Specific Inhibition Of Ectodomain Shedding Of GPIba By Targeting Its Shedding Cleavage Site
Authors: Liang Xin , Russell Susan R. , Estelle Sandra , Jones Limei H. , Cho Sungyun , Kahn Mark L. , Berndt Michael C. , Bunting Silvia T. , Ware Jerry , Li Renhao 
Published: October 2019

Background

Ectodomain shedding of GPIbα, a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has been obtained from animal studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17. We report herein novel monoclonal antibodies (MAbs) that specifically inhibit shedding of human GPIbα in platelets and may be potentially developed into an additive to improve platelet storage.

Methods

A synthetic peptide that corresponds to a human GPIbα sequence containing the shedding cleavage site was used as the antigen for mouse immunization. Hybridoma clones obtained from immunized mice were screened in ELISA assays for binding activities to the shedding-site peptide, and purified human GPIb-IX complex. Flow cytometry-based assays and Western blotting were used to screen for direct binding to washed platelets and inhibition of GPIbα shedding in washed platelets. Platelet aggregometry using different agonists was performed on MAb-treated platelets.

Results

Six MAbs were obtained and characterized for their abilities to bind GPIbα and inhibit its shedding. The purified antibodies bind, with varying affinities, to immobilized monovalent shedding-site peptide, but all exhibit strong binding to ovalbumin-conjugated shedding-site peptide. Similarly, these antibodies bind with varying affinities to immobilized GPIb-IX, but all exhibit strong binding to human platelets. Considering the multivalent nature of ovalbumin-conjugated peptide and the abundance of GPIbα on the platelet surface, these results indicate the divalent binding of GPIbα to these MAbs on the platelet surface. The prototypic clone, designated 5G6, and its monomeric Fab fragment, bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. It is specific to human GPIbα, as it does not bind mouse platelets. 5G6 exhibits similar inhibitory potency as the broad shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not inhibit shedding of other platelet receptors, such as GPVI and GPV. Treatment of washed human platelets with 5G6 or other MAb shedding inhibitors for an hour does not induce platelet activation or aggregation, and does not exhibit detectable effects on agonist-induced platelet aggregation. Consistently, infusion of 5G6 into human transgenic mice does not induce acute thrombocytopenia, unlike other MAbs targeting the N-terminal domain of GPIbα.

Conclusion

We have obtained, for the first time, reagents that specifically inhibit ectodomain shedding of human GPIbα with little effect on platelet functions. These reagents will be useful to define the functional significance of GPIbα shedding. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding.

Disclosures:

No relevant conflicts of interest to declare.