Targeting CD148 for Antithrombotic Therapy: Functional and Molecular Evaluation of AKB-9778.

Abstract

CD148 is the primary receptor-type protein tyrosine phosphatase (PTP) regulating platelet activation, and its inhibition has been proposed as a novel anti-thrombotic strategy with potentially lower bleeding risk than current therapies. However, selective and potent inhibitors of CD148 are currently lacking. AKB-9778 (razuprotafib), a potent inhibitor of the closely related vascular endothelial-PTP (VE-PTP), has also been reported to inhibit CD148. This study investigated the effect of AKB-9778 on in vitro whole-blood thrombogenicity, examined the molecular basis of its interaction with the CD148 catalytic domain using molecular docking, and assessed inhibitor selectivity with an in vitro phosphatase assay. In a total-thrombus formation analysis system (T-TAS), AKB-9778 partially inhibited thrombus formation, reducing AUC₁₀ by 28%, without significantly affecting occlusion start time or occlusion time. The inhibitor did not alter collagen-stimulated CD62P platelet surface expression or PAC-1 binding to the activated integrin αIIbβ3. Molecular docking using CB-Dock predicted a binding configuration in which the phenylsulfamic acid group of AKB-9778 is orientated towards the catalytic cysteine, with additional conformations reflecting ligand flexibility. Site-directed mutagenesis of Tyr1071, Gln1283, His1206, and Asn1073 to alanine reduced the inhibitory effect in phosphatase assays, supporting interaction with these residues. Notably, the IC₅₀ of AKB-9778 against CD148 was approximately 300-fold higher than previously reported values, consistent with the wide variability across studies. Despite this reduced potency, these findings support the concept of CD148 inhibition as a potential anti-thrombotic strategy and suggest that AKB-9778 may serve as a useful prototype for developing more selective and efficacious CD148 inhibitors.