MSACL 2018 US Abstract

Topic: Proteomics

Differential Processing of High-Molecular-Weight Kininogen Throughout Normal Pregnancy

Stephenie Droll (Presenter)
National Institutes of Health

Bio: I graduated from Purdue University with a Bachelor of Science in genetics in 2016. Shortly after graduation, I accepted a postbaccalaureate intramural research training award at the National Institutes of Health in the labs of Dr. Zhen Zhao and Dr. Katherine Calvo.

Authorship: Stephenie Droll (1), Weixing Wang (1), Steven Drake (1), Ashley Kim (1), Zhe Cheng (2), Guoan Zhang (2), Zheng Cao (3), Katherine Calvo (1), Tony Hu (4) and Zhen Zhao (1,2)
(1) National Institutes of Health (2) Weill Cornell Medicine (3) Beijing obstetrics and gynecology hospital (4) Arizona State University

Short Abstract

Preeclampsia is a serious disorder of pregnancy resulting in adverse maternal and neonatal outcomes. Aberrant processing of high-molecular-weight kininogen(HK) in early pregnancy has been indicated preceding the onset of preeclampsia. HK derived peptides are possible biomarkers for the early detection of preeclampsia. The present study characterized the cleavage pattern of HK in longitudinal serum samples during normal pregnancy. Western blotting analysis demonstrated significantly decreased inactive HK and significantly increased active heavy chain (HC) and light chain (LC). LC-MS/MS analysis revealed significantly increased concentrations of HK peptide fragments during pregnancy. A large number of the cleavage peptide fragments mapped to LC-HK Domain 5, which down-regulates angiogenesis, inhibits endothelial cell proliferation and migration, and induces apoptosis of endothelial cells.

Long Abstract

Introduction

Preeclampsia is characterized by elevated blood pressure and proteinuria during pregnancy and is a major cause of adverse maternal outcomes. High-molecular-weight kininogen (HMWK) and its activated form (HKa) play a crucial role in coagulation and inflammation pathways associated with maternal physiological changes during pregnancy. HK remains inactive until plasma kallikrein cleaves kininogen to generate HKa, which is comprised of a heavy chain and a light chain linked by a disulfide bond. A further cleavage step releases bradykinin from the HC. Previous proteomics studies have identified kininogen derived peptides as possible biomarkers for the early detection of preeclampsia and gestational hypertension. The aim of the present study was to investigate the cleavage pattern of HK and HKa in longitudinal samples during normal pregnancy, which may provide insight into understanding the pathogenesis of pre- eclampsia.

Methods

Serum samples were longitudinally collected from sixty healthy pregnant women in each trimester (T). Western blotting was performed on 180 longitudinal serum samples from 60 participants using an antibody against high molecular weight kininogen (GeneTex). One hundred and fifty longitudinal serum samples from 50 participants were processed for circulating peptide enrichment using our well-designed nanoporous silica thin films (NanoTraps). A Thermo Fisher Scientific EASY-nLC 1000 coupled on-line to a Fusion Lumos mass spectrometer (Thermo Fisher Scientific) was used for comprehensive peptide analysis. Proteins and peptides were identified by database searching. The quantification was performed by MaxQuant and in-house scripts. Potential contaminants were filtered out. Statistical tests were performed in Prism 6 using the Friedman test, and p < 0.05 was considered significant.

Results

Western blotting of longitudinally collected serum samples revealed that the level of inactive HK significantly decreased during the course of normal pregnancy (T1 vs T2, p< 0.05; T1 vs T3, p < .0001). In contrast, an intermediate cleavage product increased during pregnancy (T1 vs T2, p< 0.005; T1 vs T3, p<0.01). Moreover, active HC (T1 vs T2, p< .0001; T1 vs T3, p < .0001; T2 vs T3, p<0.01) and LC (T1 vs T2, p< 0.0001; T1 vs T3, p < .0001; T2 vs T3, p<0.05) significantly increased during the normal pregnancy. LC-MS/MS analysis identified a total of 146 peptides derived from Kininogen 1, which is the precursor protein to HMWK, low molecular weight kininogen, and bradykinin. The majority of the identified peptides were mapped to HMWK. Approximately half of the identified peptides presented complete sets of data in 10 or more patients. More than 60 peptides were mapped to domain 5. Furthermore, a large number of Domain 5 peptides significantly increased during pregnancy (all p-values < .05).

Conclusions & Discussion

The present study demonstrated significantly decreased inactive HK and significantly increased active HC and LC of HK during pregnancy using longitudinal maternal serum samples. LC-MS/MS analyses revealed significantly increased concentrations of HK peptide fragments in T2 and T3, suggesting differential processing of HMWK in different phases of the normal pregnancy. A large number of the cleavage peptide fragments identified by LC-MS/MS mapped to LC Domain 5, which down-regulates angiogenesis, inhibits endothelial cell proliferation and migration, and induces apoptosis of endothelial cells. Future studies will attempt to reveal if the cleavage peptide fragments of HK can serve as non-invasive biomarkers for preeclampsia.


References & Acknowledgements:

This work was supported by the intermural program of the NIH clinical center.


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