A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis.

Mathivanan Chinnaraj,Nicola Pozzi,Vittorio Pengo

doi:10.3389/fimmu.2021.741589

Mathivanan Chinnaraj, Nicola Pozzi + Show 1 more

Open Access

https://doi.org/10.3389/fimmu.2021.741589

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Abstract

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R2 = 0.85) but not with aPT-A (R2 = 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.

Highlights

Antiphospholipid syndrome is a debilitating condition characterized by vascular thrombosis in the presence of antiphospholipid antibodies, which persist >12 weeks in patients’ plasma [1]
Recent studies from our laboratory have documented that aPS/ PT, despite being heterogeneous, preferentially bind to the N-terminal portion of prothrombin, known as fragment-1 [8]
We hypothesized that detection of Autoantibodies targeting prothrombin (aPT) in correlation with thrombosis might be possible by using a prothrombin derivative that can be immobilized to ELISA plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent

Summary

Introduction

Antiphospholipid syndrome is a debilitating condition characterized by vascular thrombosis in the presence of antiphospholipid antibodies (aPL), which persist >12 weeks in patients’ plasma [1]. Among aPL, anti-prothrombin antibodies (aPT) are believed to be clinically relevant since they are linked to thrombosis [2]. APT are detected using two methods [3]. Prothrombin is directly immobilized onto a hydrophilic plastic plate, in which case aPT are named aPT-A. Prothrombin is bound, in the presence of calcium ions, to plastic wells pre-coated with phosphatidylserine (PS), in which case aPT are referred as to anti-phosphatidylserine/ prothrombin antibodies (aPS/PT).

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