INTRODUCTIONC- type lectin related proteins(CL Ps) is a group of snakevenom proteins that are structurally homologous to thecarbohydrate- recognition domain (CRD) of animal C- typelectins<1 > .Most of these proteins exist as heterodim ers linkedby a single inter- chain disulfide bond,with molecular massesof～ 30 k Da.Despite their striking structural similarity,thisgroup of proteins has different effects on blood coagulationand platelet aggregation.Some of these proteins exhibitanticoagulant activities by binding to the coagulant factors Xand/ or IX<2～ 4> ;whereas other CL Ps induce varied effects onplateletfunctions by modulating the interactions between vonWillebrand factor (v WF) and platelet glycoprotein Ib(GPIb) <5～ 9> .For example,botrocetin from Bothrops jararacavenom binds to v WF and forms an activated complex thatinduces platelet agglutination<5 > .On the other hand,alboaggregins from Trimeresurus albolabris venom<6～ 7> ,echicetin from Echis carinatus venom<8> and agkicetin fromAgkistrodon acutus venom<9> all bind to platelet GPIb andfunction as receptor blockers for v WF binding.However,alboaggregins induce direct platelet agglutination whereasechicetin and agkicetin inhibit platelet agglutination.Recently,several higher molecular weight multimers ofCL Ps with different effects on platelet aggregation have alsobeen reported<7,1 0 ,2 2 ,2 6 > .For example,convulxin from theSouth American rattlesnake Crotalus durissus terificus is a72 - k Da protein that consists of three heterodim ers forming anα3β3com plex<1 2 > .Furthermore,in marked contrast to otherCL Ps that act on platelets by modulating the interactionsbetween v WF and GPIb,convulxin was reported to induceplatelet aggregation via GPVI collagen receptor<1 1 ,1 3> .Similarly,the 5 0 k Da- alboaggregin was found to potentlyinduce platelet aggregation of platelet- rich- plasma<2 2 > .On theother hand,flavocetin- A and - B,with native molecularmasses of 1 4 9and 1 39k Da,respectively,inhibit plateletaggregation at high shear stress<2 6 > .In the present study,we report the isolation and partialcharacterization of rhodoaggregin,a potent plateletaggregation inducer from the venom of Calloselasmarhodostom a (Malayan pit viper) .N- term inal amino acidsequence analysis showed that rhodoaggregin belonged to theCL P superfamily and it probably exists as aα2 β2 tetramericcom plex in the native state.MATERIAL SAND METHODSMaterialsCrude venom of Calloselasma rhodostoma was purchasedfrom Sigm a Chemical Co. (St.L ouis,MO) .FPL C andHPL C columns were from Amersham- Pharm acia Biotech andVydac,respectively,and peptide sequencing chemicals/reagents were from PE- ABD (Foster City,CA ) .All buffersalts and organic solvents were from standard commercialsources and of the highest quality available.Purification of rhodoaggreginCalloselasma rhodostoma crude venom (1 0 0 mg) wasdissolved in 3.0 m l of 0 .1 M ammonium hydrogen carbonate(p H8.0 ) and centrifuged at1 2 0 0 0 rpm for1 0 min at4℃ torem ove particulate material.The supernatant was thenfractionated by a Hi L oad Superdex 75 column (2 .6 cm×6 0 cm) equilibrated with the same buffer using a FPL Csystem .The fractions from peak I (containing proteins ofinterest based on platelet assays) were pooled and loadeddirectly onto a Mono Q HR5 / 5 column pre- equilibrated with2 0 m M Tris- HCl,p H 8.2 .Elution was perform ed with alinear gradient of 0 - 0 .5 M Na Cl in 2 0 m M Tris- HClbuffer,p H 8.2 over 30 min.Separation of subunitsPurified rhodoaggregin was reduced and s- pyridylethylated(s- PE) based on the method of Polgar et al.<1 4 > .The subunitswere subsequently separated by RP- HPL C using a p H stable C8Vydac column(4.1 mm× 2 5 0 mm) .Molecular weightdeterminationMolecular weights of the native protein and its subunitswere determined by gel filtration,SDS- PAGE and electrospraymass spectrometry.(A ) Gel filtration. The native molecular weight ofrhodoaggregin was estimated by gel filtration chromatographyon a Superose1 2 HR1 0
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