作用於醣蛋白VI引發血小板凝集之一種台灣龜殼花蛇毒蛋白的特性和作用機轉之探討

Characterization and Mechanism of Action of a GPVI-targeting Platelet Aggregation Inducer from Snake Venom of Trimeresurus mucrosquamatus

楊舒雅(Shu-Ya Yang)

指導教授 ： 黃德富

國立臺灣大學/醫學院/藥理學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00745

摘要

台灣龜殼花(Trimeresurus mucrosquamatus) 原毒粉末經離子交換樹脂與膠體過濾法，再經由FPLC 膠體過濾層析法純化出一能引發血小板凝集之成分，並命其為TMF。經SDS-PAGE 分析，在還原狀態下，其分子量則為26 kDa 與23 kDa。並且在二維電泳還原狀態下，可得知在26 kDa 與23 kDa 位置分別有四個點等電點相近，在26 kDa 位置等電點為5.8~6.5;而在23 kDa 位置等電點為 6.7~~7.2。經LC/MS/MS 分析比對後，發現的TMF-b (26 kDa) 與β-fibrinogenase 約有42%相似性，而TMF-a (23 kDa)與metalloprotease (α-fibrinogenase) 約有25%相似性。
TMF 在人類血小板，包括富血小板血漿(PRP)與血小板懸浮液(PS)的功能分析
上，其所引起凝集的程度皆呈濃度相關性。其中在PRP 中引起血小板凝集的EC50
為0.3 μg/ml，而在PS 中的EC50 則為1.8±0.32 μg/ml。TMF 也會依濃度相關地誘發血小板表面分子P-selectin 表現，同時引發TxB2 釋放及細胞內的Ca2+增加。PGE1、EDTA 與BAPTA/AM 皆能有效抑制由TMF 所引起的血小板凝集，而heparin、hirudin 和aprotinin 則不影響。另外，apyrase 與MEK 抑制劑(PD98059) 不影響TMF引發的血小板凝集；indomethacin, CP/CPK、Tyrosine kinase 抑制劑 (genistein) 能稍抑制; Ro-31-7549 與PLC 抑制劑 (U73122) 分別抑制99% 與 45%; 而cytochalasin D 與 PI3K 抑制劑 (LY294002)分別有20% 與62% 的抑制作用外，使用Syk 抑制劑 (piceatannol) 抑制80%，並且 Src family kinase 抑制劑 (PP2) 全面性抑制TMF 所引發血小板的形變 (shape change)與凝集反應。GPIb 拮抗劑 (6D1)，GPIa/IIa 拮抗劑(6F1)，僅能些微抑制TMF 引起的血小板凝集反應; IIb/IIIa 拮抗劑 (7E3) 雖能抑制血小板凝集，卻無法壓制血小板凝活化過程的形變;低濃度GPVI 拮抗劑 (326E12) 能有效抑制血小板凝集反應，由此可知TMF 誘發血小板凝集是透過醣蛋白VI。以流式細胞儀來分析TMF 在血小板表面分子的結合性，TMF 稍微影響6D1、6F1 結合至GPIb 與GPIa/IIa，卻不影響7E3結合IIb/IIIa，而TMF 可以依濃度相關性抑制326E12 結合到GPVI。利用西方點墨法，我們也發現TMF 引起血小板凝集的訊息傳遞可引起血小板內tyrosine kinases(包含Syk、Src、LAT、PLCγ2 等)活化，並且使用piceatannol 與PP2 會全面性抑制TMF 所引發血小板凝集的訊息傳遞。
在酵素活性方面，以fibrinogen 當作受質與TMF 於37 ℃作用後，我們觀察
到 TMF 分解fibrinogen 的酵素活性，呈現濃度與作用時間相關性，因此推測TMF可能具有proteinase 活性。預先以EDTA 處理，不影響TMF 的血小板凝集活性和β-fibrinogenolytic activity，卻可抑制 α-fibrinogenolytic activity。另外，使用serineprotease 抑制劑PMSF 預先和TMF 作用，PMSF 可抑制其分解Bβ活性，但仍不影響其活化血小板活性。因此推測TMF 可能含有可分解Aα之金屬依賴性蛋白脢(metalloprotease) 和可分解Bβ的serine protease，而TMF 活化血小板活性與其酵素活性不相關。
總結上述，TMF 在等張溶液下，可能呈現TMF-b (β-fibrinogenase) 和TMF-a(α-fibrinogenase)的複合體。除了具有分解fibrinogen 活性，於PRP 中不會引起血液凝固，為非典型serine proteinase，於活體中更引發thrombocytopenia 現象。TMF藉由作用於血小板表面受體GPVI 引發許多訊息分子，包括Syk、Src、LAT、Fyn、PLCγ2、PI3K、Akt 之磷酸化; 經由細胞內游離鈣離子增加，誘發TxB2 的形成和P-selectin 的表現，最後引發IIb/IIIa 的活化進而使血小板凝集。藉由TMF 對於血小板表面醣蛋白VI 的專一性，相較於已知的結合蛋白(例如蛇毒蛋白convulxin 與trowagerix)之間差異，可用以探討TMF 與GPVI 之分子作用機制，有利於小分子GPVI 拮抗劑的研發，以做為新一代的抗血栓藥物。

關鍵字

蛇毒 ； 台灣龜殼花 ； 醣蛋白VI ； 血小板 ； 纖維蛋白原

並列摘要

TMF, a platelet aggregation inducer, was purified from Trimeresurus
mucrosquamatus venom. By means of cationic exchange, gel filtration and FPLC
column chromatography, two bands were detected on SDS-PAGE with apparent
molecular weights of 26 kDa and 23 kDa, respectively, under reducing condition. On
2-D PAGE under reduction, there were four bands with different pI ranges 5.8~6.5 withsimilar mass of 26 kDa; pI 6.7~7.2 with similar mass of 23 kDa. Through LC/MS/MS
analysis, the 26 kDa bands exhibit high similarity to β-fibrinogenase and the 23 kDa
bands was similar to metalloprotease (α-fibrinogenase).
TMF concentration-dependently induced platelet aggregation both in platelet-rich
plasma (PRP) and platelet suspension (PS), with EC50 0.3 and 1.8±0.32 μg/ml,
respectively. TMF also concentration-dependently caused P-selectin expression,
increase of intracellular Ca2+ concentration, and release of TxB2. PGE1, EDTA and
BAPTA/AM effectively inhibited TMF induced platelet aggregation, but heparin,
hirudin, aprotinin did not. TMF specifically inhibited the binding of 326E12, mAbs of
GPVI to platelet measured by Flow cytometry. TMF activated the signaling molecules
involved in GPVI signal pathway, including Syk, Src, PLCγ2, LAT, which were
completely inhibited by PP2. Regarding enzymatic activity, TMF concentration-dependently degraded α- and β-chain of fibrinogen. After incubation with
serine protease inhibitors, PMSF or metalloprotease inhibitor, EDTA, its activities of
β-fibrinogenase and α-fibrinogenase were respectively inhibited while its aggregation
was not affected. These results suggest that its platelet aggregating activity is not related
to the intrinsic enzymatic activities.
Taken together, TMF might exist as a complex under isotonic condition, consisting
of two proteins, namely serine proteinase (i.e.β-fibrinogenase) and metalloprotease
(i.e.α-fibrinogenase). It activates platelets and the downstream signaling molecules
mainly through the specific biding of platelet GPVI. By phosphorylation of Syk, Src,
LAT, Fyn, PLCγ2, PI3K and Akt, TMF induced expression of P-selectin and TxB2
biosynthesis, and finally activated integrin IIb-IIIa, resulting in platelet aggregation.
However, its platelet aggregating activity is not related to the proteolytic activity. This
snake venom protein may provide a useful tool for studying how it interacts with
platelet GPVI at a molecular level. The information may provide insights for designing
the novel small-mass antagonist of platelet GPVI, a new class of antithrombotic agent.

並列關鍵字

snake venom ； Trimerserurus mucrosquamatas ； GPVI ； platelet ； fibrinogen

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