Kooistra T, Toet K, Kluft C, VonVoigtlander P F, Ennis M D, Aiken J W, Boadt J A, Erickson L A
IVVO-TNO Gaubius Laboratory, Leiden, The Netherlands.
Biochem Pharmacol. 1993 Jul 6;46(1):61-7. doi: 10.1016/0006-2952(93)90348-z.
In our search for compounds that can stimulate endogenous fibrinolysis, we have found that certain triazolobenzodiazepines enhance the production of tissue-type plasminogen activator (t-PA) by vascular endothelial cells maintained in vitro, with no or even a lowering effect on plasminogen activator inhibitor type-1 (PAI-1) production. The most active compounds tested, U-34599, U-46195 and U-51477, were studied in more detail and showed a time- and dose-dependent increase in the production of t-PA by human umbilical vein endothelial cells. At optimal stimulatory concentrations (about 10 microM), the three compounds stimulated t-PA expression about 2-fold after 24 hr and maximally about 4-fold after 48 hr of incubation; this maximal increase in t-PA synthesis was sustained at prolonged incubations of 72 or 96 hr. The triazolobenzodiazepine effects on t-PA production were accompanied by parallel increases in t-PA mRNA levels, without marked changes in PAI-1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA concentrations. Numerous analogues of the three lead compounds were then tested to determine the relationship between benzodiazepine structure and the ability to stimulate t-PA production. No positive correlation was found between the ability of the various triazolobenzodiazepines to stimulate t-PA production and their affinity for the benzodiazepine receptor. In agreement with this, no specific binding of [3H]flunitrazepam, a specific ligand for benzodiazepine receptors, to endothelial cell membrane preparations was observed. Thus, it is unlikely that the triazolobenzodiazepines act through central-type benzodiazepine receptors to stimulate t-PA production. Similarly, no evidence was found for the presence of peripheral-type benzodiazepine receptors on endothelial cell membranes. The ability of the benzodiazepines to stimulate t-PA production, however, appeared to be related to their platelet-activating factor (PAF) antagonist activity. Despite this finding, several non-benzodiazepine PAF antagonists did not stimulate t-PA production. While the precise mechanism of action is not yet clear, selected benzodiazepine analogues possessing PAF antagonist activity stimulate the production of t-PA by endothelial cells in vitro.
在寻找能够刺激内源性纤维蛋白溶解的化合物的过程中，我们发现某些三唑并苯二氮䓬可增强体外培养的血管内皮细胞组织型纤溶酶原激活物（t-PA）的产生，而对1型纤溶酶原激活物抑制剂（PAI-1）的产生没有影响甚至有降低作用。对测试的活性最强的化合物U-34599、U-46195和U-51477进行了更详细的研究，结果显示人脐静脉内皮细胞产生t-PA的量呈时间和剂量依赖性增加。在最佳刺激浓度（约10微摩尔）下，这三种化合物在孵育24小时后可刺激t-PA表达约2倍，在孵育48小时后最大可刺激约4倍；t-PA合成的这种最大增加在72或96小时的延长孵育中得以维持。三唑并苯二氮䓬对t-PA产生的影响伴随着t-PA mRNA水平的平行增加，而PAI-1或甘油醛-3-磷酸脱氢酶（GAPDH）mRNA浓度没有明显变化。然后测试了这三种先导化合物的众多类似物，以确定苯二氮䓬结构与刺激t-PA产生能力之间的关系。未发现各种三唑并苯二氮䓬刺激t-PA产生的能力与其对苯二氮䓬受体的亲和力之间存在正相关。与此一致的是，未观察到苯二氮䓬受体的特异性配体[3H]氟硝西泮与内皮细胞膜制剂有特异性结合。因此，三唑并苯二氮䓬不太可能通过中枢型苯二氮䓬受体来刺激t-PA的产生。同样，也没有证据表明内皮细胞膜上存在外周型苯二氮䓬受体。然而，苯二氮䓬刺激t-PA产生的能力似乎与其血小板活化因子（PAF）拮抗剂活性有关。尽管有这一发现，但几种非苯二氮䓬类PAF拮抗剂并未刺激t-PA的产生。虽然确切的作用机制尚不清楚，但具有PAF拮抗剂活性的特定苯二氮䓬类似物可在体外刺激内皮细胞产生t-PA。
