Runhua Wang

    ( Bioscience Centre,Department of Biological Sciences¹ , )

    Thiang Max C.M.Chung

    ( Department ofBiochemistry² , )

    Karen S. C. Max C.M.Chung

    ( BioprocessingTechnology Centre, National University of Singapore³ , Singapore)

Abstract ： By means of Superdex 75gel filtration and Mono Q ion exchange chromatography, we have isolated a novel plateletaggregation inducer, termed rhodoaggregin, from the crude venom of Calloselasma rhodostoma(Malayan pit viper). The native molecular mass of rhodoaggregin (as estimated by gelfiltration chromatography) was 66 kDa while its isoelectric point (pI) was determined tobe 3.45. Under reducing conditions of SDS-PAGE, rhodoaggregin exhibited two distinctivebands with molecular masses of 18 kDa ( α subunit) and 15 kDa ( β subunit). In the absence of reducing agents, however, twobands were also observed, but with apparent molecular masses of 28 (major) and 52 (minor) kDa, respectively. Furthermore, mass spectrometric analysis also showed that rhodoaggreginhad a molecular mass of 30155.39±3.25. These molecular weight data suggest thatrhodoaggregin probably exists as a tetrameric structure consisting of two disulfide-linked heterodimers. N-terminal amino acid sequence analysis showed that the two subunits ofrhodoaggregin exhibit a high degree of homology with each other and with those of theC-type lectin related proteins (CLPs) from other snake venoms. Functional platelet assaysshowed that rhodoaggregin induced platelet aggregation in rabbit and human whole blood,platelet-rich-plasma (PRP) and washed platelets with a lag period and in an all-or-nonemanner. The concentration of rhodoaggregin that induced maximal aggregation is estimatedat about 0.04 μ g/ml(or 0.7 nM).

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