Measurement of Active Thrombin Bound to Circulating D‐Dimers as a Sensitive Biomarker for Prothrombotic Conditions

BACKGROUND: Thrombin activity plays a crucial role in hemostasis, and it is tightly regulated by a system of activators and inhibitors. Active, unregulated thrombin may initiate clotting without previous vessel injury, causing thrombosis. Thrombin bound to fibrin participates in fibrinogen transformation, while its inhibition by the naturally occurring heparin-antithrombin complex is impeded. Bound thrombin activity has been detected on fibrin degradation products. METHODS: D-dimers were isolated using a commercial ELISA kit. Mass spectrometry was used to confirm the presence of thrombin on captured D-dimers. D-dimer-bound thrombin activity was evaluated using specific substrate and inhibitors: hirudin-PPACK and heparin-antithrombin. Groups of 72 patients and 159 controls were analyzed. RESULTS: The activity of thrombin was assessed utilizing our system, demonstrating stability throughout the procedures. The thrombin concentration during fibrin polymerization was reflected in the thrombin activity levels detected on D-dimers. Analysis of patient plasma samples indicated the feasibility of detecting low levels of active thrombin. Significant differences in D-dimer-bound thrombin activity were observed between subjects with chronic (controls) and acute conditions (patients). CONCLUSIONS: We present a proof-of-concept method for the evaluation of thrombin activity on circulating D-dimers that may aid in the diagnosis of thrombotic events and the monitoring of anticoagulant therapy.