Ο ρόλος των ουδετερόφιλων στην θρόμβωση. Δημήτριος Στάκος, FESC, FSCAI Επ. καθηγητής Καρδιολογίας Δ.Π.Θ. Αλεξανδρούπολη

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1 Ο ρόλος των ουδετερόφιλων στην θρόμβωση Δημήτριος Στάκος, FESC, FSCAI Επ. καθηγητής Καρδιολογίας Δ.Π.Θ. Αλεξανδρούπολη

2 Αιμόσταση vs θρόμβωση

3 Αιμόσταση vs θρόμβωση Αγγειακός ιστικός παράγοντας (TF) και blood-born TF Η συμμετοχή ανοσιακών διαδικασιών βασική διαφορά μεταξύ αιμόστασης - θρόμβωσης

4 Ποια είναι η σημασία των κυττάρων της φυσικής ανοσίας στη θρόμβωση?

5 Hematopoietic TF, but not vessel wall TF is responsible for initiation of DVT ctrl ctrl +BM (low TF) Control mice transplanted with low-htf bone marrow cells Pawlinski R, et al. Blood, 2010

6 Neutropenic mice and thrombus size * anti-ly6g antibody* * Ab to deplete neutrophils von Brühl, et al. J Exp Med. 2012

7 How innate immune cells initiate DVT development? The significance of TF ctrl Fibrin formation ctrl Furie B. J Thromb. Haemost Manly DA. Annu. Rev. Physiol 2011

8 What signals supporting thrombogenesis are delivered by neutrophils? PMNs expressing TF is the main source of TF needed for platelet thrombus formation and fibrin generation following a laser-induced injury in vivo. Darbousset R, et al. Blood 2012

9 Encrypted TF

10 Brinkmann V, et al. Neutrophil extracellular traps kill bacteria. Science 2004 Neutrophil-derived structures composed of extruded DNA, decorated with antimicrobial proteins

11 NET s function High local concentration of antimicrobial components Kill microbes extracellularly independent of phagocytic uptake Physical barrier that prevents further spread of the pathogens Delivery of granule proteins into NETs (keep injurious proteins - proteases from diffusing)

12 Less energy consuming PMN Pathogen

13 Brinkmann V. J Cell Biol NET formation Firm attachment to the substratum Disintegration of membranes - mixture Cell contraction membrane rupture

14 Our hypothesis

15 NETS in STEMI Normal artery of the same patient Culprit lesion in Lcx (STEMI)

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17 MPO/DNA complex ELISA

18 Στην ένοχη αρτηρία υπάρχει σημαντική απελευθέρωση ΝΕΤs (τοπικά)

19 Έκφραση ιστικού παράγοντα στα STEMI NETs

20 Ο ιστικός παράγοντας στα NETs είναι ενεργός και η ενεργότητα του εξαρτάται από το ΝΕΤ Παραγωγή θρομβίνης (ενεργότητα TF)

21 Ο ιστικός παράγοντας στα NETs είναι ενεργός και η ενεργότητα του εξαρτάται από το ΝΕΤ Ενεργοποίηση αιμοπεταλίων (ενεργότητα TF)

22 ΝΕΤs με ιστικό παράγοντα μέσα στο θρόμβο

23 Intracellular TF upregulation in circulatinh PMNs of STEMI Circulating PMNs STEMI Western TF mrna (PCR)

24 1 st stimulus intracellular TF upregulation (systemic) 2 nd stimulus (highly local) release of NETs expressing TF thrombosis

25 A mechanism for TF-bearing NET release in STEMI A substance in STEMI plasma (inflammatory) is responsible for the 1 st hit

26 NETs in arterial thrombosis Platelet aggregation (RED)

27 Activated platelets induce the formation of NETs not expressing TF in the absence of the 1 st hit (STEMI plasma)

28 IRA-derived platelets In vitro reproduction of double hit phenomenon

29 Conclusions 1. Abundant NET release in IRA of STEMI patients 2. Local phenomenon 3. Functional TF is expressed on NETs able to induce thrombin generation and PLT activation into thrombus 4. Thrombin-activated platelets induce more NET release in a vicious cycle 5. NET (DNA) scaffold in imperative for TF functionality 6. TF-bearing NET release is a double-hit mechanism initial intracellular TF upregulation is followed by TF-bearing NET release 7. DNase treatment may potentiated the effects of current antithrombotic treatment 8. NETs may represent another mechanism that link thrombosis-inflammation and innate immunity

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31 Do PMNs regulate the delivery of intracellular substances on their NETs depending on the circumstances? NETs decorated with anti-microbial enzymes (NE, MPO) Elimination PMNs Pathogen invasion NETosis Sterile inflammation NETs decorated with prothrombotic material (TF) Thrombosis

32 NETs as a possible link between innate immunity and Thrombosis Vasculitis INFLAMMATION Ruptured atherosclerotic plaque Vascular wall Sterile PMNs TF gene DVT/arterial thrombosis/stemi Large vessels Microcirculation Thrombosis Intracellular TF Platelets Pathogen invasion NETosis NETs exposing TF Thrombosis Microcirculation Sepsis

33 Sh2-199, Soul Nebula IC NASA Baboon DVT Immunostaining DNA/Histone and VWF NGC 6334 "Cat's Paw Nebula", Scorpius Constellation - NASA Baboon DVT Staining for extracellular DNA