genetic and biochemical approaches. My dissertation consists of four chapters: The chapter I describes the physiology and pathophysiology of the fibrinolysis system, focusing on regulation of fibrinolysis and progresses in thrombolytic agents. The chapter II describes the verification of negative regulatory roles of PAI-1 and TAFI on fibrinolysis in vivo using a newly developed rat model. The chapter III describes the biochemical characterization of DSPA, providing a molecular explanation for the fibrin-selectivity of DSPA. The chapter IV describes my initial attempt to develop a new-type of thrombolytic agent by targeting the highly-selective DSPA to freshly formed arterial thrombi.In the chapter I, we developed a batroxobin-induced rat lung fibrin deposition model. In this model, we observed a dramatic fibrin deposition in the lungs (elevated radioactivity) at 5 min following batroxobin administration, and the fibrin deposition in the lungs was clear up by endogenous fibrinolysis within 30 min (decreased radioactivity). We used this model to determine whether elevated levels of PAI-1 and TAFIa affect endogenous fibrinolysis. We either intravenously injected recombination PAI-1 or gave LPS to increase endogenous PAI-1 expression. In bothcases, the elevated level of PAI-1 prevented the reduction of radioactivity in the lungs at 30 min. Similarly, increased amounts of activated TAFI (TAFIa) through intravenous administration also prevented the fibrin clear -up in the lungs. These results provide direct evidence that an increase of PAI-1 and TAFIa in circulation impaired endogenous fmbinolysis, suggesting that by inhibiting PAI-1 or TAFI may be a new avenue for thrombolytic therapy.DSPA is a plasminogen activator originally isolated from the saliva of the vampire bat Desmodus rotundus. It consists of finger-, EGF-, kringle- and protease-domains, and exhibits a remarkable fibrin-specificity, which is several hundred-fold better than that of tPA. However, the molecular mechanism for such a highly fibrin-selectivity has not been elucidated, although previous studies suggest that the finger domain is involved in the fibrin selectivity. In the chapter II, I construct and express deletion mutant DSPA that only consists of the kringle- and pro tease-domains. I compare the enzymatic and fibrinolytic properties of DSPA and DSPK. In the absence of fibrin, the kinetic parameters of S2765 hydrolysis by DSPA and DSPK were similar. By contrast, in the presence of fibrin, the kcat/Km ratio of DSPA increases 3.9 fold, whereas the kcat/Km ratio of DSPK increases only 1.6 times. Furthermore, the fibrinolytic activities of DSPK decrease dramatically as compared those of DSPA. In a microtiter plate clot lysis assay, the potency of DSPK was decreased 48 times compare to that of DSPA. In 12S I-labled human plasma clot lysis assay, there was no thrombolytic activity was detected when DSPK was tested at concentrations up to 50 nM. The data indicate that DSPK retains a similar enzymatic activity towards substrate S2765 as with DSPA. However, DSPK lacks fibrin-stimulatory effects on its catalysis towards fibrin, which is in a striking contrast with DSPA. The lack of fibrin-stimulatory effect towards fibrin is not due to the inhibitory effect of PAI-1, beca更多还原