The effect of PROTACs upon platelet functionality and thrombi formation. Platelets in PRP were incubated with vehicle (DMSO) or the indicated concentration of PROTAC TL12-186, DD-04-015, and DD-03-171 at 3 μM for 20 h/30°C (A-C,E). Alternatively, PRP was incubated with the indicated inhibitors for 10 min (D,E). Platelets were washed and resuspended at 2 × 10⁷/ml for FACS analysis (A-D). Washed platelets were stimulated with 2 μg/ml CRP-XL (A-D) or 10 μM TRAP-6 (B-D) for 10 minutes in the presence of PAC1-FITC/CD62P-PE to assess integrin α_IIbβ₃ activation and P-selectin expression, respectively. Alternatively, platelets were stimulated by a combination of 2 μg/ml CRP and 1 U/ml thrombin in the presence of Annexin-V-488 fluorescent dye to measure levels of PS exposure (A). Results are expressed as mean % of maximal normalized signal intensity ± SEM, n = 4 (A-D). For in vitro flow thrombosis experiments PRP was treated with vehicle (DMSO), DD-03-171, CGI-1764, and ibrutinib and recombined with the red cell layer of fresh blood from the same donor. Whole blood was then flown over a collagen Vena8 GCS Cellix biochip using a laminar flow rate of 3.07 ml/h for a sheer rate of 1000/s. Statistical analysis was performed using a repeated measures two-way ANOVA, ∗P ≤ .05, ∗∗P ≤ .01 (B-D); or two-way ANOVA, ∗∗∗∗P < .0001, ∗∗∗P ≤ .001 (E). Shown are representative blots and graphs (n = 3-4; A:4, B:4, C:4, D:4, E: 3). Max, maximum; PAR-1, protease activated receptor-1.