- 學位論文
探討重組人類凝血第九因子338L及TripleL之功能
Functional Characterization of Human Coagulation Factor IX-338L and Factor IX-TripleL
摘要
B型血友病是一種性聯遺傳的出血性疾病，病人主要由於缺乏第九凝血因子的功能所造成。目前B型血友病的治療方式多採用替代性療法，即病人接受注射自血漿純化或重組合成的第九因子。本實驗室研發出一個具有三個單點突變(V86A/E277A/R338A)的第九因子Factor IX-Triple (簡稱FIX-Triple)，FIX-Triple在in vitro比野生型第九因子高出13倍專一活性。先前文獻報導一位靜脈血栓病人具有第九凝血因子的單點突變(R338L)，導致雖然第九凝血因子的表現量正常，但凝血活性卻高出正常人8倍(Simioni et al, 2009)。本論文的研究目標是希望顯示具338L變異型的高活性第九因子可能造成的病症，與比較FIX-Triple與338L的差異及病理機制。 初步我建構兩種表現質體，其一是胺基酸第338位置單點突變(FIX-338L)，而另一是將FIX-Triple 胺基酸第338位置的精胺酸(Arginine, R)置換成白胺酸(Leucine, L)，命名為FIX-TripleL (V86A/E277A/R338L)。純化的FIX-TripleL具有比野生型第九因子(FIX-WT)高的專一活性，FIX-338L為8倍，FIX-TripleL為15倍，且FIX-TripleL的内源凝血酶生成能力(Endogenous Thrombin Potential)也是野生型的1.7倍，透過血栓彈力圖(TEG)分析儀發現FIX-TripleL具有最快速的血栓形成速率。而在第九因子輸注至血友病小鼠體內評估治療狀況的實驗，發現FIX-TripleL在小鼠體內具有比野生型高8倍的活性，FIX-338L及FIX-Triple為4倍。當使用腺病毒相關病毒載體(AAV)，感染血友病小鼠與野生型小鼠(C57BL/6)，發現接受FIX-TripleL的血友病小鼠測得第九因子專一活性是接受FIX-WT的小鼠的14倍，又帶有FIX-TripleL腺病毒相關病毒載體感染野生型小鼠初步評估並無提高血栓風險。使用抗凝血酶III (Antithrombin III)定量活化態凝血第九因子時發現，當338位置突變成白胺酸(L)時，第九因子較不受抗凝血酶III抑制，且此情形與肝素(Heparin)存在與否無相關性。而變異型與野生型的第九因子皆能結合TFPI (Tissue factor pathway inhibitor)。 總結本論文的研究可發現，就補充第九因子蛋白及基因療法策略而言，FIX-TripleL的確能有效提高凝血能力及血纖維蛋白(Fibrin)形成速率。而在血栓風險探討結果發現FIX-TripleL可與抗凝血酶III形成複合體，且在FIX-TripleL治療組小鼠發現凝血酶/抗凝血酶複合物偵測結果上是安全的，且不影響與組織因子路徑抑制因子結合能力。因此上述結果支持FIX-TripleL具有臨床治療血友病的應用潛力。
並列摘要
Hemophilia B is an X-linked bleeding disorder that results from a deficiency or dysfunction of factor IX (FIX). Conventional treatment involves the replacement of the deficient factor. We previously reported a FIX variant containing three point mutations (V86A/E277A/R338A, FIX-Triple) with 13-fold higher specific clotting activity compared with FIX wild-type (FIX-WT). Simioni et al. reported a case of thrombophilia associated with a substitution of leucine for arginine at position 338 (R338L) with 8-fold higher specific clotting activity than FIX-WT. This thesis compared differences between 338L and the FIX-Triple as well as investigated the pathological mechanism of 338L. To this end, I generated novel FIX variants with leucine replacement at the 338 residue of FIX-Triple and of FIX-WT. These Variants are designated FIX-TripleL and FIX-338L, respectively. Purified FIX-R338L and FIX-TripleL proteins revealed 8 and 15-fold higher specific clotting activity than FIX-WT. Furthermore, FIX-TripleL had the greatest improvement of endogenous thrombin potential and maximum rate of thrombus generation among the FIX variants assessed by the calibrated automated thrombogram and thromboelastography. In protein replacement therapy for hemophilia B mice, FIX-TripleL respectively resulted in 8-fold greater clotting activity than FIX-WT. Additionally, HB mice treated with AAV particle expressing either FIX-R338L, FIX-Triple, or FIX-TripleL exhibited a respective 7, 9, and 14-fold higher specific clotting activity than FIX-WT. C57BL/6 mice injected with different doses of virus particles carrying FIX-WT, FIX-R338L, FIX-Triple, or FIX-TripleL did not show increased risk of thrombosis compared with FIX-WT. The results indicate that the FIX-TripleL variant exhibits significantly enhanced clotting activity relative to FIX-WT and the other gain-of-function FIX variants studied in this work. FIX-TripleL neither showed thrombogenic with the mouse models nor effected binding ability with TFPI. These data support the potential for therapeutic use of FIX-TripleL for treating HB.
參考文獻
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