蛇毒蛋白C 型凝集素在血栓和發炎之研究

Abstract

蛇毒中含有許多具有活性的蛋白質和胜肽分子，經過純化後的成分有特定的作用標的並具有獨特的作用，藉由研究這些分子結構和活性之間的關係，它們不僅能夠當作診斷的工具更可以發展成新的治療藥物。其中C型凝集素蛋白(snaclecs)家族會作用在血小板上不同的受體，包括醣蛋白Ib、醣蛋白VI和黏著蛋白α2β1，進而影響血小板的功能。本論文中我們從Tropidolaemus wagleri原毒中純化出一個新的蛇毒蛋白trowaglerix，利用此蛇毒蛋白和另一個已知的蛇毒蛋白Aggretin當作模板來研究新藥物發展的契機。這兩種蛇毒蛋白皆屬於C型凝集素蛋白家族，並具有造成血小板凝集的活性。 當血管受傷時，內皮細胞層會產生破裂，使得平常隱藏在下的基質蛋白膠原蛋白(collagen)暴露出來，提供了血小板吸附的受質並造成血小板活化，進而吸引更多的血小板到受傷的血管壁，最終造成血栓的形成。血小板分別透過醣蛋白Ib和醣蛋白VI、黏著蛋白α2β1與vWF和膠原蛋白結合，來調控血栓形成初期的血小板活化。本論文中我們純化出一個新的蛇毒蛋白trowaglerix，由兩個不同的次單元組成，屬於C型凝集素家族成員。Trowaglerix在人類血小板懸浮液（PS）和富含血小板血漿（PRP），皆會呈現濃度相關性地引起血小板凝集。接有生物素(biotinylated)的trowaglerix會結合到血小板膜上的醣蛋白VI，但不會結合到醣蛋白Ib或是黏著蛋白α2。在人類血小板中給予trowaglerix會造成醣蛋白VI的消失，而在小鼠注射給予trowaglerix後，其血小板失去對膠原蛋白引起凝集的活性，但對於二磷酸腺苷(ADP)所引起的血小板凝集則沒有影響。然而投予基質金屬蛋白酶(MMP)抑制劑GM6001能夠抑制trowaglerix所造成的醣蛋白VI分裂，同時恢復小鼠血小板對於膠原蛋白的反應。因為血小板上的醣蛋白VI對於暴露出來的膠原蛋白而言是重要的受體，加上之前的研究顯示，缺少醣蛋白VI的病人會失去對於膠原蛋白引起的貼附和凝集反應，然而卻只有輕微的出血症狀。近年來在急性冠狀動脈症狀(短暫缺血或是中風)中發現血小板醣蛋白VI的表現會增加，顯示醣蛋白VI可以當作急性冠狀血栓的指標。在我們之前未發表的數據中，我們發現C型凝集素蛋白結合到受體的位置主要位於C端。因此我們定出trowaglerix的部份胺基酸序列，並且依據這些C型凝集素蛋白的C端序列合成數個六胜肽。我們發現從trowaglerix α次單元序列中合成了六胜肽會專一性地作用在醣蛋白VI上，進而抑制膠原蛋白所引起的血小板凝集，在動物模式中同時也顯示有抗血栓的活性。 巨噬細胞是主要的免疫細胞並且在免疫防禦機制中扮演重要的腳色，當外來病原菌入侵時會啟動發炎反應，巨噬細胞活化並且產生許多發炎物質。C型凝集素蛋白會作用在不同的標的，包括表現在血小板、內皮細胞或是骨髓細胞上的各種受體(醣蛋白VI、醣蛋白Ib、黏著蛋白α2β1或是CLEC-2)。在本篇論文中我們觀察許多C型凝集素蛋白在單核白血球和巨噬細胞上的反應，我們發現在數種C型凝集素蛋白當中，唯有aggretin會增加人類單核白血球細胞株和老鼠巨噬細胞株中TNF-α和IL-6的產生。在巨噬細胞中，aggretin會促使ERK1/2和JNK磷酸化。事先給予各種激酶(kinase)抑制劑能夠抑制aggretin所造成的細胞素(cytokine)釋放。流式細胞實驗中顯示aggretin會呈現濃度相關性地結合到單核球表面，並且能夠減少CLEC-2抗體的結合；實驗也顯示immobilized aggretin會專一性地結合到血小板和單核球上的CLEC-2受體。此外，肌肉注射aggretin的小鼠，其血漿中IL-6的量也會增加。 對於這兩種C型凝集素蛋白在血栓和發炎上仍有許多值得研究的部份，目前實驗室針對trowaglerix和aggretin也正在進行抗血栓和抗發炎小分子的藥物研發。

Snake venoms contained various bioactive proteins and polypeptides, and some purified components are specific for their substrates and have unique mode of action. Based on the structure-activity relationship of these unique molecules, they may be used as tools for diagnosis of hemostatic disorders and for development of new classes of therapeutics. For example, the snaclec family has diverse targets including platelet glycoprotein (GP) Ib, GPVI and integrin α2β1, and affect platelet function in a various way. In this report, we purified a novel snake venom protein, trowaglerix from venom of Tropidolaemus wagleri and used this protein and another well-known snake venom protein, aggretin from Calloselama rhodostoma venom as the tools for investigating their opportunity as new drug-designing candidates. Both of these snake venom proteins belong to snaclec family and exhibit platelet-aggregating activity. Exposure of matrix protein collagen after vessel injury provides a substrate for platelet adhesion and triggers platelet activation, which recruits additional platelets to area of injured vessel wall, thereby initiating thrombus formation. Binding of von Willebrand factor (vWF) to platelet GPIb complex and collagen to platelet GPVI and integrin α2β1 both mediate platelet activation in the early stage of thrombus formation. Trowaglerix has two distinct subunits and belongs to snaclec family. Trowaglerix induced platelet aggregation of washed human platelets and platelet-rich plasma (PRP) in a concentration-dependent manner. Biotinylated trowaglerix specifically bound to platelet membrane GPVI, but not to GPIb or α2 integrin. Treatment with trowaglerix induced GPVI loss in human platelets in vitro, and specifically impaired the platelet aggregation of mouse PRP ex vivo in response to collagen. However, GM6001, a matrix metalloproteinase (MMP) inhibitor, inhibited trowaglerix-induced GPVI cleavage and restored the platelet responsiveness of PRP to collagen. Because platelet surface GPVI is an important receptor for platelet adhesion and activation to exposed collagen, and previous study has shown that patients with GPVI-deficient platelets lack response to collagen-induced aggregation and adhesion and only have a mild bleeding disorder. Recently, elevated platelet GPVI expression is found in acute coronary syndromes, transient ischemic attack and stroke, indicating that GPVI may provide as a biomarker for acute atherothrombotic events. Our previous data showed that the binding sites of snaclecs toward their receptors are different and probably located in C-terminal. Therefore, we determined the partial amino acid sequences of trowaglerix and synthesized several hexapeptides derived from these snaclecs C-terminal. We found that the hexapeptide of trowaglerix α subunit specifically exhibited marked inhibitory activity on platelet aggregation caused by collagen via interacting with GPVI in vitro and also displayed antithrombotic activity in animal model. Macrophages are major immune cells and play an important role in modulating homeostasis and the immune defense mechanism. In inflammatory responses to the infection of pathogens, macrophages are activated, producing various inflammatory mediators. Snaclecs have diverse targets, including platelet GPVI, GPIb, integrin α2β1 or CLEC-2 expressed in platelets, endothelial cells or myeloid cells. In this study, we evaluated the effects of various snaclecs on monocytes and macrophages. We found that aggretin increased the production of TNF-α and IL-6 in both RAW264.7 and THP-1 cells; however, the other snaclecs did not. Aggretin induced extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) tyrosine phosphorylation of RAW264.7 cells. Pretreatments with inhibitor of ERK, JNK, p38 or NF-κB abolished cytokine release caused by aggretin. Aggretin bound to THP-1 cells in a concentration-dependent manner and it displaced the CLEC-2 mAb binding to THP-1 cells, and the immobilized aggretin selectively bound to CLEC-2 of both platelets and THP-1 cell lysates. Furthermore, aggretin elevated the plasma level of IL-6 in ICR mice as it was administered intramuscularly. Some perspective studies regarding their detail mechanism of action and signal transduction pathway involved are still under investigation. Furthermore, small molecules derived from aggretin and trowaglerix are being explored as anti-inflammatory and anti-thrombotic agents.