HEMOSTATIC EFFECTS OF FIBRINOLYTIC THERAPY

纤溶治疗的止血作用

• 批准号: 2857850
• 负责人: Barbara Rita Alevriadou
• 金额: $ 12.21万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2857850
• 关键词: blood chemistry; digital imaging; drug screening /evaluation; fibrinogen; fibrinolysis; fibrinolytic agents; fibrinolytic therapy; flow cytometry; fluorescence microscopy; hemostatics; human tissue; plasminogen activator inhibitors; platelet aggregation; protein degradation; streptokinase; thrombosis; tissue /cell culture; transfection /expression vector; umbilical cord; vascular endothelium permeability; video microscopy; von Willebrand factor; western blottings

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.

纤溶(溶栓)疗法已被用于治疗动脉和 静脉血栓形成。溶栓剂的使用，如 链激酶型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 导致纤溶酶原裂解为纤溶酶。纤溶酶降解纤维蛋白 缠绕并溶解血栓。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)。这两个 通过纤维蛋白的分解分散血小板聚集体，以及 纤溶酶诱导的全身性效应可能是抑制 附壁血栓的形成及术后血管通畅的维持 成功的再通畅。非介入相关出血也可能是 与纤溶酶引起的止血缺陷有关。分子 血小板黏附/聚集的机制在以下情况下有所不同 流动与静止状态：VWF启动血小板黏附 在高流量条件下的内皮下，通常在 动脉。纤维蛋白原在接近停滞的低流速下很重要。 该项目的目的是调查 体外条件下的纤溶治疗，模拟血液流动 船只。设计了一个实验模型，它允许：(A)实时 全血荧光血小板血栓形成的成像研究 在局部损伤部位使用添加试剂的确定的流变学条件 培养的人脐静脉内皮细胞(HUVEC)单层 (B)测量血浆蛋白质的降解程度和 血小板受体。纤维蛋白原降解产物和vWF多聚体将 用免疫印迹法定量，用流式细胞仪检测血小板受体。 T-PA变异体，它更具纤维蛋白特异性或抵抗失活 纤溶酶原激活物抑制物-1，将检测其 干扰血栓形成过程和全身副作用。这个 内皮细胞分泌的t-PA调节血栓的能力 将通过以下方式评估形成和系统性缺陷：(A)抑制EC-t- 通过反义寡核苷酸合成PA，以及(B)感染内皮细胞 重组t-PA腺病毒载体。每种纤溶试剂或方法 有参与的生化反应将用计算机模拟使用 用有限元方法模拟血栓形成部位的血流。 纤溶酶和纤维蛋白原的浓度分布和峰值将 与溶栓剂、室壁切变率相关。 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输送方法奠定了基础 将抑制血栓形成或支持溶栓治疗 血栓特异性好，止血不良反应少。