# In Vivo Regulation of Factor IXa by Protein S in Hemophilia and Systemic Hypercoagulability

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2021 · $366,434

## Abstract

Protein S (PS) is a critically important natural anticoagulant as demonstrated by fatal consumptive
coagulopathies in homozygous deficiency for mouse and man. Originally discovered as a cofactor for activated
protein C (APC), subsequent studies have demonstrated a plethora of APC-independent anticoagulant
mechanisms for PS without clear demonstration of their respective physiologic relevance. Recently, genetic
“rebalancing” studies in the mouse and direct biochemical evidence suggest an important interaction between
PS and factor IXa (FIXa) The objective of this proposal is to define the role of PS in the in vivo regulation of FIXa
activity. The central hypothesis is that PS is an important physiologic regulator of FIXa in both hemostasis and
systemic hypercoagulable states. The central hypothesis will be tested by pursuing three specific aims: 1) Identify
the predominant mechanism(s) for regulation of FIXa activity in human plasma, 2) Determine the contribution of
protein S affinity to the in vivo activity of recombinant FIX(a), and 3) Determine the contribution of protein S
deficiency to in vivo regulation of FIX(a) activity. To pursue these aims, we have developed a panel of
recombinant human FIX variants that possess reduced affinity for antithrombin (AT), heparin and PS. In the first
aim, inhibition of FIXa activity in the intrinsic Xase complex by PS, PS-TFPI and PS-APC will be examined
using purified components and the FIXa variants. Likewise, the contribution of AT- and PS-dependent
mechanisms to the regulation of plasma thrombin generation will be examined with the FIXa variants and
inhibitory antibodies in immunodepleted plasma. In the second aim, the human FIXa variants will first be
employed to evaluate the impact of AT, heparin and PS affinity on zymogen and protease recovery and
clearance. Secondly, dose-dependent in vivo hemostatic and thrombotic activity will be examined using
established models in the hemophilia B mouse to evaluate the contribution of AT and PS-dependent
mechanisms. In the third aim, Pros1 specific antisense oligonucleotides (ASO) will be employed to create PS
deficiency in wild type and hemophilia B mice. The impact of the ASO-mediated PS deficiency on bleeding and
thrombosis phenotypes will be evaluated using established models, including the ability to ameliorate the
baseline bleeding phenotype in hemophilia B mice. Further, the effect of ASO-mediated PS deficiency on the in
vivo hemostatic and thrombotic activity of injected FIXa variants will be examined in hemophilia B mice, including
the ability of human PS to “rescue” the effects of PS deficiency. Completion of these aims will elucidate the
critical role of the PS-FIXa interaction in the physiologic regulation of FIXa procoagulant activity within the intrinsic
Xase complex. The proposed research is innovative because it addresses the in vivo significance of a novel
mechanism for regulating the rate-limiting step in the coagulation response. This contribu...

## Key facts

- **NIH application ID:** 10069397
- **Project number:** 5R01HL149855-02
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** JOHN Patrick SHEEHAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $366,434
- **Award type:** 5
- **Project period:** 2020-01-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10069397

## Citation

> US National Institutes of Health, RePORTER application 10069397, In Vivo Regulation of Factor IXa by Protein S in Hemophilia and Systemic Hypercoagulability (5R01HL149855-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10069397. Licensed CC0.

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