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Lab notebook

Investigating Collagen-Induced Fibrin Fibrillogenesis in the Absence of Thrombin

Garcia Center for Polymers at Engineered Interfaces, Stony Brook University · Jul 10 – Aug 7, 2026

Day 0 Fri 7/10/26

  • Read literature & discuss
    • Coradin, Wang, Law & Trichet (2026): Review of collagen-fibrin mixed hydrogels
    • Cummings, Gawlitta, Nerem & Stegemann (2004): Engineered vascular constructs
    • Hansen et al. (2026): Unraveling the molecular mechanism of in situ surface-initiated thrombogenesis

Day 1 Mon 7/13/26

  • Read literature & discuss
    • Luo et al. (2020): Dental pulp stem cell mineralization on collagen-fibrin scaffolds
    • Lai, Frey, Kerandi, Lake, Tranquillo & Barocas (2012): Microstructural/mechanical differences in co-gels
    • Kim, Litvinov, Chen, Chen, Weisel & Alber (2017): Compression behavior of fibrin-collagen composites

Day 2 Tue 7/14/26

  • Sliced silicon [100] wafers into ~1 cm × 1 cm pieces
  • Collagen solution prep (Shifu)
    • Solute: type I telocollagen, only soluble in acid; stock received at pH 4
    • Neutralized to pH 7 with NaOH so collagen comes out of solution onto the wafer
    • Diluted to 5 µg/mL and 50 µg/mL

Day 3 Wed 7/15/26

  • Deposited collagen thin films: wafers left in 5 and 50 µg/mL collagen for 18 h

Day 4 Thu 7/16/26

  • Rinsed collagen wafers (collagen-only procedure)
    • Aspirate collagen/PBS solution
    • Cover wafers with PBS and aspirate, ×3
  • Fibrinogen dilution calc: 100 µg/mL → mix 290 mL PBS with 7.5 mL fibrinogen stock (FS)
  • Deposited 4 mg/mL fibrinogen on 5 µg/mL collagen wafers, 18 h adsorption

Day 5 Fri 7/17/26

  • Rinsed fibrinogen-on-collagen wafers
    • PBS ×3, then DI water ×3
    • Aspirated all water off to prep for AFM
  • AFM: 4 mg/mL fibrinogen, 18 h, on 5 µg/mL collagen
    • Large, irregular fibres (~500 µm) formed on collagen without thrombin
    • Optical follow-up of the same sample on 7/18
    AFM, 21.9 µm scan: thick fibres several µm wide cross the collagen film, with finer fibrils branching off them.
    AFM, 21.9 µm scan: thick fibres several µm wide cross the collagen film, with finer fibrils branching off them.
    AFM, 22.9 µm scan of the same 4 mg/mL, 18 h sample: away from the large fibres, the surface is a rough, densely textured protein layer.
    AFM, 22.9 µm scan of the same 4 mg/mL, 18 h sample: away from the large fibres, the surface is a rough, densely textured protein layer.
    Optical, 10×: 4 mg/mL fibrinogen after 18 h on 5 µg/mL collagen. A branching network of large fibres formed on the collagen film with no thrombin added.
    Optical, 10×: 4 mg/mL fibrinogen after 18 h on 5 µg/mL collagen. A branching network of large fibres formed on the collagen film with no thrombin added.
  • Deposited 100 µg/mL fibrinogen on 5 µg/mL collagen for 18 h adsorption

Day 6 Mon 7/20/26

  • AFM: collagen-only control, 5 µg/mL (negative control for fibrin–collagen images)
  • AFM: 100 µg/mL fibrinogen, 18 h, on 5 µg/mL collagen
    • No fibres comparable to the 4 mg/mL sample → fibre formation looks concentration-dependent
    AFM, 25.8 µm scan: at 100 µg/mL there are only thin, scattered fibrils, nothing like the thick fibres at 4 mg/mL.
    AFM, 25.8 µm scan: at 100 µg/mL there are only thin, scattered fibrils, nothing like the thick fibres at 4 mg/mL.
  • Deposited 100 µg/mL and 4 mg/mL fibrinogen on 50 µg/mL collagen, 18 h

Day 7 Tue 7/21/26

  • Optical: fibrinogen, 18 h, on 50 µg/mL collagen
    • 4 mg/mL vs 100 µg/mL fibrinogen
    Optical, 10×: 4 mg/mL fibrinogen, 18 h on 50 µg/mL collagen. Thick, irregular aggregates (rainbow thin-film colours) joined by fibrous strands.
    Optical, 10×: 4 mg/mL fibrinogen, 18 h on 50 µg/mL collagen. Thick, irregular aggregates (rainbow thin-film colours) joined by fibrous strands.
    Optical, 10×: 100 µg/mL fibrinogen, 18 h on 50 µg/mL collagen. Only a sparse web of fine strands and small clusters, far less material than at 4 mg/mL.
    Optical, 10×: 100 µg/mL fibrinogen, 18 h on 50 µg/mL collagen. Only a sparse web of fine strands and small clusters, far less material than at 4 mg/mL.
  • Open question: are the fibres just the known hydrophobic-surface mechanism, or is the collagen film itself hydrophilic? → tested with contact angle on Day 8

Day 8 Wed 7/22/26

  • Contact angle of collagen-only films (CAM 200 Optical Contact Angle Meter)
    • Answers Day 7's open question
TrialLeft angleRight angle
123.27°30.49°
220.47°22.46°
332.49°30.12°
  • Mean 26.55° (SD 5.06°, SE 2.07°)
  • Well below the Berg limit of 65° → collagen film is hydrophilic, so fibres are not explained by hydrophobic adsorption alone

Day 9 Thu 7/23/26

  • Deposited fibrinogen on 5 µg/mL collagen
    • 1 h adsorption: 20 µg/mL and 100 µg/mL
    • 18 h adsorption: 100 µg/mL
  • Deposited 4 mg/mL fibrinogen on 50 µg/mL collagen, 18 h
  • Stained 1 h samples with anti-Bβ122–134, then fluorescent secondary

Day 10 Fri 7/24/26

  • 1 h adsorption, 100 µg/mL on 5 µg/mL collagen
    • Imaged with optical microscopy and AFM
  • Optical: 100 µg/mL, 18 h, on 5 µg/mL collagen
  • AFM: 4 mg/mL, 18 h, on 50 µg/mL collagen

Day 11 Mon 7/27/26

  • Canoe trip!

Day 12 Tue 7/28/26

  • Reviewed AFM/optical results across collagen (5 vs 50 µg/mL) and fibrinogen (20 µg/mL–4 mg/mL) concentrations
  • Planned controls (don't need all)
    • Bare silicon + PBS only
    • Bare silicon + fibrinogen
    • Collagen-coated silicon + PBS only
    • Collagen-coated silicon + fibrinogen + thrombin (positive control)
  • Planned fluorescent staining: αC (fibrin structure, polymerization) and FpA/FpB (fibrinogen cleavage to fibrin)
  • Antibody check: mouse anti-FpA (Novus NB100-73043, clone 49D2)
    • Binds fibrinopeptide A (Aα1–16) on the fibrinogen α chain, plus free FpA
    • FpA is cleaved off when fibrinogen converts to fibrin, so this stain marks fibrinogen and isn't specific to fibrin

Day 13 Wed 7/29/26

  • Prepared confocal samples
    • Collagen-only control, 50 µg/mL, collagen stain
    • 4 mg/mL fibrinogen, 18 h, on 5 µg/mL collagen, mouse anti-FpA
  • Each stain left to cover the wafer surface for 1 h

Day 14 Thu 7/30/26

  • Confocal: collagen-only control, 50 µg/mL, two scales
  • Confocal: 4 mg/mL fibrinogen, 18 h, on 5 µg/mL collagen, anti-FpA
    • Imaged at 7 scales, including two Airyscan super-resolution images
    Widefield fluorescence (EVOS): 4 mg/mL fibrinogen on 5 µg/mL collagen, anti-FpA. Long bundled fibres light up along their whole length. Scale bar 250 µm.
    Widefield fluorescence (EVOS): 4 mg/mL fibrinogen on 5 µg/mL collagen, anti-FpA. Long bundled fibres light up along their whole length. Scale bar 250 µm.
    Confocal close-up of a single anti-FpA-stained fibre; the stain outlines the fibre along its length. (Day 15: this stain alone can't tell fibrin from fibrinogen.)
    Confocal close-up of a single anti-FpA-stained fibre; the stain outlines the fibre along its length. (Day 15: this stain alone can't tell fibrin from fibrinogen.)

Day 15 Fri 7/31/26

  • Analyzed confocal images; anti-FpA alone can't separate fibrin from fibrinogen → co-stain with anti-Bβ14–15 (knob B)
  • Deposited fibrinogen on collagen for 18 h: 4 mg/mL for co-staining, plus samples for the αC time series

Day 16 Mon 8/3/26

  • Co-stained 4 mg/mL samples, 1 h per stain
    • Anti-Aα1–16 (anti-FpA) → fibrinogen (red)
    • Anti-Bβ14–15 (anti-knob B) → fibrin
    • Confocal: fibres are polymerized fibrin lying in cavities within a bed of fibrinogen
    Confocal co-stain, 5 µm scale bar: a fibrin fibre (green, anti-knob B) lies in a dark cavity cut into the surrounding fibrinogen layer (red, anti-FpA).
    Confocal co-stain, 5 µm scale bar: a fibrin fibre (green, anti-knob B) lies in a dark cavity cut into the surrounding fibrinogen layer (red, anti-FpA).
  • αC time series: deposited matching 1 h samples; stained 1 h and 18 h samples with anti-Bβ122–134 (open αC)
    • Fluorescence increased with adsorption time → layers of fibrinogen with open αC domains build up on collagen, similar to the hydrophobic-surface mechanism
  • P12 setup: 50 µM P12 (fibronectin-derived antithrombotic, binds N-terminal subdomain of αC) mixed with 3.96 mg/mL fibrinogen, deposited on collagen for 18 h

Day 17 Tue 8/4/26

  • Rinsed and imaged P12-treated samples vs untreated 4 mg/mL
    • Reduced fibrillogenesis with P12 → assembly is mediated by the αC domain
    AFM, 111.7 µm scan: 4 mg/mL fibrinogen + 50 µM P12 on 5 µg/mL collagen, 18 h. Only short, thin fibrils and small clumps, none of the large fibres seen without P12.
    AFM, 111.7 µm scan: 4 mg/mL fibrinogen + 50 µM P12 on 5 µg/mL collagen, 18 h. Only short, thin fibrils and small clumps, none of the large fibres seen without P12.
  • Started presentation/abstract outline

Day 18 Wed 8/5/26

  • Truncated αC assay
    • 50 µg/mL fibrinogen des Aα414–610 and des Aα505–610, plus negative control, deposited 30 min on silicon
    • 5 µg/mL collagen adsorbed on the fibrinogen films for 30 min
    • Rinsed and stained with anti-collagen type I

Day 19 Thu 8/6/26

  • Last images: confocal of truncated αC samples
    • Anti-collagen type I intensity significantly lower on des Aα414–610 than on negative control and des Aα505–610
    • → Aα414–505 (N-terminal subdomain of αC) is responsible for collagen adsorption, consistent with P12
  • Finished presentation and extended abstract

Day 20 Fri 8/7/26

  • Gave final presentation

Current Ongoing

  • Accepted & presenting @ Materials Science Research Conference Dec '26 Boston conference for SB07, Emerging Materials for In Vitro Diagnostics
    • Framing: malignant clot formation (stroke thrombi with lysis-resistant shells; collagen in clots impedes thrombolysis)
    • Key points: thrombin-free fibrin on collagen (AFM); fibrin in a cavitated fibrinogen bed (confocal); αC untethering like hydrophobic surfaces; binding region Aα414–505; inhibited by P12
    • Refining poster
  • Writing paper for Journal of Thrombosis and Haemostasis