Detection of α-Thrombin with Platelet Glycoprotein Ibα (GP1bα) for the Development of a Coagulation Marker

Abstract

The detection of prothrombotic markers is crucial for understanding thromboembolism and assessing the effectiveness of anticoagulant drugs. alpha-Thrombin is a marker that plays a critical role in the coagulation cascade process. However, the detection of this enzymatic molecule was hindered by the absence of an efficient modality in the clinical environment. Previously, we reported that one alpha-thrombin interacts with two alpha-chains of glycoprotein Ib (GPIb alpha), i.e., multivalent protein binding (MPB), using bioresponsive hydrogel nanoparticles (nanogels) and optical microscopy. In this study, we demonstrated that GPIb alpha-mediated platforms led to the highly sensitive and quantitative detection of alpha-thrombin in various diagnostic systems. Initially, a bioresponsive nanogel-based surface plasmon resonance (nSPR) assay was developed that responds to the MPB of alpha-thrombin to GPIb alpha. The use of GPIb alpha for the detection of alpha-thrombin was further validated using the enzyme-linked immunosorbent assay, which is a gold-standard protein detection technique. Additionally, GPIb alpha-functionalized latex beads were developed to perform latex agglutination (LA) assays, which are widely used with hospital diagnostic instruments. Notably, the nSPR and LA assays exhibited a nearly 1000-fold improvement in sensitivity for alpha-thrombin detection compared to our previous optical microscopy method. The superiority of our GPIb alpha-mediated platforms lies in their stability for alpha-thrombin detection through protein-protein interactions. By contrast, assays relying on alpha-thrombin enzymatic activity using substrates face the challenge of a rapid decrease in postsample collection. These results suggested that the MPB of alpha-thrombin to GPIb alpha is an ideal mode for clinical alpha-thrombin detection, particularly in outpatient settings.