Regulation of platelet plug formation by von Willebrand factor and its modulator proteins

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1) Structure and function of C-type lectin purified from snake venom of Crotalus ruberWe have purified the C-type lectin(CRL) from the snake venom of C.ruber and determined the primary structure of CRL. CRL showed high similarity with the lectin from C. atrox (CAL). CRL and CAL showed similar sugar specificity and molecular mass, but showed different divalent cation-requirement. Homology modeling suggested that the amino acid substitution found in CRL does not affect sugar recognition of the lectin but might alter the conformation and influence the sugar binding pocket induced by the metal-ion binding.2)Cloning and expression of botrocetinBotrocetin cDNAs were cloned from cDNA library of venom gland by PCR. cDNAs were subcloned into the expression vector and 293T cells were transfected with the expression vector containing botrocetin cDNAs. The cell culture medium was concentrated and subjected to SDS-PAGE. The band corresponding to botrocetin was observed and the concentrates showed the platelet agglutination activity in the presence of von Willebrand factor (VWF).3)Isolation and characterization of VWF-binding protein from the venom of king cobraVWF-binding protein was isolated from the venom of king cobra (O.hannah) by several steps of chromatographies. The VWF-binding activity of the protein was diminished after reduction of the protein. The protein inhibited the platelet agglutination induced by ristocetin calcium ion-dependently.4)Carbohydrate analysis of human ADAMTS13Sugar chain moiety of ADAMTSI3 was analyzed by lectin blotting. SSA and MAM showed binding to ADAMTS13, which was diminished after sialidase-digestion. RCA120 and PNA bound to ADAMTS13 after sialidase digestion suggesting the presence of N-linked and 0-linked sugar chain with sialic acid residues at the non- reducing terminal. Human ADAMTSI3 did not have ABO(H) blood group antigenicity.