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 produ ction of tissue-type plasminogen activator (t-PA) by vascular endothel ial cells maintained in vitro, with no or even a lowering effect on pl asminogen activator inhibitor type-1 (PAI-1) production. The most acti ve compounds tested, U-34599, U-46195 and U-51477, were studied in mor e detail and showed a time- and dose-dependent increase in the product ion of t-PA by human umbilical vein endothelial cells. At optimal stim ulatory concentrations (about 10 muM), the three compounds stimulated t-PA expression about 2-fold after 24 hr and maximally about 4-fold af ter 48 hr of incubation; this maximal increase in t-PA synthesis was s ustained at prolonged incubations of 72 or 96 hr. The triazolobenzodia zepine effects on t-PA production were accompanied by parallel increas es in t-PA mRNA levels, without marked changes in PAI-1 or glyceraldeh yde-3-phosphate dehydrogenase (GAPDH) mRNA concentrations. Numerous an alogues of the three lead compounds were then tested to determine the relationship between benzodiazepine structure and the ability to stimu late t-PA production. No positive correlation was found between the ab ility of the various triazolobenzodiazepines to stimulate t-PA product ion and their affinity for the benzodiazepine receptor. In agreement w ith this, no specific binding of H-3!flunitrazepam, a specific ligand for benzodiazepine receptors, to endothelial cell membrane preparatio ns was observed. Thus, it is unlikely that the triazolobenzodiazepines act through central-type benzodiazepine receptors to stimulate t-PA p roduction. Similarly, no evidence was found for the presence of periph eral-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) antag onist activity. Despite this finding, several non-benzodiazepine PAF a ntagonists did not stimulate t-PA production. While the, precise mecha nism of action is not yet clear, selected benzodiazepine analogues pos sessing PAF antagonist activity stimulate the production of t-PA by en dothelial cells in vitro.