| Summary: | 碩士 === 臺灣大學 === 藥理學研究所 === 98 === By using column chromatography of Sephadex G-75, FPLC Superdex75 10/30 GL, and FPLC Mono-S 5/50 GL in sequence, a novel snake venom protein ,P2F4 was purified form Deinagkistrodon acutus venom. It can inhibit ristocetin-induced aggregation in human platelet suspension and human platelet- rich plasma in a concentration dependent manner. P2F4 contains two subunits with molecular weight 10kDa and 11kDa,respectively. Its native molecular was estimated to be 21kDa by gel filtration. The reduced and alkylated α and β subunits were obtained and sequenced, showing the N-terminal sequence of P2F4 was partially similar to those of C-type lectin. However, the N-terminal sequence of P2F4 differed from the conserved sequence (CXXXWXXXXXXC ) of known C-type lectins. The N-terminal sequence of P2F4 was similar to Botrocetin (α50%；β35%)and Bitiscetin (α50%；β50%) two vWF-mediating agonist.
P2F4 exhibits high potency in blocking ristoction-induced aggregation. Its IC50 was estimated to be 2.3nM and 23nM in human platelet-rich plasma and in platelet suspension, respectively. P2F4 inhibited agglucetin-induced aggregation with higher concentration required in blocking ristocetin-induced aggregation. P2F4 can not reverse agglucetin-induced aggregation but can prevent further progression of platelet aggregation. On the other hand, P2F4 can reverse ristocetin- induced aggregation. P2F4 did not inhibit platelet aggregation induced by ADP, or thrombin, P2F4 at higher concentration (920nM) had slight inhibition on collagen-induced aggregation .
In flow cytometric assay, P2F4 specifically interrupted GPⅠb mab ( AP1 and SZ2) binding to GPⅠb, while P2F4 did not interfere with the binding of other receptor specific antibody such as 11A12(GPⅥ), 204.11(GPⅥ), 6F1(α2β1), or 7E3(αⅡbβ3). P2F4(4.6nM) reduced TxA2 formation caused by ristocetin about 50% and reduced agglucetin-induced P-selectin expression. As P2F4 was administered intravenously it prolonged the tail bleeding time, and did not cause thrombocytopenia syndrome within 30 min.
Taken together, P2F4 is a highly specific GPⅠb antagonist. It is very unique that it exhibits a higher activity in platelet-rich plasma than in platelet suspension. Whether it is a potent antithrombotic agent in vivo needs further investigation.
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