在動脈硬化斑塊破裂或高剪力血流處的血栓形成中,血小板凝集的發生是由醣蛋白(GP)Ib-IX-V複合體與von Willebrand factor (vWf)結合而啟動。此與在高剪力血流處的止血反應中,血小板GPIb-IX-V複合體黏附於受傷血管壁之vWf的過程是相仿的,而後促使血小板活化與黏著分子αIIbβ3 (GPIIb/IIIa)相關之血小板凝集;然於低剪力血流處,則是由黏著分子α2β1 (GPIa/IIa)或GPVI,來媒介血小板黏附於collagen。目前已知許多作用於GPIb-IX-V、GPIIb/IIIa、GPIa /IIa、GPVI、vWf與collagen的蛇毒蛋白,都屬於C-type lectin-like與metalloproteinase-disintegrins等兩大類蛋白家族,由於它們具有特異生物活性,現已充作研究「止血與血栓形成」的工具。
GPIb-IX-V複合體是一種在血小板表面持續表現的受體,由四種貫穿胞膜之醣蛋白Ibα、Ibβ、IX與V所組成,其中GPIbα在胞外分別以雙硫鍵與GPIbβ聯結,以非共價鍵與GPIX及GPV聯結;有證據顯示,vWf在GPIbα最N端的282個胺基酸內,至少有兩處以上的結合位。其實在體外透過非生理性調節劑,如抗生素ristocetin與蛇毒蛋白botrocetin結合於vWf-A1 domain,亦可促使vWf結合至GPIbα,而造成血小板凝聚(agglutination)。此外,在眾多屬於二元體C-type lectin-like之蝮蛇蛋白,業已證實可直接結合至GPIbα的N端可能重疊但不相同的部位,而阻斷vWf結合。亦即目前發現的醣蛋白Ib結合蛋白(GPIb-BPs),大多是GPIb拮抗劑,不會誘發血小板凝聚,但分別從T. albolabris與A. halys blomhoffii分離的alboaggregin-B和mamushigin,則可直接凝聚血小板,功能上為醣蛋白Ib致效劑;此外,另一分離自T. flavoviridis的Flavocetin-A,亦可藉交錯聯結血小板,造成血小板凝聚。相較之下,從T. albolabris分離之alboaggregin-A,除了能凝聚血小板外,尚能作用於GPVI,而誘導凝集與釋放反應。
最近,我們自臺灣百步蛇(Agkistrodon acutus)原毒,分離出一嶄
新血小板凝聚誘導劑agglucetin。利用凝膠電泳分析,在非還原條件下,此蛋白呈單一色帶,其估算分子量約58.8 kDa;還原後則出現兩個色帶,分子量分別為16.2 與14.5 kDa。再經rp-HPLC、電噴離子化質譜儀(ESI-MS)與二維電泳(2D-PAGE)分析,我們證實native agglucetin是一個由雙硫鍵聯結之 α1、 α2、β1與 β2 等次單元所組成之四元體蛋白。其四個次單元之N端部份序列, 均與GPIb-BPs有高度相似性。功能性研究顯示, Agglucetin於vWf不存在下,在人類血小板懸液中,能以劑量相關性誘導血小板凝聚,並且引起微量胞內鈣離子濃度升高,與血栓素(thromboxane) B2生成。醣蛋白Ib單株抗體AP1或LJ-Ib1,能以劑量相關型式,抑制agglucetin所誘發之血小板凝聚;然而EDTA、arietin (一種長鏈含RGD之抗黏著蛇毒蛋白)、7E3 (一種GPIIb/IIIa單株抗體)、heparin、hirudin、PGE1、indomethacin或醣類等試劑,則不具抑制作用。我們進一步藉流式細胞儀分析,發現螢光標定(FITC)之agglucetin,能以劑量相關方式結合至福馬林(甲醛)固定之血小板,並且可達飽和型式,然此作用會被醣蛋白Ib專一作用抗體阻斷。
另外, Agglucetin也可交錯連接血小板GPIb,而促使血小板凝聚。我們藉分子選殖技術,解讀其兩個β次單元(β1/β2)的全部cDNA與推算胺基酸序列,發現其先導胜肽(23/23胺基酸)及其成熟蛋白(123/126胺基酸),與GPIb-BPs有極高相似性,且兩個β次單元都具有保留性的7個Cys及兩個hydrophilic patch,最近有文獻指出這兩個hydrophilic patch可能就是蛇毒蛋白用來與醣蛋白Ibα結合的部位;然而在β2次單元分子中,可見有額外的Cys3與在第二個patch的Gly105,這暗示兩個β次單元在與GPIb作用方面,分別扮有不同角色。
在人類富含血小板血漿(PRP), Agglucetin可先後引發凝聚與凝集反應,我們證實這兩相反應分別與GPIb及GPIIb/IIIa有關;以不同血小板製備(甲醛固定與等張液清洗之血小板)來觀察比較,Agglucetin確實可直接凝聚血小板,而無須任何輔因子;此外,利用流式細胞儀分析,我們初步認為agglucetin能專一作用於GPIb來增強血小板GPIIb /IIIa複合體的表現,是因為這種受體表現增強情形,會因crotalin (一種能切除醣蛋白Ibα之蛇毒金屬蛋白)的前處理而抑制;另外,以staurosporine、BAPTA-AM與PGE 1等前處理,亦可完全阻斷此作用,這表示在GPIb相關的inside-out訊息中,蛋白激C與胞內鈣離子移動會引起GPIIb/IIIa暴露及血小板凝集。
蛇毒金屬蛋白酶(SVMP)以其分子中有保留性之鋅結合序列-HEXXHXXGXXH而隸屬鋅蛋白中metzincin家族的成員,目前已知此特色構型(motif)與其蛋白分解活性有關。SVMP可依其結構,分成P-I、P-II、P-III與P-IV等四群; 這四群SVMP的metalloproteinase domain都具有一個鋅結合序列; P-I class僅含一個metalloproteinase domain,例如HR2a與trimerelysin Ⅱ等;P-II class 含metalloproteinase與 disintegrin-like domain,例如trigramin 及 rhodostomin的前驅蛋白;P-III class (高分子量金屬蛋白酶)含metalloproteinase、disintegrin-like domain與high-cysteine domain,例如mocarhagin與jararhagin等; P-IV class (具C-type lectin domain之SVMP)除擁有P-III class的三種結構域外,另含一個lectin-binding domain,例如russellysin 與 carinactivase-1等。SVMP除了會分解血漿蛋白,例如纖維蛋白原與交錯連接之纖維蛋白外,也能分解許多胞外基質,例如vWf、collagen與laminin等,故能影響結締組織的重建以及凝血過程;此外, SVMP也被認為具有溶解血栓之臨床運用潛力。
我們採用陰離子交換與厭水性交互作用等管柱層析法,由台灣百步蛇原毒中純化出一高分子量金屬蛋白酶acurhagin。凝膠電泳分析顯示其還原態儗似分子量約51.4 kDa,質譜分析顯示其精確分子量為48,133 Da,故acurhagin係單元體蛋白。經序列分析得知,其分子中metalloproteinase domain的部分序列,非常類似第三群蛇毒金屬蛋白;Acurhagin依序可切割纖維蛋白原Aα、Bβ鏈,但幾乎不影響γ鏈;利用 rp-HPLC觀察其切割片段,我們發現它能強力分割纖維蛋白原成許多胜肽碎片。在37 ℃溶液中,它會自我裂解出30 kDa的片段,然此片段之N端序列,極類似含有disintegrin-like 與cysteine-rich domain的金屬蛋白;經酪蛋白分解活性檢測,Acurhagin之蛋白分解活性,會被Ca2+ 與Mg2+ 等離子些微增強,但卻會被Zn2+完全抑制,當我們用金屬螯合劑預處理時,Acurhagin會完全喪失酵素活性;此外,它除了能以溫浴時間相關方式,於PRP能抑制ADP引發之凝集反應外,我們也發現acurhagin具有切割collagen與vWf的活性,可分別抑制collagen與 ristocetin所誘發之血小板凝集。
總結:agglucetin專一作用於血小板GPIb,而誘發血小板凝聚,其結合部位與單株抗體AP1重疊,然此作用與鈣離子、醣類及GPIIb /IIIa無關。另外,我們解讀出agglucetin兩個β次單元全長cDNA與推算蛋白序列,此完全序列有助於說明其不具鈣離子及醣依賴性的結合特性;Agglucetin藉由專一性連接GPIb而凝聚血小板,並隨後引發功能性αⅡbβ3的趨增表現;其間訊息包括有蛋白激酶C活化與胞內鈣離子濃度增高;然在完整agglucetin分子中, 其α與β次單元究竟如何與GPIb做最適切的交互作用則尚待釐清。咸信務需完全揭曉其另兩個α次單元的全長序列,始能有助探討agglucetin與GPIb作用之生物活性;此外,我們亦證實acurhagin係P-III class蛇毒金屬蛋白,可有效切割胞外基質如collagen、fibrinogen與 vWf等。我們期望日後能利用X光晶體繞射與推理式site-directed mutagenesis等實驗,來進一步探討agglucetin與acurhagin之完整分子結構與活性的關係,並釐清相關問題。

In thrombosis, platelet aggregation is initiated by a specific membrane glycoprotein (GP) Ib-IX-V complex binding to its ligand, von Willebrand factor (vWf), in the matrix of ruptured atherosclerotic plaques or in plasma exposed to high shear stress. This process closely resembles normal haemostasis at high shear stress, where GPIb-IX-V-dependent platelet adhesion to vWf in the injured blood vessel wall initiates platelet activation and integrinαⅡbβ3 (GPIIb/IIIa)-dependent platelet aggregation. At low shear stress, other receptors such as integrinα2β1 (GPIa/IIa) or GPVI that bind collagen mediate platelet adhesion. It is well-known that snake venoms contain a variety of GPIb-IX-V, GPIIb/IIIa, GPIa/IIa, GPVI, vWf and collagen targeting proteins typically belonging to one of two major protein familys, the C-type lectin-like proteins and the metalloproteinase-disintegrins. Based on their biology activities, some of them have been utilized as useful probes for the basic studies of haemostasis and thrombosis.
The GP Ib-IX-V complex constitutively expressed on the platelet membrane is made up of four transmembrane glycoproteins: GPIba disulfide-linked to GPIbb and the non-covalently associated subunits, GPIX and GPV. There are accumulating evidences revealing that vWf binds to at least two sites within the first 282 residues of GPIba. In vitro, vWf is activated to bind GPIb-IX-V complex in the absence of shear by the non-physiological modulators, such as an antibiotic, ristocetin, or a viper venom protein, botrocetin. Ristocetin and botrocetin all bind to specific sequences within the A1 domain of vWf and induce platelet agglutination. Viper venom proteins of the C-type lectin-like family consisting of heterodimers have been shown to bind to overlapping, but not identical, sites within the N-terminal ligand-binding domain of GPIba, thus blocking vWf binding. Meanwhile, most snake venom GPIb-BPs, functioning as GPIb antagonists, do not induce platelet agglutination. However, alboaggregin-B (AL-B) from the venom of Trimeresurus albolabris and mamushigin from that of Agkistrodon halys blomhoffii, directly agglutinate platelets without the need of any cofactor, functioning as GPIb agonists. Otherwise, flavocetin-A, from Trimeresurus flavoviridis venom, induces platelet aggregate formation by cross-linking platelets. Alboaggregin-A (AL-A) from the same snake venom with AL-B also can agglutinate platelets. However, AL-A has an additional GPVI effect in inducing platelet aggregation and release reaction.
In this thesis, we purified a novel platelet agglutination inducer, agglucetin, from the Formosan Agkistrodon acutus (A. acutus) snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked α1, α2, β1 and β2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like GPIb-BPs. Functional studies showed that agglucetin, in the absence of vWf, dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca2+ mobilization and thromboxane B2 formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, indomethacin or carbohydrate exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb.
On the other hand, we also found that agglucetin triggers platelet agglutination through the cross-linking of platelet membrane GPIb. Structurally, we resolved the cDNA sequences of two agglucetin β subunits (β1 andβ2) by molecular cloning and found that the leader peptides (23/23 amino acid residues) and the mature subunits (123/126 amino acid residues) share a high degree of homology to those of GPIb-BPs. In human platelet-rich plasma, agglucetin elicited the sequential biphasic responses of agglutination and aggregation in a GPIb- and GPIIb/IIIa-dependent manner, respectively. Agglucetin directly agglutinated platelets in the absence of any cofactor as observed with fixed platelets by microscopy and washed platelets by flow cytometry. Furthermore, agglucetin specifically enhanced the surface expression of GPIIb/IIIa complex in a GPIb-dependent manner because the enhanced expression of functional GPIIb/IIIa complex on agglucetin-aggregated platelets was abolished as platelets were pretreated with a GPIb-cleaving metalloproteinase, crotalin. Pretreatments with staurosporine, BAPTA-AM or PGE1 also completely blocked such effects, suggesting that protein kinase C activation and intracellular calcium mobilization are involved in the GPIb-dependent inside-out signaling, i.e. the subsequent GPIIb/IIIa exposure and platelet aggregation.
Snake venom metallopoteinases (SVMPs) belong to the metzincin family among several zinc-containing metalloproteinases. They are characterized by the presence of a conservative zinc-binding sequence, HEXXHXXGXXH, as an essential motif for the proteolytic activity. SVMPs are classified according to their domain structure into four basic groups, P-I (protein class I) to P-IV. All four groups share a metalloproteinase domain containing the Zn2+-binding motif. The P-I class only has a metalloproteinase-domain structure, such as HR2a and trimerelysin II. The P-II class has an additional domain carboxy to the proteinase domain, a disintegrin or distintegrin-like domain, such as precusor proteins of trigramin and rhodostomin. The P-III class (high-molecular mass metalloproteinase) has both a disintegrin-like domain and a high-cysteine domain carboxy to the proteinase domain, such as mocarhagin and jararhagin. The P-IV class has a similar domain structure to the P-III class, but with an additional lectin-binding domain, such as russellysin and carinactivase-1. In addition to degradation of plasma proteins such as fibrinogen and cross-linked fibrin, metalloproteinases are able to degrade many constituents of the extracellular matrix such as vWf, collagen and laminin. Thus, they are implicated in connective tissue remodeling and blood coagulation processes. Furthermore, these proteins have potential clinical use in dissolving thrombi.
By means of anion-exchange and hydrophobic interaction chromatography, acurhagin, a high-molecular mass hemorrhagic metalloproteinase, was also purified from the crude venom of A. acutus. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other P-III class metalloproteinases from snake venoms. It preferentially cleaved Aa chain of fibrinogen, followed by Bβ chain, while g chain was minimally affected. Monitored by rp-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa-fragment at 37℃. The N-terminal sequence of the 30 kDa-fragment of acurhagin showed a high homology to those proteins consisting of disintegrin-like and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of platelet-rich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWf, respectively.
It is concluded that agglucetin derived from Formosan A. acutus venom acts specifically on an epitope of platelet membrane GP Ib overlapping with that of AP1, causing platelet agglutination in a Ca2+-, carbohydrate- and GPIIb/IIIa-independent manner. In addition, we further resolved the full-length cDNA and deduced protein sequences of both β subunits of agglucetin. The complete sequences also help to explain its lack of binding capacity to Ca2+ and carbohydrate. Moreover, agglucetin specifically and directly agglutinates platelets via ligating GPIb and triggers a sequential upregulation of functional αIIbβ3. The activation of protein kinase C and elevation of intracellular Ca2+ level are involved in agglucetin-induced αIIbβ3 activation. However, the precise role of the α or β subunit within an intact molecule for the optimal interaction with GPIb remains to be investigated. Information regarding the complete sequences of α subunits of agglucetin are needed to fully explore its molecular interaction with GPIb. On the other hand, we also isolated a novel P-III class hemorrhagic metalloproteinase, termed acurhagin, and found that it is an effective enzyme in cleaving many extracellular matrices, such as collagen, fibrinogen and vWf. In order to clarify associated issues, we expect to perform further studies on the structure-function relationship of intact agglucetin and acurhagin by X-ray crystallography analysis and rational site-directed mutagenesis in the future.

縮寫表........................................................1

中文摘要......................................................4

英文摘要......................................................9

第一章 緒論..................................................15
1-1 文獻回顧..........................................16
1-2 研究動機與目的....................................25

第二章 一嶄新百步蛇毒之四元體醣蛋白Ib致效劑agglucetin的精製..35
2-1 前言..............................................36
2-2 實驗材料與方法....................................39
2-3 實驗結果..........................................47
2-4 討論..............................................52

第三章 Agglucetin之分子結構與作用機轉的探討..................76
3-1前言...............................................77
3-2實驗材料與方法.....................................79
3-3實驗結果...........................................85
3-4討論...............................................91

第四章 高分子量金屬蛋白acurhagin之精製與作用機轉的探討......114
4-1前言..............................................115
4-2實驗材料與方法....................................118
4-3實驗結果..........................................124
4-4討論..............................................130

第五章 結論與展望...........................................152

參考文獻....................................................165

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