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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. Microplastic Effects on Thrombin–Fibrinogen Clotting Dynamics Measured via Turbidity and Thromboelastography

   https://doi.org/10.3390/biom12121864  

   Tran, Daniela Q; Stelflug, Nathan; Hall, Abigail; Nallan_Chakravarthula, Tanmaye; Alves, Nathan J (December 2022, Biomolecules)

   Micro/nanoplastics, whether manufactured or resulting from environmental degradation, can enter the body through ingestion, inhalation, or dermal pathways. Previous research has found that nanoplastics with diameters of ≤100 nm can translocate into the circulatory system in a dose-dependent manner and potentially impact thrombosis and hemostasis. To investigate the direct effects of microplastics on fibrin clot formation, a simplified ex vivo human thrombin/fibrinogen clot model was utilized. The 100 nm polystyrene particles (non-functionalized [nPS] and aminated [aPS]) were preincubated (0–200 µg/mL) with either thrombin or fibrinogen, and fibrin clot formation was characterized via turbidity and thromboelastography (TEG). When the particles were preincubated with fibrinogen, little effect was observed for aPS or nPS on turbidity or TEG up through 100 µg/mL. TEG results demonstrated a significant impact on clot formation rate and strength, in the case of nPS preincubated with thrombin exhibiting a significant dose-dependent inhibitory effect. In conclusion, the presence of microplastics can have inhibitory effects on fibrin clot formation that are dependent upon both particle surface charge and concentration. Negatively charged nPS exhibited the most significant impacts to clot strength, turbidity, and rate of fibrin formation when first incubated with thrombin, with its impact being greatly diminished when preincubated with fibrinogen in this simplified fibrin clot model. 

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3. Fibrin-modulating nanogels for treatment of disseminated intravascular coagulation

   https://doi.org/10.1182/bloodadvances.2020003046  

   Mihalko, Emily P.; Sandry, Megan; Mininni, Nicholas; Nellenbach, Kimberly; Deal, Halston; Daniele, Michael; Ghadimi, Kamrouz; Levy, Jerrold H.; Brown, Ashley C. (February 2021, Blood Advances)

   null (Ed.)

   Abstract Disseminated intravascular coagulation (DIC) is a pathological coagulopathy associated with infection that increases mortality. In DIC, excessive thrombin generation causes symptoms from formation of microthrombi to multiorgan failure; bleeding risks can also be a concern because of clotting factor consumption. Different clinical events lead to DIC, including sepsis, trauma, and shock. Treatments for thrombotic episodes or bleeding presentation in DIC oppose each other, thus creating therapeutic dilemmas in management. The objective of this study was to develop fibrin-specific core-shell nanogels (FSNs) loaded with tissue-type plasminogen activator (tPA) to treat the microcirculatory complications of DIC, which would facilitate targeted clot dissolution to manage microthrombi and the potential consumptive coagulopathy that causes bleeding. FSNs enhance formation of actively polymerizing clots by crosslinking fibrin fibers, but they can also target preexisting microthrombi and, when loaded with tPA, facilitate targeted delivery to lyse the microthrombi. We hypothesized that this dual action would simultaneously address bleeding and microthrombi with DIC to improve outcomes. In vivo, tPA-FSNs decreased the presentation of multiorgan microthrombi, recovered platelet counts, and improved bleeding outcomes in a DIC rodent model. When incorporated with human DIC patient plasma, tPA-FSNs restored clot structure and clot growth under flow. Together, these data demonstrate that a fibrinolytic agent loaded into fibrin-targeting nanogels could improve DIC outcomes. 

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4. Fibrin protofibril packing and clot stability are enhanced by extended knob-hole interactions and catch-slip bonds

   https://doi.org/10.1182/bloodadvances.2022006977  

   Asquith, Nathan L.; Duval, Cédric; Zhmurov, Artem; Baker, Stephen R.; McPherson, Helen R.; Domingues, Marco M.; Connell, Simon D.; Barsegov, Valeri; Ariëns, Robert A. (July 2022, Blood Advances)

   Abstract Fibrin polymerization involves thrombin-mediated exposure of knobs on one monomer that bind to holes available on another, leading to the formation of fibers. In silico evidence has suggested that the classical A:a knob-hole interaction is enhanced by surrounding residues not directly involved in the binding pocket of hole a, via noncovalent interactions with knob A. We assessed the importance of extended knob-hole interactions by performing biochemical, biophysical, and in silico modeling studies on recombinant human fibrinogen variants with mutations at residues responsible for the extended interactions. Three single fibrinogen variants, γD297N, γE323Q, and γK356Q, and a triple variant γDEK (γD297N/γE323Q/γK356Q) were produced in a CHO (Chinese Hamster Ovary) cell expression system. Longitudinal protofibril growth probed by atomic force microscopy was disrupted for γD297N and enhanced for the γK356Q mutation. Initial polymerization rates were reduced for all variants in turbidimetric studies. Laser scanning confocal microscopy showed that γDEK and γE323Q produced denser clots, whereas γD297N and γK356Q were similar to wild type. Scanning electron microscopy and light scattering studies showed that fiber thickness and protofibril packing of the fibers were reduced for all variants. Clot viscoelastic analysis showed that only γDEK was more readily deformable. In silico modeling suggested that most variants displayed only slip-bond dissociation kinetics compared with biphasic catch-slip kinetics characteristics of wild type. These data provide new evidence for the role of extended interactions in supporting the classical knob-hole bonds involving catch-slip behavior in fibrin formation, clot structure, and clot mechanics. 

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5. Microplastic Effects on Thrombin-Fibrinogen Clotting Dynamics Measured via Turbidity

   https://doi.org/10.18060/26812  

   Stelflug, Nathan; Tran, Daniela Q; Hall, Abigail; Chakravarthula, Tanmaye Nallan; Alves, Nathan J (December 2022, Journal of Clinical Prosthodontics and Implantology)

   Background/Hypothesis:Widespread use of plastic has created a world where exposure to microplastics is inevitable leading to their presence in our circulatory system. This raises questions about microplastics impact on thrombosis. Aminated polystyrene (aPS) plastics have been shown to increase platelet aggregation and thrombus formation in animal models. Given this, we hypothesized that aPS administration will increase the rate of fibrin clot formation in a simple thrombin-fibrinogen clot model. Project Methods:We evaluated how concentrations of 25-200 μg/mL of 100 nm aPS particles affect fibrin clot formation using turbidity assays. To determine the effect of surface charge, experiments were also performed with non-modified polystyrene (nPS) particles. Microplastics were pre-incubated either with physiological concentrations of fibrinogen or thrombin and the clot formation was measured using turbidity at 405 nm every 10 seconds over 45-minutes. Clotting parameters such as maximum turbidity (TurbMax), time to 90% maximum turbidity (TurbTime), and clot formation rate (Vmax) were determined and compared to controls without microplastics. Results:When increasing concentrations of aPS were preincubated with thrombin or fibrinogen, there was less than a 2-fold change in Vmax, TurbMax, and TurbTime. When increasing concentrations of nPS were preincubated with thrombin, there was up to a 27-fold decrease in Vmax, 2.4-fold decrease in TurbMax, and 4.36-fold increase in TurbTime compared to the control. Whereas preincubation of nPS with fibrinogen resulted in 1.86-fold decrease in Vmax, 1.63-fold decrease in TurbMax, and 2.30-fold increase in TurbTime. Potential Impact:In this simplified clotting model, it was surprising to find inhibitory effects on clot formation and that they were more pronounced with nPS than with aPS. However, these results align with increase prothrombin time observed in literature in presence of aPS. Therefore, future studies with more complex clotting models need to be performed before claims can be made on the impact of microplastics on thrombosis. 

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