HEMOSTATIC EFFECTS OF FIBRINOLYTIC THERAPY

纤溶（溶栓）疗法已用于治疗动脉和 静脉血栓形成。给予溶栓剂，例如 链激酶、尿激酶或组织型纤溶酶原激活剂 (t-PA) 是 急性心肌梗死患者的标准治疗。巴赫代理人 导致纤溶酶原裂解为纤溶酶。纤溶酶降解纤维蛋白 绞碎并溶解血栓。此外，纤溶酶还能降解血小板 受体 (GP Ib) 和血浆蛋白在 止血，例如。纤维蛋白原、血管性血友病因子 (vWF)。两者都 通过纤维蛋白溶解，血小板聚集体分散， 纤溶酶诱导的全身效应可能部分解释了抑制 附壁血栓形成和术后血管通畅的维持 成功的再通。非干预相关的出血也可能是 与纤溶酶诱导的止血缺陷有关。分子 血小板粘附/聚集的机制在不同情况下是不同的 流动与静态条件：vWF 启动血小板粘附 内皮下细胞在高流量条件下通常会遇到 动脉。纤维蛋白原在低流速、接近停滞状态下很重要。 该项目的目的是调查止血后果 在模拟血流的体外条件下进行纤溶治疗 船只。设计了一个实验模型，允许：（a）实时 全血荧光血小板血栓形成成像 在局部损伤部位确定流变条件，并添加药物 培养的人脐静脉内皮细胞（HUVEC）单层，以及 (b) 血浆蛋白降解程度的测量和 血小板受体。纤维蛋白原降解产物和 vWF 多聚体将 通过免疫印迹定量，血小板受体通过流式细胞术定量。 t-PA 变体，更具纤维蛋白特异性或抗失活 通过纤溶酶原激活剂抑制剂-1，将测试其 干扰血栓形成过程和全身副作用。的 内皮细胞 (EC) 分泌的 t-PA 调节血栓的能力 将通过以下方式评估形成和系统缺陷： (a) 抑制 EC t- 通过反义寡核苷酸合成 PA，以及 (b) 用 重组t-PA腺病毒载体。每种纤溶药物或方法 参与的生化反应将使用计算机模拟 有限元方法对血栓部位的血流进行建模。 纤溶酶和纤维蛋白原的浓度分布和峰值将是 与溶栓剂和壁剪切率相关。 这些研究的结果将进一步加深我们对 血栓形成和溶栓过程之间的动态相互联系 流动环境。此外，所获得的信息可能形成 为设计更好的分子或递送方法奠定了基础 会抑制血栓形成或支持溶栓作用更大 血栓特异性好，对止血的不良影响较小。

Fibrinolytic (thrombolytic) therapy has been used in treating arterial and venous thrombosis. Administration of thrombolytic agents, such as streptokinase, urokinase or tissue-type plasminogen activator (t-PA), is standard treatment for acute myocardial infarction patients. Bach agent results in cleavage of plasminogen to plasmin. Plasmin degrades the fibrin strands and dissolves thrombi. In addition, plasmin degrades platelet receptors (GP Ib) and plasma proteins that play a pivotal role in hemostasis, eg. fibrinogen, von Willebrand factor (vWF). Both the dispersion of platelet aggregates, through the lysis of fibrin, and the plasmin-induced systemic effects may partly account for inhibition of mural thrombus formation and maintenance of the vessel patency after successful recanalization. Non intervention-related hemorrhage may be also associated with the plasmin-induced hemostatic defects. Molecular mechanisms employed for platelet adhesion/aggregation are different under flow vs. static conditions: vWF initiates platelet adhesion to subendothelium under the high flow conditions typically encountered in arteries. Fibrinogen is important at low flow rates, close to stasis. The aim of this project is to investigate the hemostatic consequences of fibrinolytic therapy under in vitro conditions that mimic blood flow in a vessel. An experimental model is designed that allows both: (a) real-time imaging of fluorescent platelet thrombus formation from whole blood under defined rheologic conditions, with added agents, at localized injury sites of cultured human umbilical vein endothelial cell (HUVEC) monolayers, and (b) measurement of the extent of degradation of plasma proteins and platelet receptors. Fibrinogen degradation products and vWF multimers will be quantified by immunoblotting, and platelet receptors by flow cytometry. t-PA variants, which are more fibrin-specific or resistant to inactivation by the plasminogen activator inhibitor-1, will be tested for their interference with the thrombotic process and systemic side effects. The ability of endothelial cell (EC)-secreted t-PA to regulate thrombus formation and systemic defects will be evaluated by: (a) inhibiting EC t- PA synthesis by antisense oligonucleotides, and (b) infecting ECs with a recombinant t-PA adenovirus vector. Each agent or method of fibrinolysis with participating biochemical reactions will be computer-simulated using finite element methods to model blood flow over a thrombotic site. Concentration profiles and peak values of plasmin and fibrinogen will be correlated with thrombolytic agent and wall shear rate. The results of these studies will further our comprehension of the interconnection between thrombotic and thrombolytic processes in a dynamic flow environment. Moreover, the information obtained may form the foundation for the design of better molecules or methods of delivery that will either inhibit thrombosis or support thrombolysis with greater thrombus specificity and with less adverse effects on hemostasis.