Brazilin isolated from Caesalpinia sappan L. acts as a novel collagen receptor agonist in human platelets.
BACKGROUND
Brazilin, isolated from the heartwood of Caesalpinia sappan L., has been shown to possess multiple pharmacological properties.


METHODS
In this study, platelet aggregation, flow cytometry, immunoblotting analysis, and electron spin resonance (ESR) spectrometry were used to investigate the effects of brazilin on platelet activation ex vivo. Moreover, fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used in in vivo study.


RESULTS
We demonstrated that relatively low concentrations of brazilin (1 to 10 μM) potentiated platelet aggregation induced by collagen (0.1 μg/ml) in washed human platelets. Higher concentrations of brazilin (20 to 50 μM) directly triggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an antagonist of ADP). It was not inhibited by yohimbine (an antagonist of epinephrine), by SCH79797 (an antagonist of thrombin protease-activated receptor [PAR] 1), or by tcY-NH2 (an antagonist of PAR 4). Brazilin did not significantly affect FITC-triflavin binding to the integrin αIIbβ(3) in platelet suspensions. Pretreatment of the platelets with caffeic acid phenethyl ester (an antagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies raised against collagen receptor glycoprotein VI and integrin α2β(1), respectively, abolished platelet aggregation stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the phosphorylation of phospholipase C (PLC)γ2 and Lyn, which were significantly attenuated in the presence of JAQ1 and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. An in vivo mouse study showed that brazilin treatment (2 and 4 mg/kg) significantly shortened the occlusion time for platelet plug formation in mesenteric venules.


CONCLUSIONS
To the best of our knowledge, this study provides the first evidence that brazilin acts a novel collagen receptor agonist. Brazilin is a plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.
Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd, Taipei 11101, Taiwan.
Yi
Y
Chang
Steven Kuan-Hua
SK
Huang
Wan-Jung
WJ
Lu
Chi-Li
CL
Chung
Wei-Lin
WL
Chen
Shun-Hua
SH
Lu
Kuan-Hung
KH
Lin
Joen-Rong
JR
Sheu
D000818:Animals; D001578:Benzopyrans / Q000737:chemistry / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology; D029911:Caesalpinia / Q000737:chemistry; D003094:Collagen / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D038981:Receptors, Collagen / Q000819:agonists / Q000378:metabolism; D013927:Thrombosis / Q000188:drug therapy*
D001578:Benzopyrans; D010975:Platelet Aggregation Inhibitors; D038981:Receptors, Collagen; D003094:Collagen; C044362:brazilin