蛇毒金屬依賴性蛋白酵素抗血栓作用及蛇毒抗黏著蛋白抗敗血症作用之研究

Abstract

蛇毒中有許多不同成份，而許多成份經純化後具有特殊作用。研究這些特殊成份分子結構和活性，不僅僅可應用為工具更可以發展成為新的治療策略。舉例來說，出血性蛇毒蛋白在血管系統中透過針對血球，血管和血中蛋白的活性而影響循環平衡。本論文中我們利用兩個純化自馬來亞腹蛇（C. Rhodostoma）中具有抗血小板活性的蛇毒蛋白，來研究其機轉成為藥物的設計策略。其中一個是蛇毒金屬依賴性蛋白酵素 Kistomin，另一個則是抗黏著蛋白 rhodostomin（Rn）。 血管壁損傷所造成胞外基質的暴露將引發血栓生成，血小板上的受體如糖蛋白（GP）Ibα和VI扮演著血小板被基質活化凝集的關鍵。vWF及膠原蛋白(collagen)分別和血小板上的GPIba與GPVI結合來調控著血栓生成初期的血小板活化。Kistomin屬於蛇毒金屬依賴性蛋白酵素，已知可以抑制vWF引發之血小板凝集。本論文中進一步證明此抑制作用乃經由kistomin結合並水解GPIbα和vWF，其中kistomin在GPIbα上分別水解兩個不同位置而產生兩個小片段（45和130kDa），而vWF經水解產生小分子的多聚體。活體外試驗中，kistomin在人類全血中仍具有水解GPIbα的活性；注射kistomin的小鼠，其血小板失去了對GPIbα致效劑引發凝集的活性。由於GPIbα和GPVI在活性機轉和結構功能的類似性，我們測試了kistomin在GPVI與膠原蛋白間交互作用的影響。研究中發現高濃度的kistomin會抑制膠原蛋白所引起的血小板凝集，也會抑制convulxin(GPVI的致效劑)和GPVI專一抗體所引發的凝集。利用流式細胞儀和西方點墨法得知kistomin可水解GPVI並產生小的片段。基因工程表現的GPVI也被kistomin作用後產生25kDa和35kDa的片段，推測其水解位置約在類mucin區域。因此我們合成此區域（Leu180到Asn249）作為kistomin受質，水解後利用飛行質譜儀（MALDI-TOF-MS）檢測發現水解位置在FSE205/A206TA和NKV218/F219TT。這種水解活性也會抑制血小板GPVI活化時的tyrosine kinase磷酸化及血小板貼附到膠原蛋白。活體試驗中更證明了kistomin注射有效延長小鼠腸繫膜微血管栓塞時間及尾巴出血時間。本研究進一步研究kistomin阻礙GPIbα和vWF交互作用的機轉，並且首次發現小分子蛇毒金屬依賴性蛋白酵素會透過水解GPVI而抑制collagen和GPVI間的交互作用。Kistomin提供了針對GPIbα/GPVI作用相關之新抗血栓的治療策略。 敗血症常造成多重器官衰竭和敗血性休克引發病人死亡，然而治療策略卻仍然有限。在抗發炎作用中，內毒素（endotoxin）注射常用於急性發炎之研究。我們評估了抗黏著蛋白 Rn 在菌血症之作用及對脂多醣（lipopolysaccharide, LPS）活化單核球之作用機轉。活體研究中發現Rn有效降低血中TNFα，IL-6，IL-1β和IL-10以及改善心血管功能衰竭和血小板低下症。組織切片中也可以發現Rn對脂多醣引發之組織發炎也有保護作用。為了了解這些作用的可能機轉，我們在試管實驗中研究Rn對脂多醣活化之人類單核球細胞株（THP-1）的影響。Rn同樣抑制了細胞激素的分泌及mitogen-activated protein kinases的活化。流式細胞儀分析顯示Rn可以濃度相關性的結合到單核球表面，並且專一性阻斷αVβ3抗體的結合。脂多醣引發之單核球對纖維結合素（fibronectin）黏著及移行均為Rn所抑制。此外，Rn也抑制了脂多醣活化單核球造成組織因子（tissue factor）的表現和促凝血活性。本研究證實除了藉由抗血小板作用外，Rn可能藉由和單核球上的αVβ3交互作用而影響脂多醣對單核球的活化，包括發炎細胞激素釋放，細胞貼附和移行以及促凝血活性。 對於蛇毒金屬依賴性蛋白酵素和抗黏著蛋白仍有許多值得研究的功能及應用，目前實驗室也繼續進行中。

Some snakes store different protein mixture of toxins in the venom gland, and purified venom components are special and specific to their substrates. Based on the molecular structure-relationship of these unique molecules, we can not only use as tool but also develop new classes of therapeutics. For example, the proteins from hemorrhagic venoms have diverse effect on the cardiovascular system by affecting blood cells, plasma proteins and vessel wall components. In these reports, we used two snake venom proteins purified from venom of Calloselasma rhodostoma, both with antiplatelet activities, as the tools for investigating the their opportunity as new drug-designing candidates. One is SVMP, kistomin, and the other is disintegrin, rhodostomin. Injuries to the vessel wall and the subsequent exposure of matrix of the subendothelial layer result in thrombus formation. Binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ib-IX-V and collagen to platelet GPVI both mediate platelet activation in the early stage of thrombus formation. Kistomin has been shown to inhibit vWF-induced platelet aggregation. In the present study, we found that kistomin specifically inhibited vWF-induced platelet aggregation through binding and cleavage of platelet GPIbα and vWF. Cleavage of platelet GPIbα by kistomin resulted in release of 45- and 130-kDa soluble fragments, indicating that kistomin cleaves GPIbα at two distinct sites. In parallel, cleavage of vWF by kistomin also resulted in the formation of low-molecular-mass multimers of vWF. In ex-vivo studies, kistomin exhibited the cleaving activity on platelet GPIbα in whole blood, and GPIbα agonist-induced platelet aggregation was inhibited. Because of the crucial roles of GPIbα-vWF and GPVI-collagen in mediating the initial platelet aggregation process and the structural similarity between GPIbα and GPVI, we further investigated the effect of kistomin on GPVI-collgen interaction. We found that kistomin also inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin, a GPVI agonist, and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased the expression of platelet GPVI but not that of integrin α2β1 or αIIbβ3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25 and 35kDa-fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu180 to Asn249 as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE205/A206TA and NKV218/F219TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin. Its in vivo antithrombotic effect was evidenced by prolonging the occlusion time in mesenteric microvessels of mice and tail-bleeding time was prolonged in mice receiving intravenous kistomin. In conclusion, kistomin, a P-I class metalloproteinase, interrupts interactions between both GPIbα-vWF and GPVI-collagen through its proteolytic activity, which is responsible for its antithrombotic activity both in vitro and in vivo. Kistomin can be a useful tool for studying metalloproteinase-substrate interactions and ligand-GPIbα/GPVI interaction at molecular basis. Sepsis cause lots of death because of multiple organs failure and septic shock, but progress in the development of better therapeutic modalities for sepsis has been slow. Endotoxin injection is widely used to study the acute inflammatory response. We evaluated the effects of rhodostomin on endotoxemia and explor the possible mechanism on LPS-activated monocytes. We found that rhodostomin significantly decreased the production of tumor necrosis factor-α, interleukin-6, -1β and -10 and improved cardiovascular dysfunction and thrombocytopenia in vivo. Rhodostomin also protected against tissue inflammation following LPS-induced endotoxemia in mice, as evidenced by histological examination. To understand the mechanism of this obvious inhibition, we evaluated the in vitro effects of rhodostomin on LPS-treated human monocyte cell line, THP-1. Rhodostomin inhibited cytokine production and mitogen-activated protein kinase activation of THP-1 induced by LPS. Flow cytometric analysis revealed that rhodostomin concentration-dependently bound to LPS-activated THP-1 and specifically blocked anti-αvβ3 mAb binding to THP-1, but not anti-β1 and anti-β2 mAbs binding. LPS-induced THP-1 adherences to immobilized fibronectin and cell migration were inhibited by rhodostomin. Moreover, rhodostomin blocked the enhanced expression and the procoagulant activity of tissue factor on THP-1 cells stimulated by LPS. These results suggest that in addition to its antiplatelet activity, rhodostomin may interact with αvβ3 integrin of monocytes leading to interfere with the LPS triggered activation of monocytes, including proinflammatory cytokines release, subsequent adhesion, and migration and the procoagulant activity. Furthermore, we are interested in some topics about kistomin and Rn and more related investigations are undergoing.