HEMOSTATIC EFFECTS OF FIBRINOLYTIC THERAPY

纤溶（血栓溶解）疗法已用于治疗动脉和 静脉血栓溶栓药物的给药，例如 链激酶、尿激酶或组织型纤溶酶原激活剂（t-PA）， 急性心肌梗死患者的标准治疗。巴赫因子 导致纤溶酶原裂解为纤溶酶。纤溶酶降解纤维蛋白 绞合并溶解血栓。此外，纤溶酶降解血小板 受体（GP Ib）和血浆蛋白，发挥关键作用， 止血，例如，纤维蛋白原、血管性血友病因子（vWF）。两者 分散血小板聚集体，通过溶解纤维蛋白， 纤溶酶诱导的全身效应可能部分地抑制了 附壁血栓形成和维持血管通畅后， 成功再通。非介入相关出血也可能是 与纤溶酶诱导的止血缺陷相关。分子 血小板粘附/聚集所采用的机制在不同条件下是不同的。 流动与静态条件：vWF启动血小板粘附， 内皮下的高流量条件下，通常遇到的， 动脉纤维蛋白原在低流速、接近停滞时很重要。 本项目的目的是调查止血的后果， 在体外条件下模拟血液流动的纤维蛋白溶解疗法， 容器。设计了一个实验模型，它允许：（a）实时 荧光血小板血栓形成的成像 在局部损伤部位，使用添加剂的规定流变条件 培养的人脐静脉内皮细胞（HUVEC）单层，和 (b)血浆蛋白降解程度的测量， 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，通过流式细胞术定量血小板受体。 t-PA变体，其更具纤维蛋白特异性或抗失活性 由纤溶酶原激活物抑制剂-1，将测试其 干扰血栓形成过程和全身副作用。的 内皮细胞分泌t-PA调节血栓形成的能力 形成和系统性缺陷将通过以下方式进行评价：（a）抑制EC t- （B）用反义寡核苷酸感染EC， 重组t-PA腺病毒载体。每种纤维蛋白溶解试剂或方法 参与生化反应将是计算机模拟使用 有限元方法来模拟血栓部位上的血流。 将测定纤溶酶和纤维蛋白原的浓度曲线和峰值。 与溶栓药物及室壁剪切率相关。 这些研究的结果将进一步加深我们对 血栓形成和血栓溶解过程之间的相互联系， 流动环境此外，所获得的信息可以形成 设计更好的分子或递送方法的基础， 将抑制血栓形成或支持血栓溶解， 血栓特异性和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.