| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 91 === By using column chromatography of CM-sephadex C-50 and Mono-S ionic exchanger in sequence, a novel snake venom protein, echiquatin, was purified from Echis multisquamatus snake venom. Echiquatin inhibited ristocetin-induced platelet agglutination mediated by von Willebrand factor (vWF)-GPIb interaction. Echiquatin was homogenous as judged by SDS-PAGE. Its apparent molecular weight was estimated to be 30,800 daltons as measured by SDS-PAGE. Upon reduction by 2% beta-mercaptoethanol, echiquatin was shown to contain two subunits with molecular weight of 14,800 (alpha-chain) and 13,100 (beta-chain) daltons, respectively. It is devoid of enzymatic activity as assayed by fibrinogenolysis and azocaseinolysis.
Echiquatin concentrationdependently inhibited ristocetin (1 mg/ml)induced platelet agglutination in the presence of plasma (20ul) as the source of vWF in platelet suspension. Echiquatin (1.5ug/ml, 49 nM) completely inhibited ristocetin-induced platelet agglutination. The antiplatelet activity of echiquatin was not inhibited by preincubating it with 10 mM EDTA, suggesting that echiquatin is not a venom metalloproteinase and its inhibitory effect is not bivalentcation dependent. Echiquatin only slightly inhibited collagen (10ug/ml), and TRAP (20uM) induced platelet aggregation, but it obviously prolonged the latent period in triggering aggregation. Echiquatin did not inhibit thrombin (0.1, 0.03 U/ml)induced platelet aggregation, but it also prolonged the latent period in triggering aggregation. Even at a high concentration of 20ug/ml, echiquatin had no significant effect on platelet aggregation caused by U46619 (1uM), and ADP (20uM). Using flow cytometry, echiquatin blocked the binding of AP1 and 6D1, mAbs against platelet GPIb, to platelet. However, it showed no significant effects on the binding of mAb, raised against platelet GPIb (CLB-MB45), GPIX, GPIIb or GPIa/IIa (6F1).
Echiquatin dosedependently prolonged the bleeding time of ICR mice after intravenous injection. When a dose of echiquatin (0.35ug/mouse) given, a decrease of platelet count (39.98%) was observed at the first 5 min, however the platelet count gradually returned to control level within 6 hr. Platelet agglutination was observed with blood smears obtained from the coincubation of the whole blood with echiquatin. Echiquatin concentrationdependently induced platelet agglutination in plateletrich plasma. Echiquatin (1.25ug/ml) induced maximum platelet agglutination, and its activity was inhibited by AP1 and 6D1, mAbs against platelet GPIb, but not by mAbs of SZ2, 6F1 and 7E3, raised against GPIb, GPIa/IIa or GPIIb-IIIa, respectively. In the presence of plasma, echiquatin induced platelet agglutination of platelet suspension, suggesting that some plasma factor(s) are involved in triggering platelet agglutination in cooperation with echiquatin. Using ELISA technique, we demonstrated that echiquatin specifically binds to IgMkappa, but not IgMlambda.
In conclusion, echiquatin inhibits ristocetin-induced agglutination in human platelet suspension, but induces agglutination in plateletrich plasma. Binding study with flow cytometry has demonstrated that echiquatin selectively inhibits AP1 and 6D1 binding to platelets, suggesting that echiquatin selectively binds to platelet surface receptorGPIb, thereby blocking the interaction between GPIb and vWF. According to ELISA experiment, echiquatin binds to plasma IgMkappa, a pentamer molecule, thus leading to a complex formation among platelet GPIb, echiquatin and IgMkappa and subsequently platelet agglutination. The phenomena may explain the in vivo thrombocytopenia effect of a GPIb binding protein, echiquatin. The elucidation of the structureactivity relationship of echiquatin and other GPIb binding proteins at a molecular level would be helpful for developing a new group of antithrombotic drugs.
|
|---|
