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1. Mathematical modeling to understand the role of bivalent thrombin-fibrin binding during polymerization

   https://doi.org/10.1371/journal.pcbi.1010414  

   Kelley, Michael A. ; Leiderman, Karin ( September 2022 , PLOS Computational Biology)

   Vavylonis, Dimitrios (Ed.)

   Thrombin is an enzyme produced during blood coagulation that is crucial to the formation of a stable clot. Thrombin cleaves soluble fibrinogen into fibrin, which polymerizes and forms an insoluble, stabilizing gel around the growing clot. A small fraction of circulating fibrinogen is the variant γ A / γ ′, which has been associated with high-affinity thrombin binding and implicated as a risk factor for myocardial infarctions, deep vein thrombosis, and coronary artery disease. Thrombin is also known to be strongly sequestered by polymerized fibrin for extended periods of time in a way that is partially regulated by γ A / γ ′. However, the role of γ A / γ ′-thrombin interactions during fibrin polymerization is not fully understood. Here, we present a mathematical model of fibrin polymerization that considered the interactions between thrombin, fibrinogen, and fibrin, including those with γ A / γ ′. In our model, bivalent thrombin-fibrin binding greatly increased thrombin residency times and allowed for thrombin-trapping during fibrin polymerization. Results from the model showed that early in fibrin polymerization, γ ′ binding to thrombin served to localize the thrombin to the fibrin(ogen), which effectively enhanced the enzymatic conversion of fibrinogen to fibrin. When all the fibrin was fully generated, however, the fibrin-thrombin binding persisted but the effect of fibrin on thrombin switched quickly to serve as a sink, essentially removing all free thrombin from the system. This dual role for γ ′-thrombin binding during polymerization led to a paradoxical decrease in trapped thrombin as the amount of γ ′ was increased. The model highlighted biochemical and biophysical roles for fibrin-thrombin interactions during polymerization and agreed well with experimental observations. 

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2. A Mathematical Model of Bivalent Binding Suggests Physical Trapping of Thrombin within Fibrin Fibers

   https://doi.org/10.1016/j.bpj.2019.09.003  

   Kelley, Michael ; Leiderman, Karin ( October 2019 , Biophysical Journal)
3. An integrated microfluidic platform for selective and real-time detection of thrombin biomarkers using a graphene FET

   https://doi.org/10.1039/D0AN00251H  

   Khan, Niazul I. ; Mousazadehkasin, Mohammad ; Ghosh, Sujoy ; Tsavalas, John G. ; Song, Edward ( June 2020 , The Analyst)

   null (Ed.)

   Lab-on-a-chip technology offers an ideal platform for low-cost, reliable, and easy-to-use diagnostics of key biomarkers needed for early screening of diseases and other health concerns. In this work, a graphene field-effect transistor (GFET) functionalized with target-binding aptamers is used as a biosensor for the detection of thrombin protein biomarker. Furthermore, this GFET is integrated with a microfluidic device for enhanced sensing performances in terms of detection limit, sensitivity, and continuous monitoring. Under this platform, a picomolar limit of detection was achieved for measuring thrombin; in our experiment measured as low as 2.6 pM. FTIR, Raman and UV-Vis spectroscopy measurements were performed to confirm the device functionalization steps. Based on the concentration-dependent calibration curve, a dissociation constant of K D = 375.8 pM was obtained. Continuous real-time measurements were also conducted under a constant gate voltage ( V GS ) to observe the transient response of the sensor when analyte was introduced to the device. The target selectivity of the sensor platform was evaluated and confirmed by challenging the GFET biosensor with various concentrations of lysozyme protein. The results suggest that this device technology has the potential to be used as a general diagnostic platform for measuring clinically relevant biomarkers for point-of-care applications. 

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4. Micellar Thrombin-Binding Aptamers: Reversible Nanoscale Anticoagulants

   https://doi.org/10.1021/jacs.7b07799  

   Roloff, Alexander ; Carlini, Andrea S. ; Callmann, Cassandra E. ; Gianneschi, Nathan C. ( November 2017 , Journal of the American Chemical Society)
5. Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

   https://doi.org/10.1038/s41598-021-88432-z  

   Peacock, Riley B. ; McGrann, Taylor ; Tonelli, Marco ; Komives, Elizabeth A. ( April 2021 , Scientific Reports)

   Serine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

    

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