Microscopy of blood clot dissolution
postgraduate seminar
Franci Bajd
advisor: doc. dr. Igor Serša
MRI Laboratory, Jožef Stefan Institute
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Outline
Introduction
Blood components
Coagulation cascade and blood clot properties
Coagulation cascade
Blood clot elastic properties
Blood clot structure
Thrombolysis at submicron and macroscopic level
Thrombolysis at sub-micron level
clinical MRI / MRI microscopy
Thrombolysis at cell level
Some recent results
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Introduction
• Blood clots (thrombi) have dual role in hemostasis:
1 . After wound injury, coagulated blood provides a blood vessel recovery
and prevents bleeding and is thus vital for preservation of life,
2. improper coagulation of fluid blood into blood clots can completely
(occlusive clots) or partially (non-occlusive clots) impede blood flow in a circulatory
system, which can seriously threatens life.
• Diseases: deep vein thrombosis, pulmonary embolism, arterial thrombosis
• Mortality due to cardiovascular diseases: > 50%
• Clot properties gain multiscale interest in both basic and clinical studies:
1. to elucidate the origin of elastic properties of blood clots,
physiological conditions of their formation, the dynamics of blood vessel restoration
2. to optimize the thrombolytic treatment (surgically inaccessible vessels)
• Thrombosis (clot formation) and thrombolysis (clot dissolution) are complex processes
• Optical microscopy experiments: thrombolysis is biochemo-mechanical process
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Blood components
- complex colloidal suspension (complex structure and functionality)
- liquid state over decades of human life vs. solidification within minutes
• Blood cells: erythrocytes (RBCs) > thrombocytes (Plt) > leukocytes (WBCs)
Plasma
White blood cells and platelets
Hematocrit level
42%
5 um
Red blood cells
23 nm
• Blood proteins (7g/dl): albumins(60%), immunoglobulins (18%),
fibrinogen (4%), regulatory proteins
• electrolyte
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Formation of blood clot (thrombosis)
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Coagulation cascade
Int.
Fibrin polymerization and crosslinking: formation of fibrin clot
F XIIIa
Crosslinked
Fibrin Meshwork
Ext.
F XIII
Fibrin
polymers
Plasmin
Thrombin
Fibrin
monomers
Fibrinogen
Plasminogen
Fibrinopeptides A,
A,BB
rt-PA
Serious bleeding problems
Fibrin Degradation
Products (FDP)
D-dimers
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Blood clot elastic properties 1
The elasticity of an individual fibrin fiber in a clot
Bending experiment : E = 1.7 MPa (14.5 MPa) in non-crosslinked (crosslinked) fiber
E=
(
Fab 2
L − a2 − b2
6 yLI
)
I = πr 4 / 4
Streching experiment: E = 1.9 MPa (11.5 MPa) in non-crosslinked (crosslinked) fiber
F L
πr 2 y
Brownian motion experiment: E = 2.3 MPa (23.1 MPa) in non-crosslinked (crosslinked) fiber
E=
Collet et al, PNAS 102, 9133 (2005)
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Blood clot elastic properties 2
Fibrin fibers have extraordinary elasticity and extensibility
Liu W. et al, Science 313, 634 (2006)
Forced unfolding of coiled-coils in fibrinogen by single-molecule AFM
Brown et al, Biophys J. 92, L39 (2007)
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Blood clot structure
• Blood cells in fibrin meshwork: blood clot
• Microscopy-based technique (electron, confocal optical)
a)
c)
b)
Platlet-poor plasma clot
Platlet-rich plasma clot
Thrombus form heart attacked patient
• Colvalent bounds within and between fibrin fibers
• Covalently bound platelets
• van der Waals interaction between red blood cells and fibrin meshwork
• Retracted vs. non-retracted blood (amount of syneresed serum)
• Blood clots are highly compact structures – influence on clot’s susceptibility to thrombolysis
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Dissolution of blood clot (thrombolysis)
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Thrombolysis at submicron level
Confocal microscopy of fibrin clot dissolution:
a) straight and sharp front of lysis in platelet-poor plasma clot
b) deformed front of lysis in platelet-rich area -> retarded fibrinolysis
a)
b)
c)
Fibrinolysis time interval: 5 min
Electrophoresis:
• mobility-based separation in external electric field: electrophoresis patterns v = µ (m) E
• determination of fibrinogen degradation products (FDPs)
Weisel JW, JTH 5, 116 (2007)
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Thrombolysis at macroscopic level
S = S 0 (1 − e −TR / T 1 ) e −TE / T 2 e − bD
ρ (r ) =
1
S (t ) e −iω ( r ) t dt
2π ∫
MRI signal
proton density
clinical MRI
MRI images of human brain: T1-/T2- weighted
MRI image of healthy human brain (first image)
vs. diffusion-weighted MRI images of stroke brain
(second and third image). Lesions are indicated
by arrows. Clot position and its size are not seen.
Image dimension is of 20 cm.
MRI microscopy
T1-/T2-weighted MRI image of a venous blood
clot ex vivo exhibits its layered structure.
The clot is mostly composed of red blood cells
with small platelet region. Image dimension is of
3 cm.
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Thrombolysis at cell level
• Aim: In vitro model of thrombolytic therapy in venous circulatory system
• Hypothesis: thrombolysis is both biochemical and mechanical process
Materials and methods:
• observation chamber with retracted model blood clot
• perfusion system mimicked rheological conditions in venous circulatory subsystem
• conventional optical microscopy (Nikon 80 Eclipse, objective: 0.3 NA, 10x magnification)
Observation region
Glue
Plasma flow
Retracted
blood clot
Microscope
objective
Plasma
Observation
chamber
Peristaltic
pump
Plasma
reservoir
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Results 1
• In fluid blood, red blood cells are arranged in rouleau-like fashion
• Blood clot is formed when thrombin was added to blood
+
fluid blood
=
fibrin fibers
blood clot
• Stretching/bending experiment:
reversible deformation of fibrin fiber in plasma flow
• Optimal imaging parameters:
frame rate: 1 frame per 15 sec
exposure time: 20 ms
rt-PA
640 x 480 @ 1.3 um/p
• Detection difficulties associated with moving objects
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Results 2
• t = 0: rt-PA was added to the plasma reservoir
• Clot fragments of several RBCs with partially degraded fibrin meshwork are released
a)
200 um
Slow flow
(3 cm/s)
10 min
13 min
16 min
19 min
rt-PA
b)
200 um
Fast flow
(30 cm/s)
4 min
5 min
7 min
8 min
Quantification of blood clot dissolution:
•Clot dissolution curves: A(t)
•Distribution of removed clot fragments
•Distribution of discrete area changes, i.e. time derivative of clot dissolution curves
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Results 3
No flow
(0 cm/s)
Slow flow
(3 cm/s)
Fast flow
(30 cm/s)
rt-PA
Complete dissolution in less than 30 min after administration of rt-PA!
rt-PA
Elastic deformations with no dissolution
Advantage: similar rheological conditions as in venous blood vessels
Disadvantage: controlled manipulation with individual fibers in plasma flow was not possible
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Results 4
b)
A(t )
A(t )
0,3
1,0
0,8
0,6
0,4
0,2
Slow flow + rt-PA
Faster flow + rt-PA
Discrete area change [a.u.]
Normalized non-lysed blood clot area [a.u.]
a)
0,0
0
5
10
15
20
25
Slow flow + rt-PA
Faster flow + rt-PA
0,1
0,0
30
0
5
10
15
20
25
30
Time [min]
Time [min]
Clot dissolution curves
0,2
dA
dt
Discrete area change
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Results 5
1
3
b)
1,0
50
Relative bin frequency [a.u.]
Normalized non-lysed blood clot area [a.u.]
a)
Discrete area change distribution
Averaged clot dissolution curves
0,8
0,6
No flow + rt-Pa
Slow flow + rt-PA
Faster flow + rt-PA
0,4
0,2
No flow
Slow flow
Faster flow
2,200 um2
40
10,600 um2
30
Slow flow + rt-PA
Faster flow + rt-PA
20
14,000 um2
160,000 um2
10
0
0,0
0
5
10
15
Time [min]
20
25
30
3
10
10
4
5
10
2
Discrete area change [µm ]
• Thrombolysis is three-step process:
1. rt-PA transport from plasma reservoir to the clot (2 - 20 s)
2. plasminogen activation
3. biochemo-mechanical degradation
• Significantly different clot dissolution curves and discrete area change distributions
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Summary
• Microscopy-based techniques provide efficient visualization of a clot structure and its dissolution
• Mechanical properties of a blood clot are defined by its structure
• Thrombolysis is a biochemo-mechanical process
• Clot degradation dynamics depends on rheological conditions (mechanical forces, rt-PA transport)
• Outlook: Mathematical modeling of blood clot dissolution
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Thank you for your attention!