Research ArticleBioengineering

A synthetic fibrin cross-linking polymer for modulating clot properties and inducing hemostasis

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Science Translational Medicine  04 Mar 2015:
Vol. 7, Issue 277, pp. 277ra29
DOI: 10.1126/scitranslmed.3010383
  • Fig. 1. PolySTAT synthesis and characterization.

    (A) The PolySTAT polymer backbone, a linear statistical copolymer of HEMA and NHSMA synthesized via RAFT polymerization, was grafted with the cyclic fibrin-binding peptide Ac-Y(DGl)C(HPr)YGLCYIQGK-Am (19) through NHS ester reaction with the lysine ε-amine. DGl, d-glutamic acid; HPr, hydroxyproline; Ac, acetylated N terminus; Am, amidated C terminus. (B) Two different polymer backbones were used, including a fluorescent FMA-modified p(HEMA-co-NHSMA) for confocal studies. Polymer backbones were grafted with the fibrin-binding peptide for PolySTAT or a nonbinding scrambled peptide for the PolySCRAM control. Polymer molecular weight (MW) and polydispersity index (PDI) were determined using gel permeation chromatography (GPC). Peptides per polymer were calculated using ultraviolet (UV) absorbance. NA, not applicable. (C) PolySTAT integration into fibrin was confirmed using confocal imaging. Pure fibrin clots were made using a solution of Alexa Fluor 546–labeled fibrinogen (red) and thrombin mixed with PBS, fPolySTAT (green), or fPolySCRAM (green). Scale bars, 10 μm.

  • Fig. 2. In vitro characterization of PolySTAT-modified fibrin architecture.

    (A) Turbidity of pure fibrin clotting solutions with PBS, PolySCRAM, PolySTAT, and hFXIIIa was measured to determine whether there were potential changes in fibrin nanostructure. Data are averages ± SD (n = 3). The P value compares PBS and PolySTAT at t = 15 min; one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc test. (B) Flow rates of water through fully formed fibrin were measured and used to extrapolate pore size using Darcy’s law and a model by Carr and Hardin (39). Data are averages ± SD (n = 3). *P < 0.05, **P = 0.01 versus PolySCRAM (one-way ANOVA with Tukey-Kramer post hoc test). (C) Fully formed fibrin clots were imaged using SEM to visualize fibrin architecture. hFXIIIa was included for a cross-linking control. Scale bars, 250 nm. Schematics (not drawn to scale) above the SEM images depict the exclusion of nonbinding PolySCRAM from fibrin, PolySTAT-induced fibrin cross-linking via binding of fibrin-binding peptides, and enzymatic cross-linking by hFXIIIa.

  • Fig. 3. In vitro characterization of fibrin polymerization kinetics, clot strength, and fibrinolysis.

    (A) Cone-and-plate rheometry was used to measure PolySTAT-induced changes in fibrin storage moduli, a measure of elasticity. Measurements were taken for PolySTAT-modified and control fibrin formed with fibrinogen (Fbg) (1.5, 2.2, and 3.0 mg/ml) with intact hFXIIIa (n = 3). The critical threshold indicates the storage moduli below which mortality increases markedly in trauma patients (33). Data are averages ± SD. ***P < 0.0001 for both effect of treatment and fibrinogen concentration (P = 0.07 for interaction; that is, whether or not the differences as a result of treatment are the same across all fibrinogen concentrations); two-way ANOVA. The effect of PolySTAT on clotting kinetics, clot strength, and extent of fibrinolysis was evaluated in a hyperfibrinolytic model using TEG. (B to E) Clotting onset time, clotting rate, maximum clot strength, and extent of clot lysis 30 min after time to maximum clot strength. Three hFXIIIa concentrations were included to compare PolySTAT activity. Data are averages ± SD (n = 3). P values determined using one-way ANOVA with Tukey-Kramer post hoc test. (F) Lysis of fibrin was visualized using confocal microscopy. Plasmin (10 μg/ml) was applied to the edge of fully formed fibrin clots, and time-lapsed images were taken. The area of the clot in images was measured to determine rate of lysis. Scale bars, 50 μm.

  • Fig. 4. Evaluation of PolySTAT in a rat model of femoral artery injury and fluid resuscitation.

    (A) Workflow schematic. Rats were normalized to the same starting blood pressure (BP), and clamps proximal and distal to the injured femoral artery were removed to allow the wound to bleed freely. After clamp release, polymer solutions were injected. The wound was allowed to bleed or clot without interference for the first 15 min, and subsequent challenge to the clot was presented in the form of 0.9% saline infusion to maintain blood pressure above 60 mmHg for 60 min. (B) Survival of animals over the 75-min protocol (n = 5 per treatment). P value determined by log-rank Mantel-Cox test. (C) Bleeding profiles for volume controls (0.9% saline) and animals injected with PolySTAT, PolySCRAM, hFXIIIa, and albumin. Boxed traces are representative of clots that experience bleeding during fluid resuscitation and clots that maintain hemostasis during fluid resuscitation. Data are single measurements per time point per animal (n = 5 per treatment group). (D) Cumulative blood loss normalized to survival time, including blood lost during catheter hemorrhage, the free bleeding period, and fluid resuscitation. (E) Saline infusion volumes normalized to survival time. Data in (D) and (E) are averages ± SD. P values determined using a one-way ANOVA and Tukey-Kramer post hoc test. (F) MAP was tracked over the protocol time to determine whether animals were hypotensive. Data are averages ± SD, with variable n depending on survival times (11 to 75 min). P values for effect of time and treatment (and interaction of time and treatment) were determined by two-way ANOVA.

  • Fig. 5. PolySTAT biodistribution.

    PolySTAT was administered via tail vein injection at a dose of 15 mg/kg in rats. Animals were euthanized at various time points to determine polymer biodistribution. (A) Percentage of the injected dose in whole organs at various time points. (B) Percentage of injected dose normalized to organ mass. Data are averages ± SD (n = 3).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/277/277ra29/DC1

    Materials and Methods

    Fig. S1. TEG measurements for fibrinogen and plasmin titration study.

    Fig. S2. In vitro comparison of PolySTAT to PolySCRAM and component controls.

    Fig. S3. TEG measurements for hemodilutions.

    Fig. S4. PT in rat femoral artery injury models.

    Fig. S5. Fibrinogen concentration in rat femoral artery injury models.

    Fig. S6. MAP in rat femoral artery injury models.

    Fig. S7. Lactate concentration of rat femoral artery injury models.

    Table S1. Comprehensive metabolic and hepatic function panel results for 1 hour, 1 day, and 1 week after PolySTAT injection in rats.

    Table S2. Pharmacokinetic constants for PolySTAT.

    Table S3. PolySTAT content in urine after tail vein injection.

    Movie S1. Lysis of control fibrin formed with PBS.

    Movie S2. Lysis of control fibrin formed with PolySCRAM.

    Movie S3. Lysis of PolySTAT-modified fibrin.

    Movie S4. Lysis of hFXIIIa cross-linked fibrin.

  • Supplementary Material for:

    A synthetic fibrin cross-linking polymer for modulating clot properties and inducing hemostasis

    Leslie W. Chan, Xu Wang, Hua Wei, Lilo D. Pozzo, Nathan J. White,* Suzie H. Pun*

    *Corresponding author. E-mail: spun@uw.edu (S.H.P.); whiten4@uw.edu (N.J.W.)

    Published 4 March 2015, Sci. Transl. Med. 7, 277ra29 (2015)
    DOI: 10.1126/scitranslmed.3010383

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. TEG measurements for fibrinogen and plasmin titration study.
    • Fig. S2. In vitro comparison of PolySTAT to PolySCRAM and component controls.
    • Fig. S3. TEG measurements for hemodilutions.
    • Fig. S4. PT in rat femoral artery injury models.
    • Fig. S5. Fibrinogen concentration in rat femoral artery injury models.
    • Fig. S6. MAP in rat femoral artery injury models.
    • Fig. S7. Lactate concentration of rat femoral artery injury models.
    • Table S1. Comprehensive metabolic and hepatic function panel results for 1 hour, 1 day, and 1 week after PolySTAT injection in rats.
    • Table S2. Pharmacokinetic constants for PolySTAT.
    • Table S3. PolySTAT content in urine after tail vein injection.
    • Movie S1. Lysis of control fibrin formed with PBS.
    • Movie S2. Lysis of control fibrin formed with PolySCRAM.
    • Movie S3. Lysis of PolySTAT-modified fibrin.
    • Movie S4. Lysis of hFXIIIa cross-linked fibrin.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1. Lysis of control fibrin formed with PBS.
    • Movie S2. Lysis of control fibrin formed with PolySCRAM.
    • Movie S3. Lysis of PolySTAT-modified fibrin.
    • Movie S4. Lysis of hFXIIIa cross-linked fibrin.

    [Download Movies S1 to S4]

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