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Physical determinants of fibrinolysis in single fibrin fibers.

Bucay I, O'Brien ET, Wulfe SD, Superfine R, Wolberg AS, Falvo MR, Hudson NE - PLoS ONE (2015)

Bottom Line: We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected.Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain.These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Fibrin fibers form the structural backbone of blood clots; fibrinolysis is the process in which plasmin digests fibrin fibers, effectively regulating the size and duration of a clot. To understand blood clot dissolution, the influence of clot structure and fiber properties must be separated from the effects of enzyme kinetics and perfusion rates into clots. Using an inverted optical microscope and fluorescently-labeled fibers suspended between micropatterned ridges, we have directly measured the lysis of individual fibrin fibers. We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected. Thrombin and plasmin dose-response experiments showed that the elongation behavior was independent of plasmin concentration, but was instead dependent on the concentration of thrombin used during fiber polymerization, which correlated inversely with fiber diameter. Thinner fibers were more likely to lyse, while fibers greater than 200 ± 30 nm in diameter were more likely to elongate. Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain. Using polymer physics- and continuum mechanics-based mathematical models, we show that fibers polymerize in a strained state and that thicker fibers lose their prestrain more rapidly than thinner fibers during lysis, which may explain why thick fibers elongate and thin fibers lyse. These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities.

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Images of elongated and lysed fibrin fibers.Epi-fluorescence microscopy of fibrin fibers suspended across structured surfaces (bar = 20 μm). Samples were labeled with 20 nm red fluorescent beads and polymerized with 0.11–11 U/mL of human thrombin. Images of fibers before (A, C, E, G, I, K, M) and 5–15 minutes after (B, D, F, H, J, L, N) addition of plasmin. Samples 1–2 were treated with 20 μL of 1.0 U/mL of plasmin and display a lysed fiber (B) or a lysed and elongated fiber in the same field of view (D). Samples 3–7 were treated with 20 μL of plasmin ranging from 0.6 U/mL–6.0 U/mL and show elongated fibers, exhibiting extensions up to 10%.
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pone.0116350.g002: Images of elongated and lysed fibrin fibers.Epi-fluorescence microscopy of fibrin fibers suspended across structured surfaces (bar = 20 μm). Samples were labeled with 20 nm red fluorescent beads and polymerized with 0.11–11 U/mL of human thrombin. Images of fibers before (A, C, E, G, I, K, M) and 5–15 minutes after (B, D, F, H, J, L, N) addition of plasmin. Samples 1–2 were treated with 20 μL of 1.0 U/mL of plasmin and display a lysed fiber (B) or a lysed and elongated fiber in the same field of view (D). Samples 3–7 were treated with 20 μL of plasmin ranging from 0.6 U/mL–6.0 U/mL and show elongated fibers, exhibiting extensions up to 10%.

Mentions: To study individual fiber behavior during fibrinolysis and the timescales within which plasmin can lyse individual fibers, we polymerized and suspended fibrin across micropatterned ridge-and-valley structures, as previously described [25]. Fibrin fibers were labeled with 20 nm red fluorescent beads and observed under an optical microscope. We then added plasmin (0.06–6 U/mL) to the fibrin samples and recorded fiber behavior for thirty minutes. Of fibers that lysed, fibers treated with the lowest plasmin concentration (0.06 U/mL) lysed within 13 minutes on average and all other plasmin concentrations resulted in more rapid fiber lysis (Figs. 2A–D and 3A).


Physical determinants of fibrinolysis in single fibrin fibers.

Bucay I, O'Brien ET, Wulfe SD, Superfine R, Wolberg AS, Falvo MR, Hudson NE - PLoS ONE (2015)

Images of elongated and lysed fibrin fibers.Epi-fluorescence microscopy of fibrin fibers suspended across structured surfaces (bar = 20 μm). Samples were labeled with 20 nm red fluorescent beads and polymerized with 0.11–11 U/mL of human thrombin. Images of fibers before (A, C, E, G, I, K, M) and 5–15 minutes after (B, D, F, H, J, L, N) addition of plasmin. Samples 1–2 were treated with 20 μL of 1.0 U/mL of plasmin and display a lysed fiber (B) or a lysed and elongated fiber in the same field of view (D). Samples 3–7 were treated with 20 μL of plasmin ranging from 0.6 U/mL–6.0 U/mL and show elongated fibers, exhibiting extensions up to 10%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340865&req=5

pone.0116350.g002: Images of elongated and lysed fibrin fibers.Epi-fluorescence microscopy of fibrin fibers suspended across structured surfaces (bar = 20 μm). Samples were labeled with 20 nm red fluorescent beads and polymerized with 0.11–11 U/mL of human thrombin. Images of fibers before (A, C, E, G, I, K, M) and 5–15 minutes after (B, D, F, H, J, L, N) addition of plasmin. Samples 1–2 were treated with 20 μL of 1.0 U/mL of plasmin and display a lysed fiber (B) or a lysed and elongated fiber in the same field of view (D). Samples 3–7 were treated with 20 μL of plasmin ranging from 0.6 U/mL–6.0 U/mL and show elongated fibers, exhibiting extensions up to 10%.
Mentions: To study individual fiber behavior during fibrinolysis and the timescales within which plasmin can lyse individual fibers, we polymerized and suspended fibrin across micropatterned ridge-and-valley structures, as previously described [25]. Fibrin fibers were labeled with 20 nm red fluorescent beads and observed under an optical microscope. We then added plasmin (0.06–6 U/mL) to the fibrin samples and recorded fiber behavior for thirty minutes. Of fibers that lysed, fibers treated with the lowest plasmin concentration (0.06 U/mL) lysed within 13 minutes on average and all other plasmin concentrations resulted in more rapid fiber lysis (Figs. 2A–D and 3A).

Bottom Line: We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected.Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain.These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Fibrin fibers form the structural backbone of blood clots; fibrinolysis is the process in which plasmin digests fibrin fibers, effectively regulating the size and duration of a clot. To understand blood clot dissolution, the influence of clot structure and fiber properties must be separated from the effects of enzyme kinetics and perfusion rates into clots. Using an inverted optical microscope and fluorescently-labeled fibers suspended between micropatterned ridges, we have directly measured the lysis of individual fibrin fibers. We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected. Thrombin and plasmin dose-response experiments showed that the elongation behavior was independent of plasmin concentration, but was instead dependent on the concentration of thrombin used during fiber polymerization, which correlated inversely with fiber diameter. Thinner fibers were more likely to lyse, while fibers greater than 200 ± 30 nm in diameter were more likely to elongate. Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain. Using polymer physics- and continuum mechanics-based mathematical models, we show that fibers polymerize in a strained state and that thicker fibers lose their prestrain more rapidly than thinner fibers during lysis, which may explain why thick fibers elongate and thin fibers lyse. These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities.

Show MeSH
Related in: MedlinePlus