具血小板醣蛋白Ib抑制性質之蛇毒C型凝集素Agkistin造成血小板低下機制之探討

The investigation of thrombocytopenia caused by Agkistin, a platelet glycoprotein Ib inhibitor with C-type lectin-like structure

10.6342/NTU201702327

陳柏榮

蛇毒C型凝集素 ； 血小板醣蛋白Ib ； agkistin ； 血小板低下 ； 肝清除 ； Ashwell-Morell受體 ； Snake C-type lectin ； glycoprotein Ib ； agkistin ； thrombocytopenia ； liver clearance ； Ashwell-Morell Receptor

臺灣大學藥理學研究所學位論文

2017年

碩士

黃德富

英文

血小板在止血功能中扮演不可或缺的角色，在一些病理因素之下，異常的血小板活化可能形成具有危險性的血栓。在往年重大疾病中，心肌梗塞與缺血性中風常伴隨著高度致死率。因此，抗血小板藥物常用來預防日常生活或心血管手術後異常血栓的生成。然而，臨床報告指出目前市面的抗血小板用藥具有增加出血的風險，因此在安全性的考量下，能抑制血栓生成且又不容易造成異常出血成為新一代抗血小板藥物的首要指標。血小板醣蛋白Ib在血小板附著與活化上為一關鍵因子，尤其在高剪切速率之下其功能愈加彰顯，因為這種對於剪切速率的選擇性，我們可以預測醣蛋白Ib的拮抗相較於健康血管，在病理情況下的動脈血管能發揮更有針對性的效果。因此，血小板醣蛋白Ib拮抗劑是一個具有前瞻性的發展目標。 近年來，數種拮抗醣蛋白Ib的蛇毒C型凝集素已透過試管試驗以及活體實驗證實其效果。意外的是，注射這類蛇毒C型凝集素進入小動物卻會造成血小板低下症狀的出現，背後的原因仍然在探索中。這篇文章將探討從大陸百步蛇Agkistrodon acutus純化出來的蛇毒C型凝集agkistin，在試管試驗中證實具有抑制醣蛋白Ib造成的血小板凝集，然而，在小鼠尾靜脈注射下卻發現會造成快速且持續的血小板低下症狀。在探討這個機制的過程中，我們評估了各式可能的因子，包含後天免疫、巨噬細胞、細胞凋亡以及肝細胞吞噬等可能性。我們發現agkistin會促使肝細胞透過Ashwell-Morell受體吞噬血小板。先前的文獻曾指出在一些具有拮抗醣蛋白Ib抗體的免疫血小板低下患者中發現其體內的血小板進行去唾液醣酸(sialic acid)後被肝細胞辨認與清除。而這些拮抗醣蛋白Ib的抗體即是造成血小板活化與醣蛋白Ibα次單元去唾液醣酸化的背後推手。然而，我們發現agkistin並不會造成血小板活化或使醣蛋白Ibα去唾液醣酸化，但發現agkistin和Ashwell-Morell受體具有高度結合能力，以及透過金屬依賴性蛋白酶kistomin切除醣蛋白Ibα次單元會抑制agkistin造成的血小板低下。這些結果顯示Ashwell-Morell受體和醣蛋白Ibα次單元在血小板清除的過程中是不可缺的。我們推測agkistin在Ashwell-Morell受體和醣蛋白Ibα次單元間扮演著「橋樑」的要角，加強肝細胞對血小板的辨識與吞噬，惟更多的結構與活性的關聯性需要進一步去釐清。 總言之，我們提出了一個agkistin對於促進肝細胞清除血小板機制的說法，這些成果對於降低出血風險的新穎血小板醣蛋白Ib拮抗劑的研發提供更多可靠的資訊。

Platelets act as an indispensable role in hemostasis. Abnormal activation of platelets may lead to formation of threatening thrombus under pathological conditions. Myocardial infarction and ischemic stroke are in high lethal rates each year. Therefore, several antiplatelet agents are indicated for preventing abnormal thrombus formation in daily life or after coronary surgery. However, current anti-platelet agents have the risk of bleeding. For safety issue, thrombus prevention with lower bleeding risk is the concerning issue of new generation antiplatelet agents. Platelet glycoprotein Ib is a key receptor in platelet adhesion, and activation during vascular injury with the immobilized vWF, especially under high shear stress. Therefore, the blockade of GPIb-mediated platelet activation would interfere more with platelet deposition in diseased arteries, than in healthy blood vessels, thus preventing thrombosis without affecting significantly physiologic hemostasis. Recently, several snake C-type lectins (snaclecs) have been reported to inhibit platelet GPIb both in vitro and in vivo. Surprisingly, intravenous injection of these snaclecs causes thrombocytopenia in small animals. The causative reasons are still under investigation. This report focuses on agkistin, a snaclec purified from venom of China Agkistrodon acutus, inhibits GPIb-mediated platelet agglutination in vitro. However, it induces a rapid and sustained thrombocytopenia upon intravenous injection to mice. To figure out the mechanism of the induced thrombocytopenia, we evaluated the causative factors including adaptive immune system, macrophage, apoptosis, and hepatocyte etc. Our results demonstrated that agkistin could promote the platelet ingestion by hepatocytes through their Ashwell-Morell Receptor (AMR). Previous studies indicated that hepatocytes recognize and internalize desialylated platelets in several immune thrombocytopenia (ITP) patients with anti-GPIbα antibodies. Additionally, these anti-GPIbα antibodies could lead to platelet activation and loss of sialic acid on GPIbα, leading to consequent clearance by hepatocytes. However, in case of agkistin, this snaclec neither activates platelet nor causes desialylation in vitro. Further, we found that agkistin has high-binding affinity to AMR and the cleavage of GPIbα by metalloproteinase kistomin would attenuate the platelet clearance. These results indicate that both GPIbα and AMR are essential factor in the agkistin-induced thrombocytopenia. We hypothesize that agkistin might play a role as the “bridge” between GPIbα and AMR, which promotes the recognition and ingestion by hepatocytes. Nevertheless, structure-activity relationships remain to be elucidated. Taken together, we provide a possible mechanism of hepatocyte in causing agkistin-induced thrombocytopenia in mice. These researches provide helpful information for designing novel GPIb-antagonists in minimizing the thrombocytopenia risk.

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