Modeling the Role of Surface Interactions in Initiation of Thrombosis

Liudi Zhang, Clement Marmorat, Jaseuong Koo, Miriam Rafailovich Department of Materials Science and Chemical Engineering

Marcia Simon, Department of Oral Biology and Pathology

Dennis K. Galanakis, Blood Bank, Stony Brook University Hospital

Brendan Casey,Shelby Skoog, and Katherine Vorvolakos, FDA

Aug 13, 2015

Biomaterials

Blood contacting biomaterials

Biomaterials for root canal therapy

A biomaterial is a nonviable material used in a medical device, intended to interact with biological systems (Williams, 1987).

Biomaterials for orthopedics

Can we provide a model to explain the risk factors?

Risk Factors for Thrombosis

Normal adult wound healing

Fibrin Clot &

Re-epithelialization:

Thrombin is released,

forming fibers out of

fibrinogen in the

blood stream.

Epithelial cells

migrate into the

wound.

(Within min / hours)

(B) Granulation Tissue

(day 4-6)

Wound remodeling

(day 7-10)

Singer AJ and Clark RAF. The NEJM, 1999, 341 (10): 738-746

and McClain SA et al. (1996) Am J Pathol. 149(4):1257-70.

Dermal fibroblasts form fibronectin

and ECM proteins that bind to the

fibrin scaffold enabling fibroblast

migration into the wound.

Epithelial cells

migrate and form the

clot (receptors for

fibrin).

Fibroblast remodel the collagen

scaffold enabling keratinocytes to

grow and the tissue to reform.

Fibrinogen molecule structure

• The fibrinogen molecule is long disulfide-linked symmetric dimmer each containing three distinct polypeptide chains, A, Bβ, and γ.

• Trinodular Structure: two D domains and one central E domain (phobic) linked by -helical coiled coil domains (philic) .

• The αC region includes: a flexible segment (Aα221-391), the “αC-connector” and an independently folded compact segment (Aα392-610), the “αC-domain”.

47 nm

1 nm

Fibrinogen assembly induced by thrombin

Koo, J.; Galanakis, D.; Liu, Y.; Ramek, A.; Fields, A.; Ba, X.; Simon, M.; Rafailovich, M. H.; Biomacromolecules. 2012, 13, 1259−1263.

(A) There are two binding sites in the E region, namely knobs ‘A’ and ‘B’. Black and orange rods at the end of knobs ‘A’ and ‘B’ represent fibrinopeptide A and B (FpA and FpB), respectively.

(B) Thrombin cleaves the FpA, and then molecules are assembled into double stranded protofibrils via interaction between exposed knob ‘A’ and hole ‘a’ in adjacent molecules.

(C) After protofibrils have formed, FpB is also released by thrombin cleavage. αC chains are then able to move to interact with another αC chains from neighboring protofibrils resulting in laterally assemble into coarse fibers.

Strong attraction between D, E domains and hydrophobic surface Strong attraction between C regions and hydrophilic surface

Surfaces effect the structure of fibrinogen molecule

Koo, J.; Galanakis, D.; Liu, Y.; Ramek, A.; Fields, A.; Ba, X.; Simon, M.; Rafailovich, M. H.; Biomacromolecules. 2012, 13, 1259−1263.

extended trinodular structure globular structure

C regions are VERY hydrophilic, D domains are VERY hydrophobic, E is neutral

I-2 I-8 ΦII

The role of C domains in self-assembly of fibrinogen fibers

TOM

A c

lay

Flu

oro

mic

a

2μm 2μm 2μm

2μm 2μm 2μm

I-2 I-2 I-2

I-8 ΦII I-2

The initial flat protein layer is determinant factor for fiber formation

2μm 2μm 2μm

Which segments are necessary to form fibers? (D. Gallenakis

collection of monoclonal antibodies )

IgG103

IgG359

A518-584

A241-476

IgG103

IgG189

A518-584

A241-476

which peptide regions in αC domain are involved in supra-molecular assembly?

0 nm

100nm

2μm 2μm 2μm

IgG103

IgG359

A518-584

A241-476

Alpha-C Domain functions: monoclonal study II

0 nm

100nm

2μm 2μm 2μm

• Note low protein coverage when D domains are blocked. • No obvious effect on protein adsorption when A-alpha is blocked. • Both Top and Mid sections of alpha-C domain are essential to form fibers. Mid section required for adsorbtion.

The proposed model of fibrinogen self-assembly on Hydrophobic surfaces

Aα518-584

A241- 476

Aα 241-476 contribute to adsorption of fibrinogen and the Aα 518-584 interact with other αC domains A knob

B knob

Knob-hole interactions between D and E domains are involved in fibrinogen assembly

Supra-molecular assembly proceeds via the C domains: Molecules are in registry.

The molecules then Align to form fibers

Soluble Fibrin Fraction is Essential

I-2+Thrombin I-8+Thrombin ΦII+Thrombin

Add thrombin to fibronigen fibers: No morphological change

2μm 2μm 2μm

2μm 2μm 2μm

Philic

Phobic

X, Drive signal

Lateral deflection

Response

ΔX Sample Buffer

Laser

Photodiode

detector

Flateral

h

Indentation

Fiber surface

Contribution of Knob-hole interaction: Shear modulus force microscopy (SMFM)

0

1

2

3

4

5

6

7

8

9

10

I-2+T

hrom

bin

I-2

I-2+G

PRP+T

hrom

bin

Rela

tive M

odulu

s

I-2+G

PRP

3 μm

Fibrinogen fiber bundle

on the TOMA clay surface

Incubated from the test solution

(I-2, 4mg/ml) at 21 oC for 18 hrs

3 μm

AFM AFM

(J. Weisel, U Penn, )

(http://biocurious.com/category/andres-research)

Material Molecular Weight

Solvent

Polystyrene (PS) 311,100 Toluene

Poly(methylmethacrylate) (PMMA)

94,100 Toluene.

Poly(4-vinylpyridine) (P4VP)

60,000 DMF.

Polylactic acid (PLA)

131,300 Chloroform.

Polymer

Substrate Oil free vacuum oven

Materials and Preparation

I. Solutions of the polymers were spun cast at 2500 RPM for 30s on cover glass, Kapton, or HF etched Si [1,0,0] wafers. The thickness of polymer films (~100nm) was measured by ellipsometery.

II. The samples were then annealed at T=130°C overnight in an oil free vacuum of 10¯̄³Torr to remove residual solvent, release the strain, and sterilize.

III. Water Contact angles independent of substrate. All polymers except P4VP satisfy Berg Hydrophobic Limit: angle>65 degrees

Polymer Substrate Water Contact Angle

(mean±s.d. n=6)

PS Si Wafer 91±1°

PS Glass 89±1°

PS Kapton film 90±2°

PMMA Si Wafer 69±1°

PMMA Glass 70 ±1°

PMMA Kapton film 70 ±1°

PLA Si Wafer 71±1°

PLA Glass 70±1°

PLA Kapton film 71±2°

P4VP Si Wafer 56±2°

P4VP Kapton film 55±2°

Adsorption of Fibrinogen on Polymers

• PLA is crystalline-more protein adsorbs on rough surfaces.

• Similar amounts of protein adsorbed on hydrophobic and hydrophilic surfaces.

• No fibers om P4VP

BCA analysis

4mg/ml fibrinogen 0.1mg/ml fibrinogen Albumin control

PS P4VP

Confocal microscopy: AαC 529-539 is available only on PS.

Identification of fibrinogen conformation: monoclonal anti-fibrinogen IgG against Aα529-539

Identification of platelet binding domain: monoclonal anti-fibrinogen IgG against γ 86-411

4mg/ml fibrinogen 0.1mg/ml fibrinogen

PS

Pre-incubated mAb-fibrinogen mixture Albumin control

P4

VP

Confocal microscopy:γ 86-411 domain exposed only on Fg fibers formed on PS and everywhere on P4VP.

Does fibrinogen fiber form in vivo?

Gerard J. Tortora, Bryan Derrickson (2012). Principles of Anatomy & Physiology, 13th. John Wiley & Sons, Inc. p. 816.

Type of blood vessels

Diameter Blood velocity

Aorta 2–3 cm 40 cm/s

Capillaries 5–10 μm 0.03 cm/s

Veins 1mm-1.5cm

15 cm/s

Closed flow loop device consisting of a 17.5 ml capacity circular 3/16” ID Tygon tube inserted into a peristalsis pump capable of circulating liquids at a constant flow rate of 100 ml/min.

Topographic SPM scans of the Kapton film inserts coated after being exposed for 12 hours (a) to a flowing solution of 4mg/ml fibrinogen and (b) whole plasma under static conditions.

Platelets preparation and incubation

• All the surfaces, including the fibrinogen-incubated and the control surfaces, were then incubated in the isolated platelets (200,000/µL) suspension for 1 h at 37°C (60 rpm).

• After washing twice with TBS, the surfaces were:

I. incubated in a 2% glutaraldehyde solution (12h, 25°C) for subsequent dehydration and SEM imaging.

II. incubated with fluorescently (FITC) labeled CD41 antibody (30 min, 21°C, 60 rpm) for fluorescent microscopy imaging.

The platelet rich plasma (PRP) was then passed through a sepharose column (2B, 60-200 µm) to separate the platelets from the plasma components.

Sepharose column gel filtration

Scanning Electron Microscopy B

lan

k

PS PMMA P4VP

0.1

mg

/ml

fib

rin

oge

n 4

mg

/ml f

ibri

no

gen

Platelets binding on adsorbed fibrinogen/fiber

Platelet Morphology as Indicator of Activation

Round (R)

Dendritic (D)

Spread-Dendritic (SD)

Spreading (S)

Fully Spread (FS)

Increasing Activation

24

10kV X3,000 5μm 12 30 SEI

PS PLA P4VP

no

fib

rin

oge

n

4

mg

/ml f

ibri

no

gen

Scanning Electron Microscopy

Flow Loop--platelets binding on adsorbed

fibrinogen/fiber

PS, PLA, and P4VP surfaces were pre-incubated with 4mg/ml fibrinogen solutions for 12 hours, then exposed to a flowing solution of isolated platelets for 1h at ambient temperature.

Endothelialization Endothelial cells: • Adhere to Fg

monolayer on PS • Do not adhere to

bare PS. • Adhere to P4VP with

or without Fg—but do not form cell-cell junctions.

• Endothelialization is favored over platelet adhesion.

SEM images of endothelial cells on PS surfaces. (a) PS

surface pre-incubated with 0.1mg/mL fibrinogen for 12

hours then exposed to endothelial cells for 24 hours, (b) PS

surface pre-incubated with 0.1mg/mL fibrinogen for 12

hours then exposed to a mixture of endothelial cells and

platelets for 24 hours.

SEM flow withP12 fibrinogen and platelets

Fg Without P12 Fg with 10μM P12

P12 blocks alpha C domain which recruits fibrin and forms fibers. Hence interferes with fiber formation and platelet adhesion BUT not with endothelialization. • Prevents micro thrombi from forming after a burn. • Mitigates necrosis following the burn, • Wounds heal faster.

Cobalt-Chromium Stents

Stents were Incubated in 4mg/ml solution of

Fibrinogen stained with Oregon Green at 37oC for 1 hr

and then imaged with confocal microscopy;

Stent coated with a PS film Uncoated stent

Stent coated with PS-r-P4VPh film (13 mole %)

Fibers are formed on PS coated and uncoated stents. Protein is adsorbed in nonfibrillar morphology

Conclusion

• Fibrinogen adsorbs on both hydrophobic and hydrophilic surfaces in different conformations.

• Understanding Fibrinogen/Materials interactions enables the design of non- thrombogenic medical devices.

• Need to develop algorithms for modeling protein adsorption on surfaces.

• Difficult, but necessary.

AFM_4mg/ml fibrinogen

Without P12 With 10μM P12 O

M

A

FM

SEM_Static_with fibrinogen and platelet

PS PMMA PLA

10

0μ

M P

12

10

μM

P1

2

n

o P

12

FIBRINOGEN STRUCTURE ON VARIOUS TYPES OF SURFACES Strong attraction between D,E domains and hydrophobic surface Strong attraction between alpha-C domains and hydrophilic surface

hydrophobic

Sibel Tunc et al. Colloids and Surfaces B: Biointerfaces 42, 219 (2005)

hydrophilic

extended trinodular structure globular structure

Immunohistochemistry Confocal microscopy:

Samples were first exposed

to (a) anti-fibrinogen alpha

chain 529-539 mAb or (b)

anti-fibrinogen gamma

chain 86-411 mAb, and

then to Alexa Fluor 594

anti-mouse IgG. (a)Note

that although the alpha

chains are present, they are

unavailable for binding to

the anti-mouse IgG on

P4VP.

(b) Note that the gamma

chains are exposed

primarily on the fibers

formed on the PS surface,

and unavailable for the

oligomers adsorbed

directly on the surface. On

the P4VP coated surface,

the oligomers are adsorbed

such that the gamma chain

is exposed.