# Thrombosuppressive mechanisms of novel mutants discovered through an ENU mutagenesis screen

> **NIH NIH R01** · OAKLAND UNIVERSITY · 2020 · $375,000

## Abstract

Abstract
Venous thromboembolism (VTE) susceptibility genes are largely unknown. We used a sensitized ENU
mutagenesis screen of 6,739 mice to identify novel dominant thrombosis suppressor genes of the
perinatal lethal F5 Leiden homozygous (F5L/L), tissue factor pathway inhibitor (Tfpi+/-) heterozygous
phenotype. These highly penetrant, dominant mutations restored viability to the F5L/L Tfpi+/- mice and
were able to be successfully passed on to offspring. We named these suppressor genes MF5L for
Modifier of Factor 5 Leiden. In order to identify the MF5L13 suppressor, we used whole exome
sequencing to directly identify a single candidate suppressor mutation in exon 7 of the Actr2 gene. This
arginine (R) to glycine (G) mutation is in the highly conserved amino acid position 258 of the ARP2
protein (Actr2+/G) and suppresses thrombosis by an as yet unknown mechanism. The studies proposed
here seek to functionally characterize the MF5L13 suppressor mutation. Preliminary experimental results
on platelet aggregation and platelet dependent thrombus formation of Actr2+/G compared to wildtype
Actr2+/+ mice have suggested a platelet functional defect in the Actr2+/G mutants. Comparative analysis of
liver Serpine2 mRNA levels via qPCR also revealed a dramatic increase in Serpine2 levels in Actr2+/G
compared to Actr2+/+ mice. In the proposed studies, we will first use CRISPR/Cas9 genome editing to re-
create the Actr2+/G in a clean (non-mutagenized) mouse genetic background. Using this coisogenic
Actr2+/G mouse, we will first test its ability to thrombosuppress F5L/L Tfpi+/-. This mouse model will then be
crossed to the Tfpi deficient background (Tfpi-/-) to genetically dissect the thrombosuppressive
mechanisms of Actr2+/G relative to F5L/L and Tfpi. To determine whether there are more global expression
changes associated with Actr2+/G or if Serpine2 alone is affected, we will perform platelet and liver
RNAseq. We will then conduct a series of experiments designed to determine the thrombosuppressive
mechanism(s) of the Actr2+/G. We will determine the effects of Actr2+/G on platelet actin filament assembly
rates, the effects on agonist induced platelet aggregation and platelet activation events,
phosphatidylserine exposure, and thrombin generation, F5 activity and in vivo arterial and venous
thrombosis induction. This work will lead to new insights into the mechanisms of action of a unique
thrombosuppressor gene and may lead to new therapeutic targets for human thrombotic disease.

## Key facts

- **NIH application ID:** 9967102
- **Project number:** 5R01HL135035-04
- **Recipient organization:** OAKLAND UNIVERSITY
- **Principal Investigator:** Randal J Westrick
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $375,000
- **Award type:** 5
- **Project period:** 2017-08-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9967102

## Citation

> US National Institutes of Health, RePORTER application 9967102, Thrombosuppressive mechanisms of novel mutants discovered through an ENU mutagenesis screen (5R01HL135035-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9967102. Licensed CC0.

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