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

Summary

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 subunits (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 specifically caused platelet agglutination and surface exposure of integrin α_IIbβ₃ with a GPIb-dependent manner in washed platelets, based on the observation that the enhanced expression of functional α_IIbβ₃ was suppressed by a GPIb-cleaving metalloproteinase, crotalin. Pretreating platelets with staurosporine or BAPTA-AM also completely blocked the exposure of functional α_IIbβ₃, 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), agglucetin elicited sequential biphasic responses of platelet agglutination and aggregation in a GPIb- and α_IIbβ₃-dependent manner, respectively, implying that other cofactors may amplify platelet activation to trigger aggregation.

The nucleotide sequences reported in Figs. 1 and 2 have deposited in the GenBank, EMBL and DDBJ Nucleotide Sequence Databases under the accession numbers: AF540645 for agglucetin-α1 subunit, AF540646 for agglucetin-α2 subunit, AF540647 for agglucetin-β1 subunit and AF540648 for agglucetin-β2 subunit.

Keywords

cDNA cloning - agglucetin - Agkistrodon acutus - glycoprotein Ib - integrin α_IIbβ₃

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