Plasma component inhibits platelet aggregation caused by the extracellulal products of Streptococus mitis

我们证明了s.m mittis菌株Nm-65培养上清浓缩液能有效聚集来自一名志愿者（YY）的血小板。采用DEAE-Sepgarose CL-6B、羟基磷灰石和Superdex 75柱序层析，从s.m ittis菌株Nm-65的细胞外产物（ECP）中纯化出一个人血小板聚集因子。一个聚集因子暂定为s.m itis衍生的人血小板聚集因子（Sm-hPAF）。一项初步研究表明，从健康成人志愿者获得的富血小板血浆（PRP）在对Sm-hPAF的易感性方面存在相当大的个体差异。来自26名健康志愿者的20份（77%）PRP标本被Sm-hPAF聚集，但其余6份（23%）没有反应。连续研究清楚地表明，在无反应供体血浆中存在抑制Sm-hPAF的因子（IF），并对IF的部分表征进行了研究。结果表明：1)在SDS-PAGE上，IF是一种分子量为55 kDa的糖蛋白，其电泳迁移率与α -球蛋白相关。2)用电泳纯化的IF制剂免疫BALB/c小鼠获得IF单克隆抗体（mAb），该单克隆抗体抑制Sm-hPAF对血小板的聚集，并与Sm-hPAF在硝化纤维素膜上直接反应。3) Western blot法测定人肝细胞系（He细胞）培养上清中IF的含量。4)Sandwich ELISA法测定血浆中IF的含量，采用单抗和多克隆Ab对IF进行测定。对Sm-hPAF无反应供者血浆中IF水平与反应供者血浆中IF水平进行比较，无反应供者血浆中IF水平明显高于反应供者。

We demonstrated that culture supernatant concentrate of S.mitis, strain Nm-65, powerfully aggregated blood platelet obtained from one volunteer (YY). A human platelet aggregation factor was purified from the extracellular products (ECP) of S.mitis, strain Nm-65, by sequential chromatography on DEAE-Sepgarose CL-6B, hydroxyapatite and Superdex 75 column. An aggregation factor was tentatively designated S.mitis-derived human platelet aggregation factor (Sm-hPAF). A preliminary study showed that there are considerable individual differences among the platelet-rich plasma (PRP) obtained from healthy adult volunteers with respect to susceptibility to Sm-hPAF.Twenty (77%) PRP specimens derived from 26 healthy volunteers were aggregated by Sm-hPAF, but the remaining 6 (23%) were not reactive. Continuous study, was clearly demonstrated the presence of an inhibitory factor (IF) against Sm-hPAF in the plasma from non -reactive donor, and partial characterization of the IF was studied. The results obtained were as follows :1)IF was a sugar protein with a molecular weight of 55 kDa on SDS-PAGE,and the electrophoretic mobility of IF was associated with alpha-globulin.2)The monoclonal antibody (mAb) to IF, obtained by immunization of BALB/c mouse with an electrophoretically purified IF preparation, inhibited the platelet aggregation by Sm-hPAF, and the mAb directly reacted with Sm-hPAF on nitrocellulose membrane.3)The IF was found in the culture supernatant of human hepatocyte cell line (He cells) on Western blot analysis using the mAb to IF.4)IF in plasms was mesured by Sandwich ELISA using the mAb and polyclonal Ab to IF.When the IF levels in plasma from non-reactive donor against Sm-hPAF were compared with that in plasma from reactive donor, the IF IN plasma of non-reactive donor had significantly higher levels than that of reactive donor.