蔡蔭和Tsai, Inn-Ho臺灣大學:生化科學研究所鄭安惇Cheng, An-ChunAn-ChunCheng2010-05-042018-07-062010-05-042018-07-062009U0001-1007200911311400http://ntur.lib.ntu.edu.tw//handle/246246/178865目前已知許多蛇毒中含有可以干擾正常凝血作用(hemostasis)的物質。很多這類的蛇毒蛋白已經被證明具有分子研究及臨床應用的價值；然而對於蛇毒中Kunitz-type蛋白酶抑制劑的功能以及毒理意義仍然有許多需要釐清並深入探討的必要性。在本研究論文中，我們從來自巴基斯坦的鎖鍊蛇(學名: Daboia russelii russelii)的毒液中分離並純化出其中兩種Kunitz-type蛋白酶抑制劑，稱之為DrKIn-I以及DrKIn-II，其分子量分別為7550以及6940。凝血試驗(APTT, PT, TT及ST tests)的實驗中我們發現到DrKIn-I以及DrKIn-II均可以明顯抑制凝血的內因途徑(intrinsic pathway)，而且使用多種缺乏單一凝血因子的血漿(factor deficient plasma)去檢驗後，我們證明了DrKIn-I以及DrKIn-II主要是透過讓FXII失去活性進而導致抑制內因途徑之進行。我們從水解酵素的活性試驗(amidolytic activity assay)的實驗中證明DrKIn-I相較於DrKIn-II，具有更高的專一性可以去抑制FXIIa以及FXIa；而DrKIn-II則是比 DrKIn-I具有較高的專一性去抑制血漿活化蛋白釋放酶(plasma kallikrein)，血纖維蛋白溶酶(plasmin)以及活化的蛋白C (activated protein C)。本研究中我們將不同量的DrKIn-I加入人類血漿中反應後，發現隨著DrKIn-I處理的濃度提高，可以更有效地降低血管舒緩激肽(bradykinin)的產生，而且這樣降低的現象同時伴隨著有更多的ADP所引起的血小板聚集以及血管通透性的降低 (以之此胜肽具抗血小板聚集作用)。我們發現血小板的促凝集效應是由於細胞內的cyclic AMP量減少所造成。另外7 nM的DrKIn-I可幾乎完全抑制thrombin-thrombomodulin所引起protein C的活化，而在小鼠的活體實驗中，同時給予RVV-X和DrKIn-I可明顯降低血中fibrinogen的濃度。當我們從另一個角度去探討DrKIn-II的功能時，雖然我們觀察到DrKIn-II只能部分降低血管舒緩激肽的產生或protein C的活化，但將DrKIn-II加入優球蛋白(euglobulin fraction)中反應後，我們觀察到血塊溶解時間(clot lysis time)有明顯延長的現象。為了更進一步確認DrKIn-II是可以抑制血栓溶解(anti-fibrinolysis)，我們進行了小鼠的活體實驗，相較只給予RVV-X處理的組別，同時給予RVV-X和DrKIn-II的組別可以更明顯地減少fibrinogen/fibrin degradation products (FDP)的產生。綜合以上實驗結果，我們認為DrKIn-I以及DrKIn-II都是重要的蛇毒促凝成分，可以協同同一蛇毒中RVV-X的促凝作用，而造成被鎖鍊蛇咬傷所引起嚴重的DIC及凝血後出血症狀。Snake venoms contain a wide variety of biologically active substances that upset the intricate balance of hemostasis. Many of these proteins have been thoroughly characterized in relation to their functions and applications to molecular and clinical research. However, the functional significance of Kunitz-type protease inhibitors remains obscure. In this study, we purified two Kunitz-type protease inhibitors, namely DrKIn-I and DrKIn-II, from the venom of Daboia russelii russelii (Pakistan), with molecular masses of 7550 and 6940 respectively. APTT, PT, TT and ST tests revealed that both DrKIn-I and DrKIn-II inhibit the intrinsic pathway of coagulation, and tests with single factor deficient plasma showed that this inhibition is mainly through the inactivation of FXII. Amidolytic activity assays revealed that DrKIn-I, compared to DrKIn-II, has higher specificities for FXIIa and FXIa, while DrKIn-II has higher specificities for plasma kallikrein, plasmin and activated protein C. Incubation of DrKIn-I with human plasma decreased the generation of bradykinin in a dose-dependent manner and that this decrease correlates well with increased ADP-induced platelet aggregation and decreased vascular permeability. The pro-aggregatory effect on platelets appears to be mediated through a decrease in intracellular cyclic AMP levels. Furthermore, 7 nM of DrKIn-I almost completely abrogated the activation of protein C by the thrombin-thrombomodulin complex, and that co-injection of RVV-X (the factor X activator in the same venom) and DrKIn-I, but not DrKIn-II, significantly decreased the level of fibrinogen in mice compared to RVV-X alone. DrKIn-II, on the other hand, has less pronounced effect on bradykinin generation and protein C activation. However, treatment of DrKIn-II with the euglobulin fraction significantly prolonged the clot lysis time, compared to DrKIn-I, and that co-administration of RVV-X and DrKIn-II reduced the generation of fibrinogen/fibrin degradation products in mice, confirming the anti-fibrinolytic role of DrKIn-II. We therefore propose that both DrKIn-I and DrKIn-II are prothrombotic agents that help to synergize the procoagulating effects of RVV-X in snakebite symptoms.口試委員會審定書……………………………………………………icknowledgement……………………………………………………ii文摘要.............................................. iiibstract…………………………………………………………… ivontents…………………………………………………………… viist of figures………………………………………………… viiist of tables………………………………………………… viiihapter 1. Introduction…………………………………………1hapter 2. Materials and methods……………………………18hapter 3. Results………………………………………………28hapter 4. Discussion………………………………………… 58eferences………………………………………………………… 67application/pdf2346302 bytesapplication/pdfen-US內因途徑優球蛋白血管舒緩激肽血塊溶解時間intrinsic pathwayeuglobulin fractionbradykininclot lysis timehttp://ntur.lib.ntu.edu.tw/bitstream/246246/178865/1/ntu-98-R96b46011-1.pdf