Inhibition of platelet kinase signaling and scaffolding activities with small molecule proteolysis-targeting chimeras (PROTACs)

Abstract Tyrosine kinase inhibitors (TKIs) play an expanding role in treating cancers and other diseases, including thromboinflammatory and vascular conditions. However, many TKIs cause off-target or toxic effects in healthy cells of the blood and vascular systems especially platelets, as bleeding and thrombotic complications are commonly associated with TKIs in and out of clinical trials. Bleeding risks are particularly notable for patients with B cell malignancies treated with TKIs such as ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor. Studies over the past decade have detailed how ibrutinib and other BTK inhibitors disrupt platelet function through direct (“on-target”) and indirect (“off-target”) mechanisms. In contrast, many TKIs targeting other kinase activities do not appear to carry the same bleeding risks as BTK inhibitors, highlighting the diversity of kinase function in blood cells. For example, Abl inhibitor asciminib does not affect platelet function and is not associated with bleeding, while other Abl inhibitors like ponatinib abrogate platelet function and increase risk of thrombosis. Beyond off-target toxicities of TKIs, evolving therapeutic resistances to TKIs present challenges in the clinic, fueling the need for novel agents to modulate signaling systems in disease. Small molecule proteolysis-targeting chimeras (PROTACs), which specifically target proteins for degradation rather than merely inhibiting their signaling activities, have emerged as agents of interest to address some limitations of kinase inhibitors. In addition, kinases like BTK and Abl are increasingly recognized to also serve structural roles not addressed by enzymatic inhibition. To this end, PROTACs hold promise in targeting pathogenic scaffolding activities of these and other proteins in diseased cells, but the effects of PROTACs targeting BTK and other signaling proteins on platelet signaling and vascular function remain mostly unexamined. Recent biochemical studies demonstrate that ex vivo treatment of purified human platelets with PROTACs can rapidly degrade targets such as BTK. Building on this potential for mechanistic studies and therapeutic applications, we examined the effects of PROTACs targeting BTK, Abl, and other kinases on platelet protein expression, signaling, and function. We found that treatment of human platelets with BTK PROTACs (DD-03-171, NX-5948) for 2 hours led to loss of BTK protein and reduced phosphorylation of the BTK substrate PLCγ2 pY1217 following stimulation of platelets with the immunotyrosine activation motif (ITAM) receptor glycoprotein GPVI. Western blot analysis similarly demonstrated that PROTACs targeting Abl1/2 (GMB475), GSK3α/β (PT-65), and p38 MAPK (SJFα) specifically degraded their respective targets in platelets. Similar to ibrutinib treatment, BTK degradation impaired platelet aggregation, substrate adhesion and spreading, as determined by Born light transmission aggregometry and differential interference contrast microscopy. Control experiments show that PROTAC reagents alone did not alter platelet function, as platelets treated with PROTACs for 10 min expressed proteins at levels similar to untreated controls, and responses remained intact. Notably, while selective Abl kinase inhibitors (e.g. asciminib) do not alter platelet function, Abl degradation with PROTAC GMB475 significantly impaired platelet spreading on fibrinogen-coated surfaces, suggesting a structural role for Abl independent of kinase activity. This finding aligns with recent studies in other myeloid cells, indicating a scaffolding role for Abl in organizing signaling and cytoskeletal components in platelets, such as the Abl interactor ABI1, which is not affected by Abl kinase inhibition. Together, our findings support mechanistic distinctions between kinase inhibition and targeted protein degradation in modulating platelet signaling and function. The disruption of platelet function resulting from PROTAC-mediated degradation of BTK and Abl suggests that these and other kinases serve structural roles independent of their enzymatic activity, highlighting therapeutic challenges and provocative translational opportunities for PROTACs in platelet research. Future studies of the effects of PROTACs relative to enzymatic kinase inhibitors will further inform scaffolding roles for these and other kinases in platelets while also supporting the development of safer and more precise strategies for managing kinase-driven diseases.