Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti-GPIbβ antibody derivative

Background Glycoprotein (GP)Ib alpha plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIb alpha and reported evidence to suggest that unfolding of the GPIb alpha MSD induces exposure of the Trigger sequence therein and subsequent GPIb-IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R-Ib alpha Tg, where the Trigger sequence is constitutively exposed, constitutive GPIb-IX-mediated cellular signals are present. Clearance of their platelets is also significantly faster than that of wild-type mice. Previously, an anti-GPIb beta antibody RAM.1 was developed. RAM.1 inhibits GPIb alpha-dependent platelet signaling and activation. Further, RAM.1 also inhibits anti-GPIb alpha antibody-mediated filopodia formation. Objective To investigate whether RAM.1 can ameliorate trigger sequence exposure-mediated platelet clearance. Methods Spontaneous filopodia were measured by confocal microscopy. Other platelet signaling events were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody. Result Transfected Chinese hamster ovary cells stably expressing the same chimeric IL4R-Ib alpha protein complex as in IL4R-Ib alpha Tg mice also constitutively exhibit filopodia, and that such filopodia could be abolished by treatment of RAM.1. Further, transfusion of a recombinant RAM.1 derivative that is devoid of its Fc portion significantly extends the endogenous life span of IL4R-Ib alpha Tg platelets. Conclusion These results provide the key evidence supporting the causative link of Trigger sequence exposure to accelerated platelet clearance, and suggest that a RAM.1 derivative may be therapeutically developed to treat GPIb-IX-mediated thrombocytopenia.

Automated summary

This study investigated the role of RAM.1 antibody in platelet clearance mediated by trigger sequence exposure. The results showed that RAM.1 can attenuate platelet clearance and prolong platelet lifespan, providing key evidence for the causative link between trigger sequence exposure and accelerated platelet clearance. This suggests the potential therapeutic development of a RAM.1 derivative for treating GPIb-IX-mediated thrombocytopenia.