Characterization and Mechanism of Action of GPVI-targeting Platelet Aggregation Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

• Main Authors: Huai-Hsien Huang, 黃懷賢
• Other Authors: Tur-Fu Huang
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/47456192928462690969

碩士 === 國立臺灣大學 === 藥理學研究所 === 103 === Snake venoms profoundly affect platelet aggregation and hemostasis. By using column chromatography of CM Sephadex C-50 cationic exchanger and Sephadex G-75 gel filtration, a novel TMV inducer was purified from Trimeresurus mucrosquamatus snake venom. Under reducing conditions, it migrates as a protein with a mass about 45 KDa on SDS-PAGE. TMV inducer-induced platelet aggregation of human washed platelets and platelet-rich plasma in a concentration-dependent manner with EC50, 54.36 ± 0.92 and 13.8 ± 0.3 ng/ml, respectively. Aggrastat, anti-integrin αIIbβ3 mAb 7E3 significantly inhibited platelet aggregation caused by TMV inducer. Anti-GPIb mAb 6D1 and anti-GPIa/IIa mAb 6F1 only slightly inhibited platelet aggregation caused by TMV-inducer. An additive inhibitory effect was observed when 6D1and 6F1 were added. However, anti-GPVI mAb 326E12 and 342D7 profoundly inhibited platelet aggregation. In pull-down assay, biotinylated TMV inducer specifically bound to GPVI, but not GPIb or integrin α2. TMV inducer did not elicit agglutination of fixed platelets. PGE1 and BAPTA/AM completely inhibited platelet aggregation induced by TMV inducer. EDTA and indomethacin also caused profound inhibition. Syk inhibitor, piceatannol had higher inhibitory effect (80%). PLC inhibitor U73122, MEK inhibitor PD98059 and PI3-K inhibitor LY294002 totally inhibited TMV-inducer induced platelet aggregation without inhibiting platelet shape change. Src inhibitor, PP2 delayed platelet shape change, and profoundly inhibited aggregation. To confirm the downstream signal transduction, TMV-inducer induced a time-dependent tyrosine phosphorylation of a number of proteins similar to those activated by convulxin, including PLCγ2, PI3K, Syk, Src, Fyn and LAT. In mice models, TMV inducer did not significantly prolonged mice tail bleeding time. However, TMV inducer caused a dose-dependent decrease of platelet counts. Taken together, TMV inducer, a monomeric platelet aggregating inducer, activates platelets mainly through GPVI, leading to phosphorylation of many signal molecules, including PLCγ2, PI3K, Syk, Src, LAT and Fcγ, finally inducing the exposure of functional αIIbβ3 and Ca2+- dependent platelet aggregation. This novel snake venom protein may provide an useful tool for studies of GPVI and signaling mechanisms involved as well as for comparative study with other C-type lectin GPVI agonist, like convulxin or trowaglerix, and its association of platelet receptors, such as GPIb and GPIa/IIa. These studies would provide new insights regarding the molecular interaction of these GPVI agonists, providing clues for the design of GPVI antagonists, a new class of antithrombotic agent.