Julie AuxierDr. Joseph McGuire, Bioengineering

Oregon State UniversityHHMI 2009

Imperfect ImplantsProblems from implanted

devices:Clotting; embolism riskBacterial adhesion; infectionOverall implant rejection

Treat with heparin and other anticoagulantsRisk of platelet depletion,

excessive bleeding

Thrombosis and Blood Proteins Thrombosis: formation of a blood clot in a blood

vessel which obstructs blood flow

Intrinsic Pathway

Extrinsic Pathway

Tissue Damage

Tissue Factor

III

Factor VII Tissue Factor

Complex

Clotting Factor VII

Activated Proenzymes, usually Factor XIII

Platelet Factor PF-3

Clotting Factors VIII,

IX

Factor X Activator Complex

Ca2+

Ca2+

Fibrinogen

Fibrin

Prothrombin Thrombin

Factor X

Prothrombinase

Common Pathway

Thrombosis and Blood ProteinsFibrin forms the

scaffolding, platelets fill the holes

Late stent thrombosis possibly caused by: Early discontinuation of

anticoagulant medication Stent fracture Abnormal reaction of tissue to

implant material Small lumen size, slow flow rate

Prevention with Pluronic® F108

HYDROPHOBIC

HYDROPHILICPEO

PEO

PPO

HYDROPHOBIC SURFACE

F108 approximate maximum length: 50nm

Approximate length of a red blood cell: 5µm

(500nm)

HYDROPHOBIC

PROTEI

N

How Brush Layer Functions

Nisin - Lantibiotic Inactivate bacteria by creating a pore and destabilizing the membrane Naturally made from bacteria Lactococcus lactisUsed in food products: preservative, making cheeseNo evidence suggests nisin induces an immunogenic reaction (based

on previous studies)

Hydrophobic Surface

Previous ResearchChange between pluronic coating with nisin before and

after challenged with fibrinogen.

Two possibilities may account for the lower signal

PurposeIdentify fibrinogen adsorption on non-

fouling, antimicrobial surfaces.

HypothesisThe pluronic layer maintains its protein

repelling nature despite nisin loading. Hence, fibrinogen more likely will not adsorb to the

surface and will displace nisin when repelled.

MethodologySurface preparation:

Silanize silica to make surface hydrophobic

Covalently attach pluronic F108 by gamma radiation

Load brush layer with nisin

Protein assay tests (ELISA)

FITC labeling fibrinogen

Parallel flow platelet adhesion tests

Inactivated Platelets

Fibrinogen

Surface

Enzyme Linked Immunosorbant Assay

Surface

Fibrinogen sticks to sample surface.

Add enzyme-linked antibody which attaches to fibrinogen.

Add colorimetric substrate to react with enzyme on antibody.

Solution changes color, read absorbance at 490nm.

Block well with bovine serum albumin (BSA) or milk.“Tagged” fibrinogen

antibody detects fibrinogen in a sample, and then a colorimetric substrate detects the antibody

Results

*Not to scale.

FITC labeling fibrinogen Fluoroscein isothiocyanate reacts with N-

terminal amines on fibrinogen.

Prepare labeled fibrinogen solution.

Contact surfaces (microspheres) with labeled fibrinogen.

Rinse thoroughly.

Dissolve microspheres with NaOH.

Read absorbance at 490nm.

Results

Parallel Flow Platelet Adhesion Flow chamber allows for evenly distributed flow at a constant rate (16 mL/min,

shear rate 480 sec-1)

Flow platelet-rich equine plasma through system.

Buffer wash.

Fix platelets with gluteraldehyde.

Buffer wash.

Dehydrate with ethanol.

Critical point dry.

Image with SEM.

Results

ConclusionsELISA and FITC-Fg results indicate:

Brush layer effectively inhibits fibrinogen adsorption.

Addition of nisin to the brush layer does not promote fibrinogen adsorption.

Platelet adhesion studies require refining before definitive results may be collected.

Future WorkContinue work with parallel flow chamber.

Repeat FITC-Fg tests.

Investigate labeling fibrinogen with trifluoroacetic anhydride which can be quantified using x-ray photon spectroscopy (XPS).

AcknowledgementsGreat deal of thanks to:Dr. Joe McGuireKarl “Rat” SchilkeDr. Karyn BirdMatt RyderLars BowlinHoward Hughes Medical InstituteAllvivo Vascular Inc.