Triazolobenzodiazepines: a new class of stimulators of tissue-type plasminogen activator synthesis in human endothelial cells.

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.