Circumventing the Platelet Storage Lesion By Relieving Platelet Inhibition Using a Novel Bi-Specific Antibody

Human platelets undergo a series of structural and functional deteriorations that begin at the time of collection, and continue during both room temperature and/or cold storage conditions awaiting transfusion into human subjects. The so-called “platelet storage lesion” is characterized by altered cellular metabolism, increased apoptosis, changes in platelet morphology, and a decline in membrane integrity, all of which contribute to reduced platelet viability and function, Upon transfusion, platelets affected by storage lesions exhibit impaired aggregation, reduced ability to form stable clots, and decreased circulating lifespan, together reducing their efficacy in controlling bleeding. To improve the reactivity of stored platelets, we have adopted the tactic of restraining the function of a naturally occurring inhibitor of platelet function, the cell adhesion and signaling molecule, Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1). PECAM-1 is a 130kDa member of the immunoglobulin gene superfamily expressed on platelets, endothelial cells, and certain leukocyte subsets. In monolayer cells like the vascular endothelium, PECAM-1 functions as a cell adhesion receptor that helps maintain cell-cell junctions that control the permeability barrier. In circulating cells, PECAM-1 functions primarily as an inhibitor of cellular activation - a function that is mediated by tyrosine residues in its cytoplasmic domain Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs), which become phosphorylated and recruit the SH2 domain-containing non-receptor protein tyrosine phosphatases, SHP-1 and SHP-2. To prevent this biochemical inhibitory reaction from occurring, we co-opted the ability of the transmembrane protein tyrosine phosphatase, CD148, to dephosphorylate phosphorylated tyrosine residues in PECAM-1. To promote cis-ligation of the phosphatase activity of CD148 with PECAM-1 cytoplasmic domain ITIMs to direct their dephosphorylation, we designed a novel bi-specific tandem single-chain antibody fragment (scFv) comprised of the variable region of an antibody specific for CD148 linked to the variable region of an antibody specific for PECAM-1. When added to human platelets, this engineered bi-specific scFv enhanced platelet secretion, and potentiated platelet aggregation and activation induced by weak platelet agonists, especially in stored platelets. Finally, tandem CD148/PECAM-1 scFv treatment also improved whole blood thrombus formation on collagen-coated surfaces under conditions of flow. Taken together, these results suggest that ITIM receptor inhibition through enforced phosphatase recruitment may be a useful strategy for increasing the functionality of donor-derived stored platelets.

Newman:Rallybio: Consultancy, Research Funding.