A tetrameric glycoprotein Ib-binding protein, agglucetin, from Formosan pit viper: structure and interaction with human platelets

Agglucetin, a tetrameric agglutination inducer from the Formosan pit viper, has been identified as a platelet membrane glycoprotein (GP) Ib agonist and directly agglutinated fixed-platelets in the absence of von Willebrand factor (vWf). Here, we resolved the complete cDNA sequences of agglucetin sub-units (α1 , α2 , β1 and β2 ) by molecular cloning. Each cloned cDNA encoding the leader peptide (23 residues) and the mature subunit (131/135/123/126 residues) shares a high degree of homology to each other and the C-type lectin-like GP Ib-binding proteins (BPs). Furthermore, agglucetin specifical-ly caused platelet agglutination and surface exposure of integrin αIibβ3 with a GPIb-dependent manner in washed platelets, based on the observation that the enhanced expression of functional αIibβ3 was suppressed by a GPIb-cleaving metallopro-teinase, crotalin. Pretreating platelets with staurosporine or BAPTA-AM also completely blocked the exposure of function-al αIibβ3, suggesting that the activation of protein kinase C and intracellular calcium mobilization are involved in the GPIb-dependent signaling. In human platelet-rich plasma (PRP), agglu-cetin elicited sequential biphasic responses of platelet agglutina-tion and aggregation in a GPIb- and αIibβ3-dependent manner, respectively, implying that other cofactors may amplify platelet activation to trigger aggregation.