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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session E

S35 - Effects of PROTAC-Mediated Degradation on Platelet Function​

Saturday, October 11, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Platelets are small, anucleate blood cells that regulate hemostasis and thrombosis. Bruton’s tyrosine kinase (BTK) plays a significant role in B cell receptor signaling and also contributes to platelet signaling and activation. Pharmacological inhibitors of BTK like Ibrutinib act by irreversibly binding to BTK active sites and blocking signal propagation. However, BTK inhibitor therapy is prone to resistance mechanisms along with substantial off-target effects, highlighting the need for novel approaches. Proteolysis targeting chimeras (PROTACs), a new type of treatment, are small molecules that target specific proteins of interest for ubiquitination, resulting in near complete proteasomal degradation, rather than inhibiting their enzymatic activity. PROTAC DD-03-171 has been developed to specifically target and degrade BTK, allowing us to better understand the signaling cascade in platelets.

Materials and

Methods: : Platelets were prepared from citrate anticoagulated blood from healthy human donors in accordance with an OHSU IRB-approved protocol.
Static Adhesion Assay:
Platelets (2 x 10^7/mL) were treated with vehicle (0.1% DMSO) or selected inhibitors and PROTAC degraders (1 µM) for 10 minutes or 2 hours on at 37°C. Platelets were then transferred to fibrinogen-coated coverslips and incubated at 37°C for 1 hour, then fixed using 4% paraformaldehyde. Coverslips were washed and were stained with antibodies and phalloidin to image BTK and actin in platelets, respectively.

Light Aggregation Assay:
Platelets (3x10^8/mL) were treated with vehicle (0.1% DMSO) or selected inhibitors and degraders (1 µM) for 10 minutes or 2 hours at 37°C, and then transferred to a CHRONO-LOG Model 490 4+4 Aggregometer. Platelets were simulated with 1 µg/mL crosslinked collagen related peptide (CRP-XL). Aggregation was measured as the percentage of light transmission over an interval of 4 minutes and 30 seconds.

Results, Conclusions, and Discussions:: We compared the effects of PROTAC DD-03-171 against the BTK inhibitor, ibrutinib, on washed platelets adhesion and spreading on fibrinogen, and through light-transmitted aggregometry. Additionally, we used fluorescent microscopy to stain BTK and analyzed the fluorescent intensity using an ImageJ macro. After 10 minutes, the platelets treated with DD-03-171 had similar surface area and slightly decreased light transmission when compared to the control group; however, after compared to vehicle control (0.1% DMSO), preincubation of platelets with DD-03-171 for 2 hours inhibited the adhesion and spreading of platelets on fibrinogen, as well as platelet aggregation in solution. Fluorescence microscopy staining and Western blot confirmed that 2 hour treatment with DD-03-171 eliminated BTK expression in platelets. The effects of BTK degradation on platelet spreading and aggregation were comparable to BTK inhibtion with ibrutinib. Short-term (10 minute) treatment of platelets with DD-03-171 had no effects on BTK expression or platelet function.

This study demonstrates PROTAC-targeted degradation of BTK effectively blocks BTK-mediated platelet function. However, further research is needed to determine whether PROTACs can overcome the potential side effects associated with pharmacological inhibition. PROTACs represent a promising tool that could lead to more precise manipulation and a deeper understanding of platelet signaling cascades.

Acknowledgements and/or References (Optional): :