臺灣博碩士論文加值系統

鏈激酶是臨床上使用的一種血栓溶解劑，相較於組織型血纖維蛋白溶酶原活化因子，它是一種不具血纖維蛋白專一性的血纖維蛋白溶酶原活化劑。理論上，鏈激酶所引發出血的副作用應該比組織型血纖維蛋白溶酶原活化因子來的高及嚴重。然而，臨床研究發現，使用鏈激酶治療動脈血栓所引發的出血副作用並不會比使用組織型血纖維蛋白溶酶原活化因子高，這個結果顯
示鏈激酶對於富含活化型血小板的血栓具有某些程度的專一性。由於動脈血栓的組成主要為活化型的血小板，因此，鏈激酶可能對於活化型血小板具有高親和力，導致溶解動脈血栓有某種程度的專一性。為了闡明鏈激酶對於活化型血小板是否具有高親和力，我們利用基因重組技術得到大量的鏈激酶後，測試它結合到活化及未活化血小板的能力。利用基因工程蛋白製造技術所獲得的鏈激酶，具有將血纖維蛋白溶酶原轉化成血纖維蛋白溶酶及溶解血纖維蛋白凝集塊的活性。接上螢光染料FITC 後的鏈激酶依然保有活化血纖維蛋白溶酶原轉變成血纖維蛋白溶酶及促進血纖維蛋白分解的能力。利用流動細胞螢光技術來看與血小板的結合情形，我們發現接有FITC 的鏈激酶對於活化血小板具有高親和力，能結合到由ADP 刺激活化的血小板，而且這種結合只能在血漿下進行，顯示血漿在此扮演重要的角色。SK 結合到活化血小板的作用能藉由阻斷血小板的GPIIb/IIIa 及纖維蛋白原鍵C 端而受到抑制，這也表示血漿中的纖維蛋白原及血小板的GPIIb/IIIa 對鏈激酶結合到活化的血小板是重要的。在加有EACA 的情況下，SK 結合到活化的血小板也會受到抑制，顯示鏈激酶必須藉由血纖維蛋白溶酶原的幫忙才能結合到活化的血小板。而血漿中沒有纖維蛋白原或血纖維蛋白溶酶原存在時，則SK 結合到活化的血小板也會明顯受到抑制。综合以上的結果，當血小板活化後，纖維蛋白原會透過GPIIb/IIIa與活化的血小板結合，而鏈激酶則會與血纖維蛋白溶酶原結合形成複合體，結合到固定在活化血小板上的纖維蛋白原，而導致動脈血栓的溶解。為了證明鏈激酶對活化的血小板具較高的親和力，因此將鏈激酶與組織型血纖維蛋白溶酶原活化因子做比較，組織型血纖維蛋白溶酶原活化因子是臨床上常用的血栓溶解劑，它對血纖維蛋白具有專一性的血纖維蛋白溶酶原活化因子。我們利用溶解血纖維蛋白的方式，取得相似的溶解劑量，以血小板凝集測定儀來測試溶解血小板凝集塊的能力，發現鏈激酶在溶解血小板凝集塊的能力比組織型血纖維蛋白溶酶原活化因子好，顯示鏈激酶對於活化型血小板具有高親和力，能較有效的作用在動脈血栓。

Streptokinase (SK), a currently used thrombolytic agent, was regarded as a fibrin-nonspecific plasminogen activator as compared with tissue-type plasminogen activator (t-PA). Theoretically, SK may result in a higher incidence of hemorrhagic events than t-PA in treating thrombotic disorders. However clinical studies found that hemorrhage accidents caused by SK were similar to that of t-PA in treating arterial thrombosis. These results imply that SK may have some specificity in lysing platelet-rich thrombi. It appears likely that SK exhibits a high affinity toward arterial thrombi, in which the activated platelets are the major components. To examine this notion, SK was produced with recombinant technology, tagged with a fluorescent dye, FITC, and tested for its ability to bind resting and activated human platelets. Both recombinant SK and FITC-conjugated recombinant SK were shown able to convert plasminogen into plasmin, leading to fibrin clot lysis. Flow cytometric analysis revealed that FITC-SK bound to ADP-activated human platelets, but not to resting platelets, in a plasma-dependent manner. Blocking either fibrinogen γ chain C-terminus or the fibrinogen receptor (glycoprotein IIb/IIIa) expressed on the platelet membrane abrogated the platelet binding activity of SK, suggesting that plasma fibrinogen plays an important role in the binding of SK to activated human platelets. We also found that ε-aminocaproic acid (EACA), a lysine analog which blocks the binding of plasminogen and plasmin to fibrin, abolished SK’s platelet binding activity. Since plasma plasminogen is the major target of SK, it was proposed that SK first combines with plasma plasminogen or plasmin, then the complex of SK-plasminogen (plasmin) binds to the fibrinogen that has been immobilized onto activated platelets. The binding of SK-plasminogen (plasmin) to activated human platelets may confer SK a more specific activity than t-PA, a fibrin specific thrombolytic agent, in lysing platelet-rich thrombi. Indeed, recombinant SK at the dose that was comparable with t-PA in lysing fibrin clot was far more potent than t-PA in causing disaggregation of aggregated platelets. These results not only demonstrate that SK exhibits a high affinity toward activated platelets they may also lead to a new action mechanism of SK in lysing arterial thrombi.

目 錄
縮寫表 --------------------------------------------------------------------------1
中文摘要 -----------------------------------------------------------------------3
英文摘要 -----------------------------------------------------------------------5
研究背景 -----------------------------------------------------------------------7
實驗材料 -----------------------------------------------------------------------13
實驗方法 -----------------------------------------------------------------------16
實驗結果 -----------------------------------------------------------------------34
討論 -----------------------------------------------------------------------------40
結論 -----------------------------------------------------------------------------44
結果附圖 -----------------------------------------------------------------------45
參考文獻 -----------------------------------------------------------------------64

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