The goal of this proposed project is to obtain a macromolecular agent that can block specifically ectodomain shedding of platelet glycoprotein (GP)Ib1. As the primary receptor in platelets for von Willebrand factor (vWF), GPIb1 is proteolyzed or shed by ADAM17 at a juxtamembrane site, resulting in the release of its N-terminal fragment, also known as glycocalicin, from platelet surface. Although the biological significance of ectodomain shedding of GPIb1 and function of glycocalicin remain to be defined, recent evidences suggest that shedding of GPIb1 plays an important role in detection and clearance of pathologically damaged platelets, and that blocking shedding of GPIb1 can hamper clearance of platelets stored in vitro. However, it is not possible at the present time to directly test these hypotheses, because it is not clear whether shedding of GPIb1 is merely an inconsequential indicator for the damaged and to-be-cleared platelets or actually the cause for platelet clearance. Such ambiguity is largely due to the usage of broad-spectrum metalloprotease inhibitors in the investigation and the lack of ability to modulate ectodomain shedding in a substrate-specific manner. Given the compelling need for a shedding inhibitor specific to GPIb1, we propose to screen the scFv phagemid library and the RNA aptamer library for binders that specifically recognize and bind to the juxtamembrane shedding cleavage site in GPIb1. Sufficiently high binding affinity should enable these binders to out-compete ADAM17 for access to, and only to, the GPIb1 shedding cleavage site, and thereby achieving specific inhibition of GPIb1 shedding. Both scFv phagemid and RNA aptamer libraries have been used successfully to produce ligands for various protein targets, some of which have been developed into FDA-approved drugs. Due to their difference in backbone structure, molecule size, folding and amenability to mutagenesis and other related properties, scFv and RNA libraries offer distinct and complementary choices for a shedding inhibitor. For the screening, recombinant proteins that contain the sequence flanking the GPIb1 shedding cleavage site in its native-like conformation will be immobilized as binding targets. Once appropriate binders are identified from the screening, they will be produced in large quantity and tested for their ability to bind the GPIb1 shedding cleavage site and inhibit shedding of GPIb1 in transfected mammalian cells and platelets. With a GPIb1-specific shedding inhibitor, we will be poised to investigate the biological significance of GPIb1 shedding and explore novel strategies to extend the shelf life of stored platelet concentrates. Moreover, our method to develop substrate-specific shedding inhibitors, if successfully developed, can be applied to many other shedding substrates in platelets or cells of interest, which may deliver significantly more membrane receptors as suitable targets for drug development.
Clearance of the stored platelet concentrates may depend on proteolytic removal of GPIb1 from the platelet surface. We plan to produce an agent that specifically inhibits the removal process. This agent is sorely needed to address the mechanism of platelet clearance and it may lead to novel strategies to extend the shelf life of stored platelet concentrates.
|Li, R; Emsley, J (2013) The organizing principle of the platelet glycoprotein Ib-IX-V complex. J Thromb Haemost 11:605-14|
|Liang, X; Russell, S R; Estelle, S et al. (2013) Specific inhibition of ectodomain shedding of glycoprotein Ib? by targeting its juxtamembrane shedding cleavage site. J Thromb Haemost 11:2155-62|