Abstract
Fibrinolysis is an integral part of the matrix remodeling process that contributes to tissue repair. Fibrin clots are broken down during fibrinolysis in a controlled process. Fibrin degradation products (FDPs) have also been shown to have a role in the regulation of cell growth and are implicated in various vascular diseases. This protocol was designed to quantitatively measure the extent of fibrin breakdown and how this can be adapted as a tool to further investigate the pathway involved in fibrinolysis or fibrin degradation products. Until now, we haven’t found an alternative method to analysis fibrinolysis.
Keywords: Fibrin, Fibrinolysis, Fibrin degradation products, Fibrin breakdown product measurement, Matrix remodeling
Background
In hemostasis, fibrin plays a crucial role as both the ultimate substrate for fibrinolysis and the primary product of the coagulation cascade (Cesarman-Maus and Hajjar, 2005; Chapin and Hajjar, 2015). In addition, fibrin has been found to mediate the acute inflammatory response to implanted biomaterials and it has been suggested that fibrin plays a prominent role in leukocyte transmigration and thereby inflammation (Yakovlev and Medved, 2018). Fibrinolysis is a tightly regulated process by which a fibrin-rich thrombus is formed and degraded, to prevent blood clots from growing (Amengual and Atsumi, 2016) and to contribute to tissue repair (Houslay et al., 2019). In Fibrinolysis, a fibrin clot (the product of coagulation) is broken down. Fibrin degradation products (FDPs), fibrous proteins that act as mitogenic factors, can promote the proliferation of endothelial cells, smooth muscle cells and fibroblasts, and cholesterol deposition (Dong et al., 2017). Numerous studies have shown that FDPs are involved in various vascular diseases. For example, FDPs induce the adhesion and gathering of leucocytes, damaging blood vessel endothelium (Dong et al., 2017). Also, FDPs have been approved that have diverse effects in inflammatory processes and acute phase responses, such as activating TLR-4 and integrin and cell adhesionor migration (Schuliga, 2015). They inhibit several neutrophil functions crucial to the bactericidal role of these inflammatory cells (Gerdin et al., 1980). It is vital that a more complete understanding of the mechanisms behind the FDPs formation and related pathway is gained. In sight of this, we present an assay called the Fibrin breakdown assay to use in laboratory research. This assay is designed to measure the extent of fibrin degradation quantitatively. Until now, we have found no alternative method to analysis fibrinolysis.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
The negative control and positive control condition should be considered, a well with fibrin matrix but no cells, or a well with cells but no fibrin matrix, and treated under the same treatment for negative control; fibrin matrices + cells + pro-fibrin degradation compound(s) that have been validated in literature, or fibrin matrix + diseased-vascular cells that are known to become fibrotic as a positive control.
Recipes
Acknowledgments
Jiayue Ling is funded by the China Scholarship Council. We thank Kirsty F. Houslay for developing this assay. This protocol described herein were originally described in Houslay et al. (2019).
Competing interests
The authors declare that they have no conflicts of interest with the contents of this article.
References
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