Snake Venom Botrocetin Induces GPIb-Independent Platelet Aggregation

Abstract

Introduction：Snake venom-derived botrocetin facilitates von Willebrand factor (VWF) binding to GPIbα, and has been used clinically for the detection of von Willebrand disease (VWD) and GPIb-related disorders. Botrocetin has also been widely used experimentally for the development and characterization of potential antithrombotic drugs targeting the GPIb-VWF axis. Although compelling evidence suggests GPIb is responsible for botrocetin-induced VWF binding and platelet aggregation, some reports suggest that botrocetin could induce platelet aggregation in some Bernard-Soulier syndrome (BSS) patients who lack a functional GPIb complex. However, the alternative mechanism for botrocetin-induced BSS platelet aggregation and the receptor(s) mediating this action are unclear.

Methods: Botrocetin was purified from the lyophilized venom of Bothrops jararaca using ion-exchange column chromatography. Light transmission aggregometry assay was performed using platelet-rich plasma (PRP) from human, wild type (WT) mice, GPIbα-deficient mice, αIIbβ3-deficient mice and VWF-deficient mice, or CHO cells stably transfected with αIIbβ3 integrin. O-sialoglycoprotein endopeptidase (OSGE) was used to cleave the N-terminal extracellular domain of GPIbα. The binding of botrocetin, VWF and fibrinogen to platelets from WT or the gene-deficient mice were measured by flow cytometry. Antibodies against GPIbα (SZ2, NIT A) and integrin αIIbβ3 (abciximab, JON/A, M1, PSI E1) were used to investigate the binding site of botrocetin. Perfusion chamber assay was used to measure thrombus formation under different shear stresses.

Results: We discovered that botrocetin induced aggregation of human platelets lacking the N-terminal extracellular domain of GPIbα and platelets from GPIbα-deficient mice in the presence of VWF. This VWF-dependent, GPIbα-independent platelet aggregation induced by botrocetin was inhibited by αIIbβ3 antagonists. Botrocetin also induced aggregation of CHO cells stably transfected with αIIbβ3 in VWF-dependent manner. Further experiments with gel-filtered platelets showed that botrocetin competitively bound to the ligand-binding area exposed on αIIbβ3 and blocked fibrinogen and other ligands from binding to the active state of αIIbβ3 in the absence of VWF. Botrocetin inhibited platelet aggregation and thrombus formation in VWF-deficient mice.

Conclusion: Integrin αIIbβ3 is the alternative receptor that mediates VWF-dependent, GPIb-independent platelet aggregation induced by botrocetin. However, via targeting αIIbβ3, botrocetin itself inhibits platelet aggregation in the absence of VWF. These results demonstrate versatility in the mechanism of botrocetin, which may provide snakes containing this toxin the adaptability necessary to aggregate platelets/thrombocytes of different prey or predators. Our data reveals a previously unknown role of botrocetin in the integrin-VWF interaction and also provides insight into developing new antithrombotic drugs that target the active conformation of integrin αIIbβ3. The target switching of botrocetin between GPIb-VWF and αIIbβ3-VWF may explain the possible misdiagnosis of the GPIb-related congenital disorders evaluated by botrocetin.