# Role of RIP3-laden extracellular vesicles in thrombosis and aortic aneurysm

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2021 · $512,637

## Abstract

Intramural or intraluminal blood clots are commonly present in human aortic aneurysms, a progressive
weakening and dilatation of aorta that is associated with depletion of smooth muscle cells (SMCs), degradation
of matrix proteins, and infiltration of inflammatory cells. Targeting constituents that are vital for thrombus
formation have been shown to reduce aneurysm severity in mouse models. However, limited knowledge exists
with regard to the molecular mechanisms that promote thrombus formation within aneurysms. In preliminary
studies, we demonstrated that extracellular vesicles (EVs) isolated from human plasma contained receptor
interacting protein kinase 3 (RIP3), an intracellular signaling protein that is critical to SMC necrosis. Extensive
preliminary studies, performed both in vivo and in vitro, demonstrated that RIP3 has a pro-thrombotic function
outside of cells. Analysis of plasma samples from aortic aneurysm patients showed a significant linear
correlation between plasma RIP3 levels and coagulation. Two related, yet independent specific aims are
proposed to test the central hypothesis that injured aortic SMCs release EVs that are rich in RIP3. When
discharged to the extracellular space, RIP3 stimulates thrombosis by interacting with coagulation
components.
In Aim 1, we will use various in vitro approaches to address mechanistic questions, including how RIP3 is
packed inside EVs and how extracellular RIP3 stimulates coagulation. Aim 1a tests whether RIP3 is sorted into
EVs by binding to proteins involved in endosomal sorting. Aim 1b seeks to demonstrate in an ex vivo model
that aneurysm-affected aortic tissues promote plasma to undergo coagulation. Aim 1c uses quantitative
proteomic analysis to determine the “protein signature” of EVs released by stressed SMCs. Aim 1d exams
whether RIP3 stimulates thrombosis at least in part by interacting with Factor IX, one of the serine proteases of
the coagulation system. In Aim 2, using a preclinical model of aortic aneurysm, we will test the hypothesis that
RIP3-carrying EVs contribute to aortic thrombosis. Aim 2a determines whether mice lacking the EV packing
factor Rab27a/b respond to angiotensin II with diminished thrombosis and aortic pathology. Aim 2b will further
establish the role of EVs in aneurysm by attempting to rescue the deficient thrombotic phenotype of Rip3-/-
mice with RIP3-carrrying EVs. As a way to translate basic findings to the clinical management of aneurysm,
Aim 2c examines the relationship between plasma RIP3 levels and clinical outcomes using an existing tissue
and data bank of human aortic aneurysm. By proving the novel extracellular function of RIP3 in coagulation,
this proposal will have a paradigm-shifting impact on the field of thrombosis and aneurysm.

## Key facts

- **NIH application ID:** 10214685
- **Project number:** 5R01HL149404-02
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Bo Liu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $512,637
- **Award type:** 5
- **Project period:** 2020-07-15 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10214685

## Citation

> US National Institutes of Health, RePORTER application 10214685, Role of RIP3-laden extracellular vesicles in thrombosis and aortic aneurysm (5R01HL149404-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10214685. Licensed CC0.

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