De l'utilisation de la microscopie intravitale pour étudier la thrombose in vivo et tester de nouveaux médicaments antithrombotiques- 1er partie Christophe Dubois INSERM UMR-S-1076, Faculté de Pharmacie, Marseille, France
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Injury creation Halogen Lamp Uniblitz Shutter Brightfield Imaging TTL syncronisation Red : Platelets Green : P-selectin Computer control
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Injury creation Halogen Lamp Uniblitz Shutter Brightfield Imaging TTL syncronisation Red : Platelets Green : P-selectin Computer control
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Injury creation Halogen Lamp Uniblitz Shutter Brightfield Imaging TTL syncronisation Red : Platelets Green : P-selectin Computer control
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Injury creation Halogen Lamp Uniblitz Shutter Brightfield Imaging TTL syncronisation Red : Platelets Green : P-selectin Computer control
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Injury creation Halogen Lamp Uniblitz Shutter Brightfield Imaging TTL syncronisation Red : Platelets Green : P-selectin Computer control
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Injury creation Halogen Lamp Uniblitz Shutter Brightfield Imaging TTL syncronisation Red : Platelets Green : P-selectin Computer control
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Injury creation Halogen Lamp Uniblitz Shutter Brightfield Imaging TTL syncronisation Red : Platelets Green : P-selectin Computer control
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Halogen Lamp Uniblitz Shutter Computer control Injury creation Brightfield Imaging TTL syncronisation
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Halogen Lamp Uniblitz Shutter Computer control Injury creation Brightfield Imaging TTL syncronisation
The Equipment Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Filter array Ablation Laser (Nitrogen) Galvinometric positioning control Halogen Lamp Uniblitz Shutter Computer control Injury creation Brightfield Imaging TTL syncronisation
The Equipment Red : Platelets Green : Fibrin Realtime Fluorescence Imaging Xenon Lamp (DG4) Fast Low Resolution Camera Image Intensifier Ablation Laser (Nitrogen) Galvinometric positioning control Injury creation Filter array Halogen Lamp Uniblitz Shutter Brightfield Imaging Piezoelectric focusing collar TTL syncronisation Slidebook (3I) and Deconvolution module Computer control
The model of laser-induced injury
The model of laser-induced injury Advantages : - No major traumatism induced (by the surgery) for the mouse, - The skin is thin enough to obtain high resolution brightfield images, independently of the weight of the mouse - Both arteries and veins are present
The model of laser-induced injury Advantages : - No major traumatism induced (by the surgery) for the mouse, - The skin is thin enough to obtain high resolution brightfield images, independently of the weight of the mouse - Both arteries and veins are present - The size, the power and the localization of the injury are controlled
The model of laser-induced injury Advantages : - No major traumatism induced (by the surgery) for the mouse, - The skin is thin enough to obtain high resolution brightfield images, independently of the weight of the mouse - Both arteries and veins are present - The size, the power and the localization of the injury are controlled - 7 15 thrombi per mouse / 2 h of experiment
The model of laser-induced injury Advantages : - No major traumatism induced (by the surgery) for the mouse, - The skin is thin enough to obtain high resolution brightfield images, independently of the weight of the mouse - Both arteries and veins are present - The size, the power and the localization of the injury are controlled - 7 15 thrombi per mouse / 2 h of experiment BUT :
The model of laser-induced injury Advantages : - No major traumatism induced (by the surgery) for the mouse, - The skin is thin enough to obtain high resolution brightfield images, independently of the weight of the mouse - Both arteries and veins are present - The size, the power and the localization of the injury are controlled - 7 15 thrombi per mouse / 2 h of experiment BUT : When shooting using the laser pulse, it s impossible to predict that one are localized in the right plane (necessary to induce an injury).the same effect may be induced by 1, 5 or 50 laser pulses.
The model of laser-induced injury Advantages : - No major traumatism induced (by the surgery) for the mouse, - The skin is thin enough to obtain high resolution brightfield images, independently of the weight of the mouse - Both arteries and veins are present - The size, the power and the localization of the injury are controlled - 7 15 thrombi per mouse / 2 h of experiment BUT : When shooting using the laser pulse, it s impossible to predict that one are localized in the right plane (necessary to induce an injury).the same effect may be induced by 1, 5 or 50 laser pulses. The B&Bs model :
The model of laser-induced injury Advantages : - No major traumatism induced (by the surgery) for the mouse, - The skin is thin enough to obtain high resolution brightfield images, independently of the weight of the mouse - Both arteries and veins are present - The size, the power and the localization of the injury are controlled - 7 15 thrombi per mouse / 2 h of experiment BUT : When shooting using the laser pulse, it s impossible to predict that one are localized in the right plane (necessary to induce an injury).the same effect may be induced by 1, 5 or 50 laser pulses. The B&Bs model : The number of pulses must be as minimal as possible to induce a thrombus (thrombi are not occlusive)
Kinetics of platelet accumulation after a laser-induced injury F platelets (Integrated Fluorescent Intensity (arbitrary units)) Median of 33 thrombi in 3 mice Elapsed time (sec)
1- an interaction between Collagen and platelets is required for thrombus formation
Accumulation of platelets in vivo after a FeCl3-induced injury -Injury induced by 10% FeCl3 for 5 min on the mesentery muscle. -Infusion of antibody directed against αiib labeled with Alexa-660. WT FcRγ -/-
Accumulation of platelets in vivo after a FeCl3-induced injury -Injury induced by 10% FeCl3 for 5 min on the mesentery muscle. -Infusion of antibody directed against αiib labeled with Alexa-660. WT FcRγ -/-
Accumulation of platelets in vivo after a FeCl3-induced injury -Injury induced by 10% FeCl3 for 5 min on the mesentery muscle. -Infusion of antibody directed against αiib labeled with Alexa-660. WT FcRγ -/-
Platelet accumulation in GPVI depleted mice after a laser-induced injury (n = 40, 3 mice) (n = 35, 3 mice) GPVI is not involved in platelet accumulation after a laserinduced injury
Collagen exposure to the blood circulation after a laser or a FeCl3-induced injury - Infusion of an antibody directed against αiib + an antibody directed against Collagen type I.
Collagen exposure to the blood circulation after a laser or a FeCl3-induced injury - Infusion of an antibody directed against αiib + an antibody directed against Collagen type I. FeCl3 WT Collagen Platelets Collagen FcRγ Platelets
Collagen exposure to the blood circulation after a laser or a FeCl3-induced injury - Infusion of an antibody directed against αiib + an antibody directed against Collagen type I. FeCl3 WT Collagen Platelets Collagen FcRγ Laser Platelets Platelets Platelets
Conclusions - GPVI is involved in vivo in the adhesion of platelets when collagen is exposed to the blood circulation.
Conclusions - GPVI is involved in vivo in the adhesion of platelets when collagen is exposed to the blood circulation. - After a laser-induced injury, GPVI-collagen interaction is not involved in thrombus formation.
Conclusions - GPVI is involved in vivo in the adhesion of platelets when collagen is exposed to the blood circulation. - After a laser-induced injury, GPVI-collagen interaction is not involved in thrombus formation. - The study of a thrombus formation in vivo using either a laser or a chemical injury is complementary and necessary to understand the different pathways involved in the thrombosis process.
Role of the coagulation cascade in the formation of a thrombus WT mouse FVIII -/- mouse
Role of the coagulation cascade in the formation of a thrombus WT mouse FVIII -/- mouse The activation of the coagulation cascade plays a key role in the thrombus induced by the dye laser
Kinetics of thrombus formation in WT mice and PAR4 -/- mice WT PAR4 Null Real-time video Real-time video
Kinetics of thrombus formation in WT mice and PAR4 -/- mice
1- an interaction between Collagen and platelets is required for thrombus formation Following a laser-induced injury thrombin but not collagen is playing a key role in thrombus formation 13
2- In vitro, addition of thrombin to plasma (washed platelets) induces a stable aggregation of platelets and immediate secretion of granule contents
Expression of P-selectin by platelets in vivo Platelets in red P-selectin in green
Before injury WT 20 s 100 s 150 s
Before injury WT 20 s 100 s 150 s Platelets do not seem to express P-selectin during the first 20 sec
2- In vitro, addition of thrombin to plasma (washed platelets) induces a stable aggregation of platelets and immediate secretion of granule contents In our model, most of the «platelet aggregate» induced by thrombin is reversible and platelets do not immediatly express P-selectin 18
3- In vitro, addition of thrombin to washed platelets induces a prolonged calcium mobilization (via a sequential activation of PAR1 and PAR4)
Importance of the Calcium Mobilization on platelet Activation Thrombin ADP Thromboxane A2 PAR, P2Y1, TxA2R G 12/13 Gq Shape Change Secretion PLC Ca 2+ αiibβ3 activation Aggregation
Infusion of 250 x10 6 (20%) platelets loaded with 3 µm Fura 2AM. Fura 2-loaded Platelets were followed by excitation at 380 nm (Fura 2 AM) and Calcium mobilization was followed by excitation at 340 nm (Fura 2-AM bound to calcium). 21
small thrombus mean size thrombus mean size thrombus Occlusive thrombus
small thrombus mean size thrombus mean size thrombus Occlusive thrombus
Real time Images of Calcium Mobilization in Platelets Pre-injury 15 sec 30 sec 45 sec 90 sec 150 sec
Real time Images of Calcium Mobilization in Platelets Pre-injury 15 sec 30 sec 45 sec 90 sec 150 sec - Calcium signal was observed in the fura-2 loaded platelets - All the platelets participating in the growing thrombus do not mobilize calcium
How long does a platelet remain in a growing thrombus? Duration of platelet residence in a growing thrombus (sec) 150 100 50 0 0 35 sec Platelets that mobilize calcium 11 sec Platelets that do not mobilize calcium 9 sec BAPTA-AM treated Platelets A platelet that mobilizes calcium remains in the thrombus longer than a platelet that does not mobilize calcium 24
200 n = 17 (events), 14 thrombi Duration of platelet adhesion (sec) 180 160 140 120 100 80 60 40 20 y = 1.0172x + 9.148; R 2 = 0.9174 0 0 20 40 60 80 100 120 140 160 180 Duration of Calcium Mobilization (sec)
10 8 F platelets 6 4 2 3 rd region 2 nd region 1 st region 0 0 50 100 150 200 250 300 350 Elapsed time (sec) 0.4 1 st region F calcium / F platelets 0.35 0.3 2 nd region 3 rd region 0.25 0 50 100 150 200 250 300 350 Elapsed time (sec)
Characteristics of Calcium Mobilization In Vivo : - By infusing Fura-2 loaded platelets we were able to follow platelet accumulation and calcium mobilization in vivo. - Platelets bind to a growing thrombus without mobilizing calcium. - Platelets that mobilize calcium after incorporation into a thrombus stay longer in the thrombus than platelets that do not mobilize calcium. - There is a direct correlation between the time that a platelet spends in a growing thrombus and the duration of the calcium spike. - Only one calcium peak was detected in our model. 28
Involvement of Thrombin in platelet activation WT FcRγ -/- WT + Hirudin PAR4 -/- Dubois C. et al. JCI, 2007
Calcium Mobilization and Thrombus formation in PAR4 null mice Platelets Calcium F platelets (arbitrary units) PAR4 WT F calcium (arbitrary units) PAR4 WT Elapsed time (sec) PAR4 null mice generate significantly smaller thrombi than wildtype mice following laser injury. Elapsed time (sec) Calcium mobilization in platelets from PAR4 null mice can be detected but is much reduced compared to wildtype. Dubois C. et al. JCI, 2007
Involvement of Thrombin on platelet activation Dubois C. et al. JCI, 2007
Involvement of Thrombin on platelet activation 0.1 F calcium / F platelets (arbitrary units) 0.08 0.06 0.04 0.02 0 WT vwf-/- FcRγ-/- WT + Lepirudin PAR4 -/- Dubois C. et al. JCI, 2007
4- In vitro, ADP is a secondary agonist playing a (limited) role in platelet aggregation induced by thrombin, mainly via P2Y12
Accumulation of platelets in vivo after gavage of mice by Clopidogrel (30 mg/kg by gavage) Control Clopidogrel Clopidogrel Mezouar S. et al., in preparation
Mezouar S. et al., in preparation
Effect of Ticagrelor on Thrombus formation : WT mouse ticagrelor 150ng/g ticagrelor 30ng/g Platelets in green ticagrelor 15 ng/g Mezouar S. et al., in preparation
Effect of Ticagrelor on Thrombus formation : N= 30, 4 mice for each condition Mezouar S. et al., in preparation
4- In vitro, ADP is a secondary agonist playing a (limited) role in platelet aggregation induced by thrombin, mainly via P2Y12
4- In vitro, ADP is a secondary agonist playing a (limited) role in platelet aggregation induced by thrombin, mainly via P2Y12 In our model ADP seems to play a key role in thrombus formation
In vitro Evidences That a pulse of a Dye Laser Activates the Endothelium HUVEC cells labeled with Fluo-3 Courtesy of Dr Ben Atkinson, 3I
In vitro Evidences That a pulse of a Dye Laser Activates the Endothelium HUVEC cells labeled with Fluo-3 Courtesy of Dr Ben Atkinson, 3I
In vitro Evidences That a pulse of a Dye Laser Activates the Endothelium HUVEC cells labeled with Fluo-3 Targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation in the targeted cell and was followed by a wave of calcium elevation in surrounding cells. Courtesy of Dr Ben Atkinson, 3I
In vitro Evidences That a pulse of a Dye Laser Activates the Endothelium HUVEC cells labeled with Fluo-3 Targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation in the targeted cell and was followed by a wave of calcium elevation in surrounding cells. Courtesy of Dr Ben Atkinson, 3I
In Vivo Evidences That a pulse of a Dye Laser Activates the Endothelium Uptake of Fluo-4AM by the endothelium Courtesy of Dr B. Atkinson, 3I
In Vivo Evidences That a pulse of a Dye Laser Activates the Endothelium Uptake of Fluo-4AM by the endothelium Courtesy of Dr B. Atkinson, 3I
In Vivo Evidences That a pulse of a Dye Laser Activates the Endothelium Uptake of Fluo-4AM by the endothelium The observed calcium elevation was rapid (within 30 s) and preceded detection of platelets in the developing thrombus. These results demonstrate that the endothelium activates rapidly prior to thrombus formation in the laser induced thrombosis model. (Atkinson et al., Blood, 2010) Courtesy of Dr B. Atkinson, 3I
In Vivo Evidences That a pulse of a Dye Laser Activates the Endothelium Uptake of Fluo-4AM by the endothelium The observed calcium elevation was rapid (within 30 s) and preceded detection of platelets in the developing thrombus. These results demonstrate that the endothelium activates rapidly prior to thrombus formation in the laser induced thrombosis model. (Atkinson et al., Blood, 2010) Courtesy of Dr B. Atkinson, 3I
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5- In vitro, negatively charged phospholipids exposed at the surface of activated platelets served as a matrix for the activation of the coagulation cascade 41
Involvement of Thrombin on platelet activation
Involvement of Thrombin on platelet activation 0.1 F calcium / F platelets (arbitrary units) 0.08 0.06 0.04 0.02 0 WT vwf-/- FcRγ-/- WT + Lepirudin PAR4 -/-
Platelet and fibrin accumulation in wild-type and PAR4 KO thrombi Wild-type PAR4 KO 150 ng/g mouse of anti-cd41 Fab-Alexa-488. 500 ng/g mouse of anti-fibrin-alexa-647. 5x real time, recording over 5 minutes Platelets: Red Fibrin: Green Platelets + Fibrin: Yellow
Maximum fibrin generation WT PAR4 KO Wilcoxon Rank Sum Test: z=0.18. No significant difference.
5- In vitro, negatively charged phospholipids exposed at the surface of activated platelets served as a matrix for the activation of the coagulation cascade
5- In vitro, negatively charged phospholipids exposed at the surface of activated platelets served as a matrix for the activation of the coagulation cascade In our model, other activated membrane surfaces (independent of the accumulated platelets) are sufficient for fibrin generation.
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In this model, how thrombin is generated? 46
J Exp Med. 2003 Jun 2;197(11):1585-98. Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin. Falati S, Liu Q, Gross P, Merrill-Skoloff G, Chou J, Vandendries E, Celi A, Croce K, Furie BC, Furie B. Center for Hemostasis and Thrombosis Research, Research East 319, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA. Abstract Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocytederived microparticles in human platelet-poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor- and PSGL-1-containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P- selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking. However : - Exogenous MPs : not a direct proof of concept 47 - P-selectin -/- and PSGL1-/- mice have a normal thrombus formation
1- Via Monocyte-derived MPs expressing TF J Exp Med. 2003 Jun 2;197(11):1585-98. Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin. Falati S, Liu Q, Gross P, Merrill-Skoloff G, Chou J, Vandendries E, Celi A, Croce K, Furie BC, Furie B. Center for Hemostasis and Thrombosis Research, Research East 319, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA. Abstract Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocytederived microparticles in human platelet-poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor- and PSGL-1-containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P- selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking. However : - Exogenous MPs : not a direct proof of concept 47 - P-selectin -/- and PSGL1-/- mice have a normal thrombus formation
2- Via PDI : involved TF generation after a laser-induced injury However : - PDI is not specific of TF and inhibition of PDI may involve much 48 more signaling pathways affecting indirectly thrombus generation
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From Bizzozero G. (1882), Virchows Arch. Pathol. Physiol. 49