Session: Research Posters

Paper Number: 173099

Effects of Mechanical Tension on Fibrin Network Structure and Enzymatic Degradation 

Introduction

Fibrinolysis is the proteolytic degradation of fibrin fibers, which are the main structural and mechanical component of blood clots. The individual fibrin fiber and network structure play a crucial role in fibrinolysis. The density, alignment, and stiffness of the fibrin network influences the accessibility of fibrinolytic enzymes. Fibrin degradation is primarily regulated by plasmin, activated from its inactive precursor, plasminogen, by tissue-type plasminogen activator (tPA), which noncovalently binds to fibrin. Mechanical tension within the fibrin network plays a critical role in modulating the accessibility of fibrin binding sites and altering the network structure. Tension can arise inherently during fibrin polymerization and network formation, or applied tension via contracting platelets or  blood flow. How tension influences blood clot breakdown and enzymatic degradation of the fibrin fiber network is understudied. We aim to investigate the role of applied tension and its effect on fibrinolysis of fibrin networks.

Contribution of the work toward advancing science and/or engineering

This research will offer novel insights of the chemical and biophysical mechanisms that govern clot degradation and the restoration of normal blood flow. Translationally, the examination of the interplay between tension and fibrinolysis may provide valuable understanding of the currently limited effectiveness of fibrinolytic treatments for thrombotic diseases, such as stroke.

Engineering-wise, this research contributes to improving in vitro studies of clot mechanics. Incorporating controlled mechanical loading when studying fibrinolysis allows more physiologically relevant modeling of the clot environment.

Methodology

Commercially available human pooled plasma from 25+ donors (Cone Biologics) was activated through the addition of 25 mM CaCl2 and 15 pM tissue factor to form an in vitro plasma clot; 275 ng/ml tissue-type plasminogen activator (tPA) was added to initiate fibrin degradation. Turbidity assays were conducted prior to mechanical testing to determine the optimal concentration of tPA, ensuring complete clot lysis within the 2-hour (7200 s) experimental timeframe.

The samples were allowed to polymerize in custom-fabricated molds for 15 minutes to ensure full crosslinking. Following polymerization, gels were mounted on the Biomomentum mechanical tester, and uniaxial tension was applied to stretch the sample at the strain rate of 0.1 mm/s to fixed strains — 5%, 25%, 50%, or 75% —simulating different degrees of applied mechanical tension. The rate of fibrinolysis was quantified by measuring the loss in force at a fixed strain over the course of time. A 2-way ANOVA was used to compare the differences between different strain and fibrinolytic rates. Samples were imaged using scanning electron microscopy (SEM) for visualization of the fibrin network at 10,000X magnification.

Preliminary results

Samples with/without tPA both exhibited relaxation — the mechanical process of fiber rearrangement caused by applied strain. The degradation rate was divided into two parts to reflect differences in the underlying mechanical processes. Part 1 involves fibrin fiber relaxation, paired with fibrinolysis leading to a greater reduction in tension over time accompanied by fiber realignment. Part 2 was characterized by continued lysis leading to clot breakdown. The overall lysis rate takes both parts into consideration.

An accelerated rate of lysis was observed during Part 2, likely due to fiber rearrangement and progressive decrease in the clot’s stiffness. Higher strain (50% and 75%) led to significantly accelerated fibrinolysis. Increasing amounts of applied strain resulted in fiber alignment and denser networks.

Preliminary conclusions

The present study identified the role of fibrin network tension on the rate of plasmin mediated degradation of fibrin fibers. It was found that applied strain changes the clot mechanics, which led to a two-part process of fibrin degradation. The first part of lysis was complicated due to ongoing active fiber relaxation, continued by steep increase of lysis rate at Part 2. Fixed strain induced fibrin thinning and diameter reduction, which are likely key factors contributing to the overall acceleration of lysis under strain. We observed that applied strain accelerates lysis and led to structural changes of the clot, as well as the expulsion of the serum.

Presenting Author: Eva Iungbliudt Rutgers, The State University of New Jersey

Presenting Author Biography: I am a PhD student in Professor Tutwiler's lab in the Department of Biomedical Engineering at Rutgers University.

Authors:

Eva Iungbliudt Rutgers, The State University of New Jersey
Andrew Gosselin Rutgers, The State University of New Jersey
Rebecca Risman Rutgers, The State University of New Jersey
Valerie Tutwiler Rutgers, The State University of New Jersey