蛇毒蛋白Agkistin對於活體內毒素血症之效應及機轉之探討

Abstract

許多研究顯示血小板在發炎反應中扮演著重要的角色，血小板醣蛋白受體GPIb-IX-V不只參與了止血機轉初期的黏附作用，而且還與leukocytes所表現的integrin Mac-1以及內皮細胞和血小板所表現的P-selectin產生交互作用。血小板醣蛋白受體GPIb-IX-V可能在發炎反應中扮演著重要的角色。 Agkistin是一個從百步蛇毒中純化而得的C型類凝血素蛋白家族之成員，其具有典型的α及β次單元。無論在含有vWF之血小板懸浮液或是富含血小板之血漿中，Agkistin皆能專一性的抑制由ristocetin所引發的醣蛋白Ib相關之血小板凝集。我們藉由這種醣蛋白Ib抑制劑Agkistin去評估其對於LPS所引發之敗血症之影響。我們觀察到投予Agkistin可以降低動物模式中LPS引發之敗血症的死亡率。然而在發炎相關的細胞激素釋放方面如TNF-α及IL-6，Agkistin並沒有顯著的抑制現象。另外，投予Agkistin會呈現更為顯著的血小板低下現象。Agkistin直接投予在正常的小鼠中，亦會發生血小板低下現象，而在其他的血球方面如白血球、紅血球並沒有顯著的影響。 我們進一步探討Agkistin引發之血小板低下的機轉，Agkistin在小鼠富含血小板血漿中沒有看到血小板凝集的現象。而且經常用於人類GPIb試驗的活化劑ristocetin也無法引起凝集現象。因此我們使用了Agglucetin 替代，Agglucetin為一種vWF非依賴性的GPIb活化劑，也是純化自百步蛇毒，希望藉此作為小鼠模式之活化劑。不論在人類血小板懸浮液或富含血小板血漿中Agkistin 皆可以有效的抑制由Agglucetin所引起的血小板凝集現象，不過在於小鼠富含血小板血漿中Agglucetin 僅呈現微小的活化現象，這一反應也可以藉由加入Agkistin 所抑制。我們也在大鼠富含血小板血漿中觀察Agkistin 及Agglucetin 之反應，Agkistin 如同在小鼠一樣沒有造成凝集現象，然而在大鼠中Agglucetin也無法產生凝集反應。在以上之試管試驗中，我們離開了血管的生理環境，且排除了血球細胞的交互作用，Agkistin所引發的血小板低下可能與血管中其他細胞所表現的受體產生的交互作用有關。 最後，適當的抑制血小板可能對於發炎性疾病是有幫助的，但是完全的排除血小板又會增加副作用的風險，如出血。專一性的阻斷血小板與中性球的交互作用，可能是對於敗血症所引起複雜的發炎性疾病一種新的治療策略。

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.