calcimycin and brazilin

Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 (6H)-tetrol), the major component of Caesalpinia sappan L., was reported to show antiplatelet activity through the inhibition of phospholipase A2 (PLA2) activity and the increase in intracellular free Ca2+ concentration ([Ca2+]i). To search more potential antiplatelet agent, brazilin derivatives were synthesized and examined for their effects on the platelet aggregation. Among those compounds, BRX-018, (6aS,cis)-Malonic acid 3-acetoxy-6a9-bis-(2-methoxycarbonyl-acetoxy)-6,6a,7,11b-tetrahydro-indeno[2,1-c]chromen-10-yl ester methylester, was confirmed as one of the potential antiplatelet agents. In the present study, we investigated the antiplatelet mechanism of BRX-018. BRX-018 inhibited the thrombin-, collagen-, and ADP-induced rat platelet aggregation in a concentration-dependent manner, with IC50 values of 35, 15, and 25 microM, respectively. BRX-018 also inhibited thrombin-induced dense granule secretion, thromboxane A2 (TXA2) synthesis, and [Ca2+]i elevation in platelets. BRX-018 was also found to inhibit A23187-induced [Ca2+]i and aggregation in the presence of apyrase (ADP scavenger) but not in the presence of both apyrase and indomethacin (a specific inhibitor of cyclooxygenase, COX). Although BRX-018 significantly inhibited arachidonic acid (AA)-induced aggregation and TXA2 synthesis, it had no significant inhibitory effect on cyclooxygenase activity in vitro. In contrast, BRX-018 inhibited the activity of purified PLA2. Dixon plot showed that this inhibition was mixed type with an inhibition constant of Ki=23 microM. Taken together, the present study suggests that BRX-018 may be a promising antiplatelet agent and that its antiplatelet activity may be based on the inhibitory mechanisms on TXA2 synthesis in stimulated platelets.

Topics: Adenosine Diphosphate; Animals; Arachidonic Acid; Benzopyrans; Blood Platelets; Calcimycin; Calcium; Cattle; Cell Degranulation; Collagen; In Vitro Techniques; Ionophores; Male; Malonates; Phospholipases A; Phospholipases A2; Platelet Aggregation Inhibitors; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Swine; Thrombin; Thromboxane B2

Brazilin increased [3H]2-deoxyglucose uptake in isolated rat epididymal adipocytes. The fact that calcium may be required for the stimulatory effects of insulin on glucose transport suggests that brazilin might also require calcium for its glucose transport-stimulating action. Changes in the concentration of extracellular calcium had no significant effect on brazilin-induced glucose transport. Nifedipine and verapamil decreased brazilin-induced glucose transport, and quin2-AM abolished the effect of brazilin on glucose transport. A23187, however, showed no effect on brazilin action. 45Ca2+ uptake into adipocytes was not influenced by brazilin treatment, and trifluoperazine significantly inhibited the effect of brazilin on glucose transport. These data suggest that calmodulin and the maintenance of the intracellular calcium concentration, rather than an increase in it, may be essential for the stimulatory action of brazilin on glucose transport.

Topics: Adipocytes; Animals; Benzopyrans; Biological Transport; Calcimycin; Calcium; Calcium Channel Blockers; Calmodulin; Cell Membrane; Dose-Response Relationship, Drug; Epididymis; Glucose; Hypoglycemic Agents; Insulin Antagonists; Male; Nifedipine; Rats; Rats, Sprague-Dawley; Trifluoperazine; Verapamil