Novel GPIb-independent platelet aggregation induced by botrocetin: implications for diagnosis and antithrombotic therapy

Background

Snake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand disease and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis.

Objectives

To explore the alternative receptor(s)/mechanisms that participate in botrocetin-induced platelet aggregation.

Methods

The effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic, ITGA2B and ITGB3-deficient mice, and Bernard–Soulier syndrome and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing enzyme-linked immunosorbent assay. Botrocetin-α_IIbβ₃ and botrocetin-GPIbα interactions were measured using enzyme-linked immunosorbent assay and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber.

Results

Botrocetin could induce aggregation of platelets from a Bernard–Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with α_IIbβ₃ and facilitated α_IIbβ₃-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting α_IIbβ₃. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to α_IIbβ₃ and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited α_IIbβ₃- and GPIb-mediated platelet aggregation, spreading, and thrombus formation.

Conclusion

Our study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both α_IIbβ₃ and GPIbα.