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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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Structure of the Fibrinogen Molecule.A structure of the fibrinogen molecule (crystal structure 3GHG) with αC domains built in using homology modeling and Discrete Molecular Dynamics Simulations [24]. Fibrinogen spans 45 nm and consists of two outer D regions, each connected by a coiled-coil segment to the central E region. Thrombin cleaves fibrinopeptides A and B from the Aα-chains and Bβ-chains, respectively, producing insoluble fibrin monomers that form fibrin networks. B A cartoon representation of the molecule, with the αC region highlighted in green. C A cartoon model of a fibrin fiber emphasizing the interaction of the αC polymer network within the fiber.
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pone.0116350.g001: Structure of the Fibrinogen Molecule.A structure of the fibrinogen molecule (crystal structure 3GHG) with αC domains built in using homology modeling and Discrete Molecular Dynamics Simulations [24]. Fibrinogen spans 45 nm and consists of two outer D regions, each connected by a coiled-coil segment to the central E region. Thrombin cleaves fibrinopeptides A and B from the Aα-chains and Bβ-chains, respectively, producing insoluble fibrin monomers that form fibrin networks. B A cartoon representation of the molecule, with the αC region highlighted in green. C A cartoon model of a fibrin fiber emphasizing the interaction of the αC polymer network within the fiber.

Mentions: The fibrinogen molecule is comprised of three pairs of distinct peptide chains: two Aα-chains, two Bβ-chains, and two γ-chains, which are interlinked by disulfide bridges (Fig. 1) [1,5–7]. Following thrombin-mediated cleavage of fibrinopeptides A and B from the N-termini of the Aα- and Bβ-chains, respectively, fibrin monomers polymerize into half-staggered, double-stranded protofibrils that bundle into fibers via interactions of the αC regions [8]. Cryptic plasminogen and plasmin binding sites in fibrinogen and fibrin monomers [9] are exposed by fibrin monomer polymerization [3]. In particular, the αC regions contain lysine-dependent tPA- and plasminogen-binding sites (Kd = 16–33 nM) within residues Aα392–610 [10,11]. During fibrinolysis, plasmin initially cleaves the αC regions, and then cleaves the three polypeptide chains connecting the central (E) and end (D) regions (Fig. 1), which contain low-affinity (Kd = 1 μM), lysine-independent plasmin and tPA binding sites [10,12–14]. These cleavages produce COOH-terminal lysine residues in fibrin, which provide additional binding sites for plasmin, and accelerate the dissolution process [10,15].


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)

Structure of the Fibrinogen Molecule.A structure of the fibrinogen molecule (crystal structure 3GHG) with αC domains built in using homology modeling and Discrete Molecular Dynamics Simulations [24]. Fibrinogen spans 45 nm and consists of two outer D regions, each connected by a coiled-coil segment to the central E region. Thrombin cleaves fibrinopeptides A and B from the Aα-chains and Bβ-chains, respectively, producing insoluble fibrin monomers that form fibrin networks. B A cartoon representation of the molecule, with the αC region highlighted in green. C A cartoon model of a fibrin fiber emphasizing the interaction of the αC polymer network within the fiber.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116350.g001: Structure of the Fibrinogen Molecule.A structure of the fibrinogen molecule (crystal structure 3GHG) with αC domains built in using homology modeling and Discrete Molecular Dynamics Simulations [24]. Fibrinogen spans 45 nm and consists of two outer D regions, each connected by a coiled-coil segment to the central E region. Thrombin cleaves fibrinopeptides A and B from the Aα-chains and Bβ-chains, respectively, producing insoluble fibrin monomers that form fibrin networks. B A cartoon representation of the molecule, with the αC region highlighted in green. C A cartoon model of a fibrin fiber emphasizing the interaction of the αC polymer network within the fiber.
Mentions: The fibrinogen molecule is comprised of three pairs of distinct peptide chains: two Aα-chains, two Bβ-chains, and two γ-chains, which are interlinked by disulfide bridges (Fig. 1) [1,5–7]. Following thrombin-mediated cleavage of fibrinopeptides A and B from the N-termini of the Aα- and Bβ-chains, respectively, fibrin monomers polymerize into half-staggered, double-stranded protofibrils that bundle into fibers via interactions of the αC regions [8]. Cryptic plasminogen and plasmin binding sites in fibrinogen and fibrin monomers [9] are exposed by fibrin monomer polymerization [3]. In particular, the αC regions contain lysine-dependent tPA- and plasminogen-binding sites (Kd = 16–33 nM) within residues Aα392–610 [10,11]. During fibrinolysis, plasmin initially cleaves the αC regions, and then cleaves the three polypeptide chains connecting the central (E) and end (D) regions (Fig. 1), which contain low-affinity (Kd = 1 μM), lysine-independent plasmin and tPA binding sites [10,12–14]. These cleavages produce COOH-terminal lysine residues in fibrin, which provide additional binding sites for plasmin, and accelerate the dissolution process [10,15].

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