Hypothesis

Senolytic removal of senescent endothelial cells diminishes pathological VWF secretion and restores ADAMTS13 activity, thereby normalizing the FVIII/VWF‑dependent prothrombotic state that accompanies aging.

Rationale

Aging endothelial cells accumulate a senescent phenotype characterized by p53/p21 upregulation, NF‑κB‑driven transcription of VWF, and release of ultra‑large VWF multimers that escape proteolysis endothelial senescence drives VWF release. Simultaneously, oxidative stress in senescent endothelium suppresses ADAMTS13 expression, impairing cleavage of these multimers oxidative stress lowers ADAMTS13. The resulting imbalance fuels thrombin generation and microthrombi formation thrombin generation and microthrombi,[https://pmc.ncbi.nlm.nih.gov/articles/PMC12889501/].

Senolytics such as dasatinib + quercetin selectively induce apoptosis in p21‑high cells, clearing senescent endothelium without affecting functional progenitors senolytic clears senescent cells. We predict that this clearance will:

• Decrease plasma VWF antigen and FVIII activity (measured by ELISA and clotting assay).
• Reduce the proportion of high‑molecular‑weight VWF multimers (detected by agarose gel electrophoresis).
• Increase ADAMTS13 activity and antigen levels.
• Lower endogenous thrombin potential (ETP) in calibrated automated thrombography.
• Reduce thrombus weight in a ferric chloride‑induced carotid injury model in aged mice.

Experimental Design

Model: C57BL/6 mice aged 20‑24 months (equivalent to ~60‑70 human years). Groups (n=12 per group):

1. Vehicle control.
2. Dasatinib + quercetin (5 mg/kg + 50 mg/kg, oral, intermittent dosing 1 day/week for 6 weeks).
3. Positive control: young mice (3‑4 months) receiving vehicle.

Endpoints (collected at week 6):

• Plasma VWF antigen, FVIII activity, ADAMTS13 activity/antigen.
• VWF multimer profile.
• Thrombin generation (ETP, peak thrombin).
• Ex vivo tail‑bleeding time (to assess bleeding risk).
• In vivo thrombosis: ferric chloride injury, thrombus weight via histology.
• Immunofluorescence of aortic endothelium for senescence markers (p16, SA‑β‑gal) and endothelial coverage (CD31).

Statistical plan: One‑way ANOVA with Tukey post‑hoc; significance set at p<0.05. Power analysis (α=0.05, β=0.2) indicates n=10 detects a 25% change in VWF antigen; we use n=12 to accommodate attrition.

Falsifiability

If senolytic treatment does not produce a statistically significant reduction in VWF antigen/FVIII activity or fails to increase ADAMTS13 activity relative to vehicle‑treated aged mice, the hypothesis is refuted. Likewise, absence of changes in thrombin generation or thrombus weight despite molecular shifts would challenge the mechanistic link between endothelial senescence clearance and thrombotic phenotype.

Potential Impact

Confirming that senolytics reset the endothelial hemostatic balance would provide a mechanistic bridge between cellular senescence and age‑related thrombotic disease, supporting repurposing of senolytics for primary prevention of VTE, stroke, and myocardial infarction in the elderly.