How does agkicetin-C bind on platelet glycoprotein Ibalpha and achieve its platelet effects?

Abstract

The platelet glycoprotein (GP) Ib-IX-V receptor complex has a central role in primary haemostasis and possesses binding sites for the plasmatic adhesive protein von Willebrand Factor (VWF) and thrombin. Several snake venom components have been identified in recent years that target this receptor complex and modulate its functionality. Among them, agkicetin-C is from Deinagkistrodon acutus and proved to be a potent antagonist of GPIb-IX-V. We further studied the structure-activity relationships of agkicetin-C in order to reveal the molecular mechanisms of its antagonistic effect. Agkicetin-C concentration-dependently inhibited botrocetin-, ristocetin- and low dose thrombin- (0.32-0.4nM) induced platelet aggregation. Moreover, it abolished platelet adhesion to collagen under high shear conditions (2600/s), while having only minor effects at low shear rate (650/s), which suggested it targets mainly GPIbalpha instead of other platelet glycoproteins. The interaction site of agkicetin-C was further refined: it recognizes a linear sequence in a recombinant GPIbalpha (AA1-289) fragment and inhibited completely the ristocetin-induced VWF binding to this fragment. Using cross-blocking studies with epitope-mapped anti-GPIbalpha monoclonal antibodies, the binding region of agkicetin-C was refined to the AA201-282 region. In conclusiong the C-type lectin agkicetin-C is a potent GPIb-IX-V antagonist, inhibiting both VWF and thrombin interaction through binding to the AA201-282 region in GPIbalpha. Another thing of interest is that, although agkicetin-C did not agglutinate platelets in all conditions tested in vitro, it caused a severe thrombocytopenia in rats, suggesting a different mechanism than with flavocetin-A or echicetin.