The effects and action mechanisms of snake venom protein Agkistin on the endotoxemic syndromes in vivo

• Main Authors: Tun-Min Lu, 呂敦民
• Other Authors: Tur-Fu Huang
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/89268517918906444569

碩士 === 國立臺灣大學 === 藥理學研究所 === 102 === Abstract Many studies show that platelets play an important role in inflammation. The platelet receptor GPIb-IX-V complex not only involves in initial platelet adhesion at high shear stress but also interacts with integrin Mac-1 in leukocytes and P-selectin in platelets and endothelial cells. This multiple interactions suggest that GPIb-IX-V complex may play an important role under inflammatory conditions. Agkistin, a heterodimer protein purified from the snake venom of Agkistrodon acutus and its amino acid sequencing of α- and β-subunits, are highly homologous to those of C-type lectin GPIb-binding proteins. Agkistin specifically inhibits ristocetin induced GPIb-dependent platelet aggregation in the presence of vWF in human platelet suspension or in platelet-rich plasma. In this study, we used Agkistin, a GPIb antagonist, to evaluate its anti-inflammatory effects in the LPS-induced sepsis model. We found that administration of Agkistin reduced mortality of endotoxemia in vivo. However, LPS-stimulated cytokine release such as TNF-α and IL-6 was not significantly inhibited by Agkistin-pretreatment, and Agkistin treatment even caused a more marked thrombocytopenic effect. Administration of Agkistin in normal mice, we found that it caused a rapid reduction in platelet count, while it did not cause a significant change in the counts of white blood cells and red blood cells. We further explored the mechanism of Agkistin-induced thrombocytopenia in mice. We tested Agkistin with mice platelet-rich plasma; however it did not cause platelet aggregation. Moreover, ristocetin, an inducer usually used in GPIb studies in humans, did not elicit the platelet agglutination in mice. Because ristocetin lost efficacy in mice, we used Agglucetin instead, a VWF independent inducer also purified from the Agkistrodon acutus snake venom, to activate platelet in mice platelet-rich plasma. In both of human platelet suspension and platelet-rich plasma Agkistin was shown to inhibit Agglucetin-induced platelet aggregation, but in mice platelet-rich plasma Agglucetin only showed slight activation, and this activation effect was still inhibited by pre-incubation of Agkistin. We also tested the effects of Agkistin in rat platelet-rich plasma. It did not cause platelet aggregation like in mice PRP. Moreover, Agglucetin did not trigger platelet agglutination in rat PRP. In our in vitro studies, we exclude the interaction of blood cells and not carry out in the vasculature, suggesting that the Agkistin-induced thrombocytopenia may involve in interaction with receptors or components present on other cells. In conclusion, the proper inhibition of platelets may present benefit in inflammatory disease, but the total depletion of platelet would increase the risk of bleeding. The specific blockage of interactions between neutrophils and platelets may offer a new strategy of therapy towards sepsis-induced inflammatory disease.