De l'utilisation de la microscopie intravitale pour étudier la thrombose in vivo et tester de nouveaux médicaments antithrombotiques- 2nd partie

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1 De l'utilisation de la microscopie intravitale pour étudier la thrombose in vivo et tester de nouveaux médicaments antithrombotiques- 2nd partie Christophe Dubois INSERM UMR-S1076, Faculté de Pharmacie, Marseille, France

2 In vivo model of thrombus formation Dye Laser Activation of the coagulation cascade ADP???? Generation of thrombin ADP???? Accumulation of Platelets: thrombus formation

3 Accumulation of platelets in vivo after injection of Ph Control Ph (1 ug/kg) Ph (10 ug/kg) Platelets : Green 2x Real time movies

4 Effect of Ti on Thrombus formation : WT mouse Ti 150ng/g Ti 30ng/g Platelets in green Ti 15 ng/g

5 Effect of Ticagrelor on Thrombus formation : Ti (30 ng/g) Ti (150 ng/g) N= 30, 4 mice for each condition

6 Accumulation of platelets in vivo after gavage of mice by Clopidogrel Control Clopidogrel (8 mg/kg) Clopidogrel (30 mg/kg)

7 In vivo model of thrombus formation Dye Laser Activation of the coagulation cascade Generation of thrombin ADP ADP Accumulation of Platelets: thrombus formation All the drugs tested inhibed thrombus formation in a dose dependent manner

8 For all the other experiments we compared drugs at a concentration used to inhibit 50% of thrombus formation

9 Effect of antip2y12 on «embolie profile» : Set-up : Injection of anti-cd41 (250 ng/g mouse) Injury induced by a dye laser Time to exposure : 10 ms during 4.5 min = 1 frame each 18 ms

10 Determination of the «embolie» profile

11 WT Ph Clopidogrel Ti N= 31, 3 mice

12 In vivo model of thrombus formation Dye Laser Activation of the coagulation cascade Generation of thrombin Ti, Clopidogrel, Ac, Ph ADP ADP thrombus formation Ti, Clopidogrel, Ac Stabilization of the growing thrombus

13 Are all the drugs affecting activation of platelets participation to a growing thrombus?

14 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

15 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). 15 Dubois C. et al. JCI, 2007

16 Effect of Ticagrelor on calcium mobilisation : WT Ti Ph Platelets in green Calcium in red Platelets and calcium in yellow

17 Ph Ac Ti

18 Ti and Clopidogrel but not Ph strongly inhibit the calcium mobilization of platelets participating to a growing thrombus Are the drugs affecting the generation of fibrin?

19 In this in vivo model, how thrombin is generated? 19

20 In vitro Evidences That a pulse of a Dye Laser Activates the Endothelium HUVEC cells labeled with Fluo-4AM 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.

21 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)

22 1- Via Monocyte-derived MPs expressing TF J Exp Med Jun 2;197(11): 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 22 - P-selectin -/- and PSGL1-/- mice have a normal thrombus formation

23 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 23 more signaling pathways affecting indirectly thrombus generation

24 From Bizzozero G. (1882), Virchows Arch. Pathol. Physiol. 24

25 Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development Peter L. Gross, Barbara C. Furie, Glenn Merrill-Skoloff, Janet Chou and Bruce Furie

26 Darbousset R. et al., in preparation

27 Visualization of red blood cells : (Ter-119 antibody) Darbousset R. et al., in preparation

28 Accumulation of leucocytes at the site of injury Injection of 2x10 6 fluorescent leucocytes (from spleen) Darbousset R. et al., in preparation

29 Accumulation of leucocytes at the site of injury Mac-1 antibody Irrelevant antibody Mac-1 : Red Platelets : Green Darbousset R. et al., in preparation

30 Accumulation of leucocytes at the site of injury Mac-1 antibody Irrelevant antibody Darbousset R. et al., in preparation

31 Depletion of platelets : Injection of an antigpib antibodies (R300) Mac-1 : Red Platelets : Green Darbousset R. et al., in preparation

32 Depletion of platelets : Injection of an antigpib antibodies (R300) Integrated)fluorescent)intensity)(arbitrary)units)) 60" 50" 40" 30" 20" 10" Accumula7on)of)Leucocytes)at)the)site)of)injury)in) presence)or)absence)of)platelets) 0" 0" 50" 100" 150" 200" 250" 300" 350" 400" 450" Before)deple7on)(n=34,)4)mice)) Elapsed)7me)(sec)) ACer)deple7on,)n=31,)4)mice) Darbousset R. et al., in preparation

33 Colocalization of platelets, endothelium and leucocytes in vivo Leucocytes : Mac-1 : Red Platelets : CD41: Green Endothelium : Isolectin GS-IB4: Blue Darbousset R. et al., in preparation

34 Population(s) of leucocytes accumulating at the site of injury CX3CR1-GFP mice : Monocytes (2%), NK and DC cells in green

35 Accumulation of monocytes at the site of injury

36 Accumulation of monocytes at the site of injury...30 min latter

37 Monocytes (or MPs-derived from monocytes) started to accumulate at the site of injury min after the injury

38 Accumulation of Neutrophils at the site of injury Injection of antibody directed against Neutrophils Green : platelets; Blue : Neutrophils

39 Neutrophils but not monocytes accumulated at the site of injury Blue : antibody (Neutrophils) Green : GFP-CXCR3 cells (monocytes and DC cells) Red: Platelets

40 Neutrophils accumulation at the site of injury In presence of bloking LFA-1 antibody Mac-1 : Red Platelets : Green Darbousset R. et al., in preparation

41 Neutrophils accumulation at the site of injury In presence of bloking ICAM-1 antibody Mac-1 : Red Platelets : Green Darbousset R. et al., in preparation

42 Kinetics of thrombus formation in presence of bloking LFA-1 antibody Integrated)fluorescence)intensity))))) (x) )) 140" 120" 100" 80" 60" 40" 20" 0" Platelets)accumula9on)at)site)of)injury) 0,4" 0,35" 0,3" 0,25" 0,2" 0,15" 0,1" 0,05" 0" 0" 50" 100" 150" 200" 250" 300" 350" 400" Elapsed)9me)(sec))

43 Inhibition of Neutrophils accumulation at the site of injury prevent fibrin generation Before injection After injection of ICAM-1 Blocking antibody Green : Fibrin Red : Leucocytes (Mac-1)

44 Integrated)Fluorescent)Intensity)(au)) Fibrin)genera6on)in)presence)or)abscence) of)leucocytes) 250" 200" 150" 100" 50" 0" 0" 50" 100" 150" 200" 250" 300" Elapsed)6me)(sec)) median)fibrin) Darbousset R. et al., in preparation

45 Conclusions : The dye laser induces an activation of endothelium Neutrophils accumulate at the site of laser-induced injury They bind to activated endothelium (and not platelets) via interactions between ICAM-1 and LFA-1 They presence is required for both accumulation of platelets and generation of fibrin at the site of injury

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