Individuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin‐III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient‐derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin‐activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule‐1 (VCAM‐1) mediated by thrombin. Our findings suggest that, by attenuating thrombin‐mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.
The maintenance of hemostasis to ensure vascular integrity is dependent upon the rapid conversion of zymogen species of the coagulation cascade to their enzymatically active forms. This process culminates in the generation of the serine protease thrombin and polymerization of fibrin to prevent vascular leak at sites of endothelial cell injury or loss of cellular junctions. Thrombin generation can be initiated by the extrinsic pathway of coagulation through exposure of blood to tissue factor at sites of vascular damage, or alternatively by the coagulation factor (F) XII activated by foreign surfaces with negative charges, such as glass, through the contact activation pathway. Here, we used transient particle tracking microrheology to investigate the mechanical properties of fibrin in response to thrombin generation downstream of both coagulation pathways. We found that the structural heterogeneity of fibrin formation was dependent on the reaction kinetics of thrombin generation. Pharmacological inhibition of FXII activity prolonged the time to form fibrin and increased the degree of heterogeneity of fibrin, resulting in fibrin clots with reduced mechanical properties. Taken together, this study demonstrates a dependency of the physical biology of fibrin formation on activation of the contact pathway of coagulation.
more » « less- Award ID(s):
- 1842580
- NSF-PAR ID:
- 10488166
- Publisher / Repository:
- Applied Rheology
- Date Published:
- Journal Name:
- Applied Rheology
- Volume:
- 33
- Issue:
- 1
- ISSN:
- 1617-8106
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.more » « less
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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.more » « less
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Abstract To regenerate functional muscle tissue, engineered scaffolds should impart topographical features to induce myoblast alignment by a phenomenon known as contact guidance. Myoblast alignment is an essential step towards myotube formation, which is guided
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1515/arh-2022-0129
