THE INTERFACE BETWEEN THROMBOSIS AND INFLAMMATION THE MULTIVALENT ROLE OF TISSUE FACTOR (TF)

THE INTERFACE BETWEEN THROMBOSIS AND INFLAMMATION THE MULTIVALENT ROLE OF TISSUE FACTOR (TF)

-1905-1995. TF/FVIIa complex is the prima ballerina in the in vivo coagulation. Rappaport and Rao, 1995-1995-2007. Cytokines and molecules TF agonists and inducers correlation and link with human clinical disorders. -2003-2008. Inflammation (???) - Immune response ΤF MF (Multivalent Factor)

TISSUE FACTOR (TF) Class-II-TYPE cytocine receptor (IFNγ receptor) ΠΩΣ ΔΡΑ Σχηματίζει καταλυτικό σύμπλοκο με τον παράγοντα VIIa εξαρτώμενο από επιφάνειες φωσφολιπιδίων αρνητικά φορτισμένες και πυροδοτεί την πήξη ενεργοποιώντας τους παράγοντες ΙΧ και Χ, με τελικό αποτέλεσμα το σχηματισμό θρομβίνης. TF is a cell receptor and cofactor of FVII

Ο TF είναι μια διαμεμβρανική γλυκοπρωτείνη 47kD, 295 a (32 leader seq) Αποτελείται από τρία τμήματα: 1. μεγάλο εξωκυττάριο τμήμα 219 2. διαμεμβρανικό τμήμα 23 3. κυτταροπλασματική ουρά 21 Το εξωκυττάριο τμήμα 219 αμινοξέων είναι σημαντικό για την αιμοστατική δραστηριότητα, το διαμεμβρανικό είναι υπεύθυνο για τη σταθεροποίηση του μορίου, ενώ η λειτουργία του ενδοκυττάριου τμήματος δεν είναι ακόμη ξεκαθαρισμένη.

Ο TF έχει υψηλή συγγένεια με τον FVII o οποίος κυκλοφορεί στο αίμα. Η ενεργοποίηση του γίνεται μετά τη σύνδεσή του με τον TF.

Αυτό το σύμπλεγμα είναι ικανό να ενεργοποιήσει τον FX και τον FIX.

ΜΟΝΤΕΛΟ ΤΡΙΠΛΟΥ ΣΥΜΠΛΟΚΟΥ

O TFPI είναι μια πρωτεΐνη του πλάσματος η οποία συνδέεται με τον FXa. Το TFPI - FXa συνδέεται με το TF-FVIIa και μέσω αρνητικού feedback αναστέλλει την ενεργοποίηση μεγαλύτερων ποσοτήτων FX.

ΙΣΤΙΚΟΣ ΠΑΡΑΓΟΝΤΑΣ (TF) ΜΟΝΤΕΛΟ ΤΡΙΠΛΟΥ ΣΥΜΠΛΟΚΟΥ TF/VIIa/Xa

Angiogenesis, cell survival and growth, Metastasis, fibrosis (CTGF)???, inflammation Thrombosis, inflammation, angiogenesis

Coagulation cascade proteases thrombin, Xa, VIIa, and Protease Activated Receptors PARs

TF inducers, pathways and cells LPS, TNFa, IFN, CRP, IL-6, +???? Monocytes, Endothelial cells, Cancer cells, +???? C5a, Leptin, Neutrophils Tissue Injury Sepsis; Inflammation TF upregulation Hypercoagulability VIIa Xa IIa THROMBOSIS INFLAMMATION platelet activation / leucocyte recruitment Coronary syndromes, atherosclerosis, DIC, APS, ARDS Lung and multi-organ failure, Cancer thrombosis and progression, procoagulant of inflammatory disorders, fibrosis

TF VIIa = unstable system Regulation of system is dependent on: TF «load, doses» (inflammatory-immune-cancer cells ImmuneTF inducers) TF isoforms System «brakes» (natural anticoagulants) Xa, Fibrin production and doses Blood flow, endothelium, platelets Fibrinolytic activity The limits of the system (comprehensive system) = desirable consequences or not : 1. Haemostasis, Thrombosis, Immune response. 2. Coagulation independent signal transduction = Immune response: Inflammation, angiogenesis, metastasis, wound healing, fibrosis.

TF expression -Brain, heart, lung, kidney, placenta, cancer cells (hemostatic envelop) -TF is not expressed in healthy endothelial cells? -TF is expressed in stimulated monocytes (peripheral blood, CD14) -Τhe role of neutrophils? Blood- borne TF The pattern of TF expression in cells is complex and is influenced by differentiation as well as various agonists. TF is subject to postranslational modifications

as-htf: ENA SPLICE VARIANT TOY TF (Bogdanov et al; April 2003, Nat Med) Σχηματική απόδοση των splice variants του TF Κόκκινο: εναρκτήρια ακολουθία, Πράσινο: εξωκυττάρια περιοχή, Μπλε: διαμεμβρανική περιοχή, Κίτρινο: ενδοκυττάρια περιοχή, Πορτοκαλί: Ίδια ακολουθία as-htf, Λευκό: Μη μεταφραζόμενες περιοχές.

Blood - borne TF or circulating TF -Membrane microparticles -astf (procoagulant activity?) -Cellurar origin of astf -Circulating TF has both coagulation dependent and independent activity? -Circulating TF signaling is only indirect (PAR1, 2)??? -Are there representative clinical models related with circulating TF? (eg. ACS, cancer, APS, ARDS)

Questions related with pro- and coagulant activity -TF INDUCERS APS-C5a (Ritis et al JI 2006 177: 4794-802 ), Leptin (Rafail et al Thromb Res 2008,), Biomaterials C5a (Kourtzelis et al, under submission) -Cells expressing TF Neutrophils (Ritis et JI 2006 177: 4794-802 ) -TF signalling JAK2, PI3 (Rafail et althromb Res 2008) -In vivo or clinical models confirming the in vitro results APS (Redecha et al Blood, 2007 7: 2423-31 ), ACS/Hyperleptinemia (Napoleone et al J Thromb Haem, 2007 7: 1462-8), Cancer (many refs) -TF / astf / Isoforms / Microparticles. - Cancer =More than one TF isoforms???

Questions related to non-procoagulant activity or properties -TF-VII / PAR1,2 signal transduction without clinical coagulation dependent events (COAGULATION BYPASS) -TF / astf / Isoforms /Microparticles. -In vivo linking with clinical models and disorders.

INFLAMMATION IMMUNOSTIMULATION CHEMOTAXIS NEUTROPHIL C5a COMPLEMENT ACTIVATION J. Immunol, 2006 Oct 177 K. Ritis, et al TF INDUCTION NEUTROPHIL

P. Redecha, R. Tilley, M. Tencati, J. E. Salmon, D. Kirchhofer, N. Mackman, and G. Girardi Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury Blood, October 1, 2007; 110(7): 2423-2431.

Neutrophil activation by the tissue factor/ Factor VIIa/PAR2 axis mediates fetal death in a mouse model of antiphospholipid syndrome Patricia Redecha, Claus-Werner Franzke, Wolfram Ruf, Nigel Mackman, and Guillermina Girardi J. Clin. Invest. 118(10): 3453-3461 (2008). doi:10.1172/jci36089.

G Girardi and N Mackman Tissue factor in antiphospholipid antibody-induced pregnancy loss: a pro-inflammatory molecule Lupus, October 1, 2008; 17(10): 931-936. A. Kinev and R. Roubey Tissue factor in the antiphospholipid syndrome Lupus, October 1, 2008; 17(10): 953-959.

Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation Busso N et al. Arthritis Research & Therapy 2008 Soluble TF induces acute inflammation through a thrombindependent pathway and both fibrin deposition and platelet activation are essential steps in this process. The activation of PAR-4 on platelets is crucial and the other PARs do not play a major role in soluble TF-induced inflammation

FIBROSIS AND POTENTIAL LINK WITH EXTRINCINC SYSTEM 1. Trauma coagulation - scar ( VIIa) 2. Cell lines (ΤΗP1, fibroblasts) ή animal models: - Correlations CCN1, CCN2 (CTGF)/PAR1,2 - Correlations TF-VIIa, Xa, Thrombin/PAR1,2. -Accumulation of cells (able to produce TF) in experimental fibrotic models. 3. Human clinical models indicating a link between coagulation and fibrosis (eg ACS, Zarnece et al Feb 2008, Circulation Research, ARDS Kambas et al JI 2008, in presss.) 4. Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Gαq and cooperation between ERK1/2 and Rho kinase signalling pathways (Chambers R, 2008)

INFLAMMATORY CYTOKINES? Is this pathway involved in any in VIVO clinical model? TF Stimulation/inhibition studies Blockage of receptors of cells followed by stimulations/inhibition studies PARs CTGF CELLS?? astf FIBROSIS!

endothelium Intervention TF TRIGGER Inflammatory - Chemotactic activation C A C = B Improvement but not crucial? Future targets TNFα, IL6, IL8, C5a etc TF TF 1 2 1. Cytokine production 2. Coagulation TF + FVIIa Thrombin TF A B 3 FIBROSIS PARs blood vessel alveolus (BAL)

ARDS ARDS BALF cytokines BALF cytokines C5a + TNFa TGF-beta1 Neutrophil Myofibroblasts Tissue Factor asma

THROMBIN PAR-1 Fibroblast CTGF + Collagen

BPD BPD RDS RDS

Fold expression Figure 1 A * B a I II BALF CELL LYSATES TF GAPDH 1 2 b I II BALF SUPERNATANS TF CTGF C i ii iii

Figure 6 Fold expression Collagen content (%) Fold expression Collagen content (%) I II III IV B TF GAPDH A i ii * * * /* / * * C iii iv i 1 2 3 ii 1 2 3 4 5 6 * * * * * * * D i ii iii E iv v vi i 1 2 3 1 ii 2 3 4 5 6

Figure 7 Tissue Injury/Inflammation Pulmonary circulation Alveolar Epithelium 1 2 Inactive coagulation factors (VII, X, prothrombin) Activation of exogenous coagulation cascade Thrombin +C5a TF Neutrophils Big Endothelin-1 ECE +Other pulmonary cells Endothelin-1 Amplification Loop PAR-1 and/or other mediators Thrombin TF 3 VIIa, Xa Migration Myofibroblasts CTGF FIBROSIS

. Blood. 2008 Jan 1;111(1):190-9.

FUTURE

Σ. Ραφαήλ Μ. Σπελέτας Κ. Καμπάς Π. Σιδεράς Γ. Κουρτζέλης Σ. Κωνσταντινίδης, Κ. Schaefer Γ. Μητρούλης Π. Πασαδάκης, Σ. Γιαγλής Γ. Πνευματικός Μ. Δούμας J. D. Labris, Κ. Ρίτης