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Anticoagulant of blood sucking insect salivary glands

吸血昆虫唾液腺抗凝剂

基本信息

批准号：
06556010
负责人：
CHINZEI Yasuo
金额：
$ 3.58万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

We have analyzed physiologically active compounds from the salivary glands of blood suckinh insect, Rhodnius prolixus. We found two different anti-coagulant activities from the extract of Rhodnius salivary glands by analyzing activated partial thromboplastin time (APTT) and prothrombin time (PT). Our analyzes showed inhibitory activities of both intrinsic pathway, Xase complex and common pathway, thrombin (factor lla) in the coagulant cascade.We focussed our analyzes on the Xase inhibitor, and tried to purify and characterize. This inhibitor is a hemeprotein with molecular weight of about 20 kDa. We named it Prolixin-S.Prolixin-S has an inhibitory activity of factor X activation, i.e.Xase complex, which include factor IX,VIII,phospholipid and Ca. We are studying on the mechanism of Xase complex blocking with Prolixin-S.Prolixin-S cDNA was cloned from Rhodnius salivary gland cDNA library with polyclonal antibody to Prolixin-S,and sequenced. Prolixin-S was expressed by baculovirus insect cell expression system and purified chromatographically. Recominant Prolixin-S had anticoagulant activity as almost same level as native Prolixin-S.rProlixin-S was easily reconstituted as heme-protein with hemine and can be bound nitric oxide (NO) reversiblly. NO bound prolixin-S has a vasodilation activity to rabbit vascular smooth muscle. This molecule, Prolixin-S,is a unique protein which has both anti-coagulant activity and vasodilating activity.

本文对吸血昆虫长喙红瓢虫（Rhodnius prolixus）唾液腺中的生理活性成分进行了分析。通过测定活化部分凝血活酶时间（APTT）和凝血酶原时间（PT），发现Rhodnius唾液腺提取物具有两种不同的抗凝活性。我们的分析表明，在凝血级联反应中，内源性途径Xase复合物和共同途径凝血酶（凝血因子IIa）都具有抑制活性。这种抑制剂是一种血红素蛋白，分子量约为20 kDa。我们将其命名为Prolixin-S。Prolixin-S具有抑制因子X激活的活性，即Xase复合物，其包括因子IX、VIII、磷脂和Ca。本研究利用Prolixin-S多克隆抗体从Rhodnius唾液腺cDNA文库中克隆了Prolixin-S cDNA，并进行了序列测定。利用昆虫杆状病毒表达系统表达Prolixin-S，并经层析纯化。重组Prolixin-S具有与天然Prolixin-S几乎相同的抗凝活性。rProlixin-S很容易与氯高铁血红素重组为血红素蛋白，并且可以与一氧化氮（NO）可逆结合。NO结合的prolixin-S对兔血管平滑肌有舒张作用。这种分子，Prolixin-S，是一种独特的蛋白质，具有抗凝活性和血管舒张活性。