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研究生:吳孟縈
研究生(外文):WU, MENG-YING
論文名稱:探討SCUBE1在血小板生理功能與血栓形成所扮演之角色
論文名稱(外文):Unravelling the role of SCUBE1 in platelet biology and thrombus formation
指導教授:楊瑞彬楊瑞彬引用關係
指導教授(外文):YANG, RUEY-BING
口試委員:徐松錕陳建璋黃彥華林恆
口試委員(外文):SHYUE, SONG-KUNCHEN, CHIEN-CHANGHUANG, YEN-HUALIN, HENG
口試日期:2017-04-17
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:78
中文關鍵詞:血小板血栓形成
外文關鍵詞:SCUBE1PlateletThrombus formation
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SCUBE1是一個在血小板活化時會分泌並且表現在血小板或是內皮細胞膜上的醣蛋白, 過去研究發現,SCUBE1會促進血小板與血小板之間的交互作用以及血小板與基質的附著。此外,血漿中SCUBE1的濃度也已經被認為在急性血栓相關疾病中,對血小板活化是一個具有潛力的生化指標。然而,血漿中SCUBE1在活體內確切的角色,尚未被釐清。在第一個部分,我們繁殖出一個SCUBE1基因上特異突變(Δ allele)的基因缺陷小鼠,這樣的小鼠無法分泌出可溶性的SCUBE1。我們測量血漿中缺乏SCUBE1的基因缺陷 (Δ/Δ)小鼠,在血球計數、凝血指標和主要的血小板接受器都與正常(+/+)老鼠沒有差異。然而在Δ/Δ小鼠的富含血小板血漿(PRP)中,我們發現,Δ/Δ PRP對於刺激血小板活化物質ADP與collagen的反應顯著下降。我們更進一步純化重組SCUBE1蛋白並加入Δ/Δ PRP中,結果顯示可以回復Δ/Δ PRP的血小板凝集作用,抑或更加強+/+ PRP凝集作用的效果。此外,利用Δ/Δ小鼠進行血管疾病模式實驗,發現有降低動脈血栓的形成以及保護小鼠對抗致命性的肺栓塞疾病模式的現象。進一步利用SCUBE1 抗體去結合+/+小鼠SCUBE1類生長因子的重複片段,影響其蛋白質間的交互作用,發現SCUBE1抗體可以保護小鼠對抗致命性的肺栓塞疾病且不會造成內出血的狀況。實驗結果顯示,血漿中SCUBE1可能透過本身類生長因子的重複片段,在血栓形成的過程中,扮演一個連接活化血小板的架橋角色。在第二部分,我們利用小鼠模式進一步探討血小板或是內皮細胞所產生的SCUBE1的類生長因子的重複片段是否參與血栓形成的過程。我們繁殖出另一個新的SCUBE1基因上特異突變(Δ2 allele)的基因缺陷小鼠,這樣的小鼠(Δ2/Δ2) 缺乏大部分SCUBE1蛋白質的片段,包含全部的類生長因子的重複片段,因此無法產生SCUBE1蛋白質。我們也利用這個小鼠模式進行+/+與Δ2/Δ2老鼠骨髓交換,分析SCUBE1的來源。Δ2/Δ2動物具有正常的血球計數與凝血指標,但Δ2/Δ2 PRP對於主要的血小板刺激物質反應顯著降低,進而削弱了Δ2/Δ2小鼠動脈血栓的形成,同時也保護小鼠對抗致命性的肺栓塞疾病模式。進一步的實驗證明,這種血小板的功能缺損與血小板內部訊號傳遞無關,而是與血小板細胞間黏著的功能有關。而利用Δ2/Δ2小鼠或是給予抗體中和+/+小鼠之SCUBE1類生長因子重複片段的血液於微流體室進行分析,發現在特定的流速下,兩者血液皆具有血小板黏附降低與血栓形成的結果。重要的是,將+/+造血骨髓前趨細胞移植到Δ2/Δ2動物體內,可使其回復動脈血栓形成的能力,也就表示影響血小板細胞間黏著功能,主要來源是由血小板所衍伸出的SCUBE1。綜合以上的實驗結果,我們首次證明了血小板衍生的SCUBE1在動物體內透過其類生長因子重複片段,參與動脈血栓形成的過程,因此,中和或者抑制血漿中SCUBE1類生長因子重複片段可作為潛在抗血栓形成療法的良好標的。
Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), carrying 9 copies of adhesive EGF-like repeats at NH2-terminus is a novel secreted and membrane protein expressed in platelets and endothelial cells. Our previous studies showed that SCUBE1 promotes platelet–platelet interaction and supports platelet–matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined.
In first part, we generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematological and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma (PRP) showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ PRP and further enhanced platelet aggregation in +/+ PRP. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. We used antibodies against the EGF-like repeats of SCUBE1, which are involved in trans-homophilic protein–protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. Together, we found that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis.
In Second part, to further elucidate whether the adhesive EGF-like repeats are truly involved and specific contribution of platelet or endothelial SCUBE1 in thrombosis in vivo. We generated a new Δ2 mutant mouse strain complete lacking these EGF-like repeats and additional chimeric mice by performing crossed bone marrow transplantation between wild-type (+/+) and Δ2/Δ2 mice. While the Δ2/Δ2 animals have normal hematologic and coagulant features, Δ2 platelet-rich plasma (PRP) showed severe decrease in platelet aggregation in response to major agonists, which manifested diminished arterial thrombosis and protection against lethal thromboembolism. The defective Δ2 PRP aggregation was not originated by abnormal intrinsic signaling rather by impaired platelet agglutination because addition of recombinant SCUBE1 protein complemented the defective PRP responses. In agreement with these findings, flow chamber assay displayed reduced platelet adhesion and thrombus formation after perfusion with the Δ2 blood or with the +/+ blood blocking with anti-SCUBE1 EGF-like repeat antibody. Importantly, hematopoietic reconstitution of +/+ bone marrow into the Δ2 mice restores normal arterial thrombosis, suggesting that the platelet-derived SCUBE1 is specifically required for platelet agglutination. We conclude that adhesive EGF-like motifs of platelet SCUBE1 are critical for platelet agglutination and thrombosis in vivo, pointing to targeting SCUBE1 may be a potential antithrombotic therapy.

Table of Contents
Chinese Abstract I
English Abstract III
Table of Contents V
List of Figures and Tables VI
Abbreviations VIII
Chapter 1. General Introduction
1. Scube gene family 2
2. Platelet and thrombus formation 4
Chapter 2. Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis
1. Introduction 7
2. Materials and methods 8
3. Results 13
4. Discussion 20
Chapter 3. The platelet, not endothelial, adhesive SCUBE1 protein is involved in platelet agglutination and thrombosis
1. Introduction 24
2. Materials and methods 25
3. Results 30
4. Discussion 36
Chapter 4. Future Perspectives 39
References 40

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