Identification of the active region responsible for the anti-thrombotic activity of anopheline anti-platelet protein from a malaria vector mosquito
Identification of the active region responsible for the anti-thrombotic activity of anopheline anti-platelet protein from a malaria vector mosquito
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DOI：
10.3109/09537104.2012.698430
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发表时间：
2013-01-01
期刊：
影响因子：
3.3
通讯作者：
Yoshida, Shigeto
中科院分区：
文献类型：
--
作者：
Hayashi, Hideki;Kyushiki, Hiroyuki;Yoshida, Shigeto
我们之前从雌性史氏按蚊（人类疟疾的蚊子媒介）的唾液腺中鉴定出一种抗血小板蛋白，即按蚊抗血小板蛋白（AAPP）。 AAPP 通过直接与胶原蛋白结合并随后引起血小板聚集，特异性地阻断血小板与胶原蛋白的粘附。本研究的目的是确定 AAPP 负责抗血栓形成活性的活性区域，因为我们假设 AAPP 可用作候选抗血小板药物。使用大肠杆菌表达系统产生了各种截短形式的 AAPP。分别使用平板测定和血小板/全血聚集研究检查每种蛋白质与可溶性/纤维状胶原的结合活性和抗血栓形成活性。在检查的截短形式中，只有外显子 3-4 (rAAPPex(3-4)) 编码的蛋白质以浓度依赖性和可饱和的方式有效结合可溶性/纤维状胶原。全长AAPP和rAAPPex(3-4)对于可溶性胶原蛋白结合的EC50值分别为35nM和36nM。与可溶性胶原蛋白相比，全长AAPP 和rAAPPex(3-4) 与纤维状胶原蛋白的结合亲和力存在差异，EC50 值分别为31 nM 和51 nM。 rAAPPex(3-4)还抑制血小板/全血的聚集，全长AAPP和rAAPPex(3-4)对血小板聚集的IC50值分别为35nM和93nM。这些结果表明，AAPP 的胶原蛋白结合和抗血栓活性的关键部分位于外显子 3-4 编码的区域，该区域在其他蚊种的对应区域中高度保守。
We previously identified an anti-platelet protein, anopheline anti-platelet protein (AAPP), from the salivary gland of female Anopheles stephensi (a mosquito vector of human malaria). AAPP specifically blocks platelet adhesion to collagen by binding directly to collagen and subsequently causing platelet aggregation. The aim of this study was to identify the active region of AAPP responsible for the anti-thrombotic activity because we hypothesized that AAPP could be used as a candidate anti-platelet drug. Various truncated forms of AAPP were produced using an Escherichia coli expression system. Each protein was examined for binding activities to soluble/fibrillar collagen and anti-thrombotic activity using a plate assay and platelet/whole blood aggregation study, respectively. Among the truncated forms examined, only a protein encoded by exon 3-4 (rAAPPex(3-4)) effectively bound to soluble/fibrillar collagen in a concentration-dependent and saturable manner. The EC50 values of full-length AAPP and rAAPPex(3-4) for soluble collagen binding were 35 nM and 36 nM, respectively. In contrast to soluble collagen, there was a difference in binding affinity to fibrillar collagen between full-length AAPP and rAAPPex(3-4), with EC50 values of 31 nM and 51 nM, respectively. rAAPPex(3-4) also inhibited aggregation of platelets/whole blood, and the IC50 values of full-length AAPP and rAAPPex(3-4) for platelet aggregation were 35 nM and 93 nM, respectively. These results indicated that the essential moiety of AAPP for collagen binding and anti-thrombotic activity was in the region encoded by exon 3-4, which is highly conserved among the counterpart regions of other mosquito species.