thromboxane-b2 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 (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 (6 H)-tetrol) inhibited thrombin-,collagen- and ADP-induced aggregation of washed rat platelets. Thrombin- and collagen-induced ATP release were also inhibited by brazilin in a concentration-dependent manner. Brazilin inhibited the formation of platelet thromboxane A2 caused by thrombin, whereas it had no effect on the prostaglandin D2 formation. Brazilin inhibited [3H]-arachidonic acid liberation from membrane phospholipids of thrombin-stimulated platelets. Brazilin inhibited the rise of intracellular free calcium caused by thrombin. These results indicate that the inhibition of phospholipase (PLA2) activity and [Ca2+]i elevation might be at least a part of antiplatelet mechanism of brazilin.

Topics: Adenosine Triphosphate; Animals; Arachidonic Acid; Benzopyrans; Blood Platelets; Calcium; Female; In Vitro Techniques; Phospholipases A; Phospholipases A2; Platelet Aggregation; Prostaglandin D2; Rats; Rats, Sprague-Dawley; Thromboxane B2