Targeting <scp>CD148</scp> for Antithrombotic Therapy: Functional and Molecular Evaluation of <scp>AKB</scp> ‐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 AUC 10 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 IC 50 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.