Microscopy of blood clot dissolution

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Brent Rodgers
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1 Microscopy of blood clot dissolution postgraduate seminar Franci Bajd advisor: doc. dr. Igor Serša MRI Laboratory, Jožef Stefan Institute 1

2 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 2

structure Thrombolysis at submicron and macroscopic level Thrombolysis at

3 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 3

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.

solidification within minutes Blood cells: erythrocytes (RBCs) > thrombocytes (Plt) > leukocytes (WBCs)

4 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 5 um Hematocrit level 42% Red blood cells Blood proteins (7g/dl): albumins(60%), immunoglobulins (18%), fibrinogen (4%), regulatory proteins electrolyte 23 nm 4

5 Formation of blood clot (thrombosis) 5

6 Coagulation cascade Fibrin polymerization and crosslinking: formation of fibrin clot Int. Ext. F XIIIa F XIII Thrombin Crosslinked Fibrin Meshwork Fibrin polymers Fibrin monomers Fibrinogen Plasmin Plasminogen Fibrinopeptides A,B B rt-pa Fibrin Degradation Products (FDP) Serious bleeding problems D-dimers 6

F XIIIa F XIII Thrombin Crosslinked Fibrin Meshwork Fibrin polymers Fibrin

7 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 ( L a b ) Fab 4 E= I = πr / 4 6yLI Streching experiment: E = 1.9 MPa (11.5 MPa) in non-crosslinked (crosslinked) fiber F = πr E 2 L y Brownian motion experiment: E = 2.3 MPa (23.1 MPa) in non-crosslinked (crosslinked) fiber Collet et al, PNAS 102, 9133 (2005) 7

5 MPa) in non-crosslinked (crosslinked) fiber 2 2 2 ( L a b ) Fab 4 E= I = πr / 4 6yLI Streching experiment:

et al, Science 313, 634 (2006) Forced unfolding of

8 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) 8

9 Blood clot structure Blood cells in fibrin meshwork: blood clot Microscopy-based technique (electron, confocal optical) a) b) c) 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 9

fibers Covalently bound platelets van der Waals interaction between red blood cells and fibrin meshwork Retracted vs.

10 Dissolution of blood clot (thrombolysis) 10

11 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 determination of fibrinogen degradation products (FDPs) v =µ( m) E Weisel JW, JTH 5, 116 (2007) 11

c) Fibrinolysis time interval: 5 min Electrophoresis: mobility-based separation in external electric field:

Lesions are indicated by arrows. Clot position and its size are not seen. Image dimension is of 20 cm.

12 Thrombolysis at macroscopic level S = S TR/ T1 0 (1 e ) e TE/ T2 e bd MRI signal ρ( r) 1 2π clinical MRI i ω( r) t = S( t) e dt proton density 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. 12

13 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 Plasma flow Glue Retracted blood clot Plasma Microscope objective Observation chamber Plasma reservoir Peristaltic pump 13

rheological conditions in venous circulatory subsystem conventional optical microscopy (Nikon 80 Eclipse, objective: 0.

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

14 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 Detection difficulties associated with moving objects 640 x 1.3 um/p 14

reversible deformation of fibrin fiber in plasma flow Optimal imaging parameters: frame rate: 1 frame per

15 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) b) 10 min 13 min 16 min 19 min rt-pa Fast flow (30 cm/s) 200 um 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 15

200 um 4 min 5 min 7 min 8 min Quantification of blood clot dissolution: Clot dissolution curves: A(t)

16 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 16

rt-pa Elastic deformations with no dissolution Advantage: similar rheological conditions

Results 4 a) b) A (t) A(t) Normalized non-lysed blood clot area [a.u.] 1,0 0,8 0,6 0,4 0,2 Slow flow + rt-pa Faster flow + rt-pa 0,0 0 5 10 15 20 25 30 Time [min] Clot dissolution curves Discrete area change [a.

17 Results 4 a) b) A (t) A(t) Normalized non-lysed blood clot area [a.u.] 1,0 0,8 0,6 0,4 0,2 Slow flow + rt-pa Faster flow + rt-pa 0, Time [min] Clot dissolution curves Discrete area change [a.u.] da dt 0,3 0,2 0,1 Slow flow + rt-pa Faster flow + rt-pa 0, Time [min] Discrete area change 17

18 Results 5 a) Averaged clot dissolution curves 1 3 b) Discrete area change distribution Normalized non-lysed blood clot area [a.u.] 1,0 0,8 0,6 0,4 0,2 No flow + rt-pa Slow flow + rt-pa Faster flow + rt-pa No flow Slow flow Faster flow 0, Time [min] Relative bin frequency [a.u.] ,200 um 2 10,600 um Discrete area change [µm 2 ] Slow flow + rt-pa Faster flow + rt-pa 14,000 um 2 160,000 um 2 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 18

three-step process: 1. rt-pa transport from plasma reservoir to the clot (2-20 s) 2. plasminogen activation 3.

19 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 19

Thrombolysis is a biochemo-mechanical process Clot degradation dynamics depends on rheological

20 Thank you for your attention! 20

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