Plasma Protein Signatures of a Murine Venous Thrombosis Model and Slc44a2 Knockout Mice Using Quantitative-Targeted Proteomics

Abstract

The plasma compartment of the blood holds important information on the risk to develop cardiovascular diseases such as venous thrombosis (VT). Mass spectrometry-based targeted proteomics with internal standards quantifies proteins in multiplex allowing generation of signatures associated with a disease or a condition. Here, to demonstrate the method, we investigate the plasma protein signatures in mice following the onset of VT, which was induced by RNA interference targeting the natural anticoagulants antithrombin and protein C. We then study mice lacking Slc44a2, which was recently characterized as a VT-susceptibility gene in human genome-wide association studies. We use a recently developed panel of 375 multiplexed mouse protein assays measured by mass spectrometry. A strong plasma protein siganture was observed when VT was induced. Discriminators included acute phase response proteins, and proteins related to erythrocyte function. In mice lacking Slc44a2, protein signature was primarily overruled by the difference between sexes and not by the absent gene. Upon separate analyses for males and females, we were able to establish a signature for Slc44a2 deficiency, in which glycosylation-dependent cell adhesion molecule-1 and thrombospondin-1 were shared by both sexes. The minimal impact of Slc44a2 deficiency on the measured plasma proteins suggests that the main effect of Slc44a2 on VT does not lay ultimately in the plasma compartment. This suggests further investigation into the role of this VT-susceptibility gene should perhaps also question the possible involvement in cellular mechanisms.

The choline transporter Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial function

“…Others have also shown that Slc44a2 increases thrombosis in various murine models 11 , 13 . Searching for mechanistic pathways through which Slc44a2 affects thrombosis, one study excluded an effect of Slc44a2 upon plasma proteins including coagulation factors, and proposed that Slc44a2 affects thrombosis through cellular pathways 12 . We confirm this prediction and extend this prior work, now demonstrating that Slc44a2 promotes thrombosis by regulating platelet metabolism.…”

“…GWAS studies have associated the SLC44A2 locus with human phenotypes including: hearing loss, Meniere's disease, and venous thrombosis 3,9 . Recent studies have explored the role of SLC44A2 in thrombosis [10][11][12][13] . Two studies found that Slc44a2 promotes thrombosis in a mouse model of laser injury or venous stenosis but did not identify the mechanisms underlying this phenomenon 11,13 .…”

“…A search for mechanisms of Slc44a2 affecting thrombosis found that Slc44a2 does not affect VWF levels in mice 13 . Another study explored the influence of Slc44a2 upon plasma proteins, and finding no difference in plasma proteins between wild-type and Slc44a2 null mice, concluded that Slc44a2 must influence thrombosis through cellular based mechanisms 12 .…”

Thrombosis and Haemostasis 2020 Editors' Choice Papers

“…In an effort to elucidate the precise mechanisms that trigger clotting in venous thrombosis, Tilburg et al 56 characterized its plasma signature in a venous thrombosis murine knockout model using mass spectrometry-based proteomics, thereby establishing a new tool to diagnose such pathologies. 57 devices.…”

Novel Approaches to Unravel Risk Factors and Mechanisms of Venous Thrombosis

“…Indeed, quantitative MS has been successfully applied to understand the pathomechanisms of multiple diseases. 2 In this issue of Thrombosis and Haemostasis, Tilburg et al attempt to uncover the plasma protein signatures upon induction of VT using state-of-the-art MS-based targeted proteomics in combination with a murine knockout model for SLC44A2, a recently identified susceptibility locus for VT. 3,4 With this elegant approach, they not only gain information about changes in the plasma proteome in VT, but also demonstrate the great advantages of MS-based proteomics such as specificity and the possibility to expand a limited sample volume to a huge dataset.…”