PROTAC-mediated, concentration-dependent degradation of target proteins of interest in human platelets. Washed platelets at 4 × 10⁸/ml (A) or platelets in PRP (B-G) were incubated with the indicated concentrations of PROTACs for 4 h/30°C or 20 h/30°C, respectively. Platelets in PRP were pretreated with DMSO, 50 μM Pom, or 10 μM NAE inhibitor for 4 h/30°C before incubation with 10 μM of DD-04-015 or 3 μM of DD-03-171 for 20 h/30°C (D) Platelets in PRP were subsequently washed in CGS and resuspended in Hepes Tyrodes at 4 × 10⁸/ml (B-G). Washed platelets were lysed in either 4× NuPage sample buffer containing 0.5M DTT and subjected to SDS-PAGE/immunoblotting with the indicated antibodies (A-D, F-G) or lysed in RIPA buffer for TMT proteomics analysis (E). Volcano plots of the TMT protein data show the log₂ fold change in protein expression of PROTAC-treated platelets compared to vehicle (DMSO) treated controls against the -Log₁₀ (P-value) from a paired t-test (E). Each dot is representative of 1 protein. The grey dots represent proteins with a P-value > .05; blue indicates proteins with a P-value < .05 that are <75% degraded; red indicates proteins with a P-value < .05 that are >75% degraded. Volcano plots were made using the “EnhancedVolcano” R package. TEC is indicated in grey as it did not pass the analytical software internal threshold for TMT quantification (E). Western blot analysis confirmed the TMT proteomics data (F). The graphs represent quantification of the results using Odyssey Licor software, expressed as percentage of vehicle control ± SEM (A,B,F-G). Curves (A,B) were fitted by non-linear regression using GraphPad Prism 9. The statistical analysis of (F,G) was performed using a repeated measures two-way ANOVA or mixed-effects analysis, ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗ P ≤ .005, and ∗∗∗∗ P ≤ .001. Shown are representative blots and graphs (n = 3-4; A:3, B:3, C:4, D:3, E:3, F:3, G:4). DMSO, dimethyl sulfoxide; PRP, platelet-rich plasma; Pom, pomalidomide; NAE, NEDD8- activating enzyme.