| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1996: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
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.
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