Tissue Factor expression

Transcription

1 Dip. Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano and Centro Cardiologico Monzino IRCCS, Milano Platelet activation and Tissue Factor expression Marina Camera

2 Conflict of interest: none.

3 Platelets as a source of TF

4 Platelets as a source of TF Fusion of TF-containing MPs with the membrane of activated platelets. The transfer of TF+ leukocyte-derived particles is dependent on the interaction of CD15 and TF with platelets. Rauch U et al, Blood 2000

5 Platelets as a source of TF However, the controversy of the presence, synthesis and functional activity of TF in platelets still persist

6 Platelets as a source of TF

7 Coagulation factors present in platelets Activated platelets rapidly release additional procoagulant moieties from their α-granules: Coagulation factor Protein Tissue Factor +

8 Coagulation factors present in platelets Activated platelets rapidly release additional procoagulant moieties from their α-granules: Coagulation factor Protein FV + FIX + FXI + FXIII + Fibrinogen + FVII + Tissue Factor +

9 Coagulation factors present in platelets Activated platelets rapidly release additional procoagulant moieties from their α-granules: n.r.: not reported Coagulation factor Protein mrna FV + n.r. FIX + n.r. FXI + + FXIII + + Fibrinogen + n.r. FVII + n.r. Tissue Factor + +

10 Platelets and haemostasis Integrin 2β1 Platelets play a critical role in the initiation and regulation of hemostasis.

11 Platelets and coagulation activation Initial phase Prothrombin Propagation phase Thrombin Fibrinogen Fibrin Final phase Platelets localize, amplify and sustain the coagulant response at the injury site.

12 Platelets and coagulation activation Extrinsic Xase Intrinsic Xase Prothrombinase

13 Platelets differently respond to agonists Individual platelets can differently respond to agonists in exposing and binding to coagulation factors and other plasma proteins. The mechanisms of origin and function of this etherogeneity is still unclear. Despite an average platelet count of /µl, platelets/µl are adequate enough to prevent bleeding.

14 Outline Heterogeneity in platelet activation Platelet phospholipid asymmetry Platelet-derived pool of FVa Platelet-derived pool of Tissue Factor Pathophysiological relevance of plateletassociated Tissue Factor

15 Outline Heterogeneity in platelet activation Platelet phospholipid asymmetry Platelet-derived pool of FVa Platelet-derived pool of Tissue Factor Pathophysiological relevance of plateletassociated Tissue Factor

16 Platelet phospholipid asymmetry Fadeel B and Xue D, Crit Rev Biochem Mol Biol. 2009

17 Phospholipid distribution in the plasma membrane of resting and stimulated platelets Resting platelets Lhermusier T et al, JTH 2011

18 Phospholipid distribution in the plasma membrane of resting and stimulated platelets Resting platelets Activated platelets Surface expression of PS provides a negatively charged membrane platform necessary for the interaction of FVa. Lhermusier T et al, JTH 2011

19 Only a fraction of activated platelets expose PS, depending on the agonist used Wolfs JLN et al, Cell Mol Life Sci 2005

20 Take home message Platelet PS exposure is a key regulator of haemostasis Only a fraction of activated platelets expose PS

21 Outline Heterogeneity in platelet activation Platelet phospholipid asymmetry Platelet-derived pool of FVa Platelet-derived pool of Tissue Factor Pathophysiological relevance of plateletassociated Tissue Factor

22

23 Coagulation Factor V (proaccelerin, labile factor) In whole blood, two pools: 80% in plasma, 20% in platelet -granules. While megakaryocytes can synthesize FV, the vast majority of platelet FV is endocytozed from the plasma pool by megakaryocytes. After endocytosis FV is modified intracellularly such that it is functionally unique compared with its plasma-derived counterpart. R. M. Camire and M. H. A. Bos, JTH 2009

24 Endocytosis of plasma-derived FV by Megakaryocytes -FV T=0 T=10 days -CD41 The endocytic process is developmentally regulated during megakaryocyte differentiation and is coagulation factor specific. Bouchard BA et al, Thromb Haemost 2005

25 Platelet-derived pool of FV Although only around 20% of the blood FV is associated with platelets, the nominal FV concentration of platelets exceeds that of plasma by an approximate factor 100. Thus its release from -granules could provide a high local concentration of FV that may be critical for the generation of platelet prothrombinase activity. Weiss HJ et al, Am J Hematol. 2001

26 Activated platelets are heterogeneous in their capacity to bind to coagulation factors Mean= 33.5 ±13.6% Fager AM et al, ATVB 2010

27 Activated platelets are heterogeneous in their capacity to bind to coagulation factors Mean= 33.5 ±13.6% Mean= 31.1 ±14.7% Fager AM et al, ATVB 2010

28 Take home message Activated platelets are heterogeneous in their capacity to bind to coagulation factors. Not all platelets express the same procoagulant potential.

29 Outline Heterogeneity in platelet activation Platelet phospholipid asymmetry Platelet-derived pool of FVa Platelet-derived pool of Tissue Factor Pathophysiological relevance of plateletassociated Tissue Factor

30 Platelets as a source of TF Fusion of TF-containing MPs with the membrane of activated platelets. The transfer of TF+ leukocyte-derived particles is dependent on the interaction of CD15 and TF with platelets. Rauch U et al, Blood 2000

31 Megakaryocytes as a source of TF F-actin Tissue Factor Nuclei Camera M et al, unpublished

32 Megakaryocytes as a source of TF.avi Camera M et al, unpublished

33 Megakaryocytes as a source of TF F-actin Tissue Factor Nuclei Camera M et al, unpublished

34 TF mrna expression in megakaryocytes and platelets megakaryocyte DCt Relative Expression (pg x 10-4 / ng total RNA) Platelets ±0.05 Megakaryocytes ±0.9 Camera et al, ATVB 2003

35 Signal-dependent splicing of tissue factor pre-mrna modulates the thrombogenicity of human platelets. Schwertz H et al, J Exp Med 2006 Human platelets synthesize and express functional tissue factor. Panes O et al, Blood 2007.

36 Platelets and Tissue Factor Camera M et al, Thromb Res 2012

37 Platelets and Tissue Factor These pathways are not mutually exclusive, and the dominant mechanism may depend on the state of platelet activation and, possibly, on other host factors that differ in physiological hemostasis versus pathological thrombosis.

38 Platelets and Tissue Factor Camera et al, ATVB 2003

39 Platelets and Tissue Factor 250 Exposure to a variety of agonists recruits TF on the platelet surface Unstimulated ADP Trap U46619 Epinephrine A TF exposure on the platelet surface is concentration- and time-dependent. Different antibodies recognize TF on the platelet surface. Camera M et al, ATVB 2003 Camera M et al, Blood 2010

40 Not all activated platelets express TF Resting Activated Tissue Factor 32.6% P-Selectin 52.4% GPIIbIIIa 77.6% Camera M et al, unpublished

41 Colocalization of P-selectin and TF positive platelets Camera M et al, Blood 2012

42 Colocalization of P-selectin and TF positive platelets Camera M et al, Blood 2012

43 Colocalization of TF, FV and PS positive platelets Camera M et al, unpublished

44 Take home message Activated platelets are heterogeneous in their capacity to express TF. Activated platelets, expressing functionally active TF, support a role not only as assembly site for coagulation enzyme complexes, but also as cells able to trigger themselves the coagulation cascade.

45 Outline Heterogeneity in platelet activation Platelet phospholipid asymmetry Platelet-derived pool of FVa Platelet-derived pool of Tissue Factor Pathophysiological relevance of plateletassociated Tissue Factor

46 Platelet-associated TF expression in pathological conditions Patients with ET (Falanga A et al 2007) Patients with cancer (Tilley RE at al Thromb Res 2008) Patients with ACS (Brambilla/Camera ATVB 2008) Patients with diabetes (Gerrits AJ et al Diabetes 2010) Patients with DES (Brambilla/Camera M et al, submitted 2012)

47 Study Aims & Design 1. To compare platelet activation in patients who underwent coronary revascularization (n=48) during dual antiplatelet therapy time (T0) and one (T1) and six (T2) months after thienopyridine discontinuation versus stable angina patients medically-treated (n=40) 2. To assess whether platelet activation markers predict the risk of revascularization in a four-year follow-up. 1 month before Clopidogrel discontinuation 1 month after Clopidogrel discontinuation 6 months after Clopidogrel discontinuation PCI Aspirin + Clopidogrel Stop Clopidogrel Aspirin

48 Platelet activation markers in DES during DAT GpIIbIIIa P-Selectin Tissue Factor

49 % positive platelets Platelet activation markers in DES during DAT GpIIbIIIa P-Selectin Tissue Factor n.s. n.s. P= ,30 n.s. 0,8 0,6 n.s. 12 n.s. 0,20 0,4 8 0,10 0,2 4 Cypher Taxus Endeav Medicallytreated SA patients 0,0 Cypher Taxus Endeav Medicallytreated SA patients 0 Cypher Taxus Endeav Medicallytreated SA patients While GpIIbIIIa and P-Selectin expression in DES on DAT is comparable to that of SA, TF expression is significantly higher in DES compared to SA patients.

50 Platelet activation at Clopidogrel suspension % positive platelets % positive platelets GpIIbIIIa P-Selectin p = p = p = p = DAT 1 month 6 months ASA only Medicallytreated SA patients DAT 1 month 6 months ASA only Medicallytreated SA patients DES-treated patients DES-treated patients

51 TF positive platelets (%) TF-positive platelets are significantly higher in DES patients RESTING p = p = 0.05 p < During DAT TF-positive platelets are significantly higher than in medically treated (MT) patients TF-positive platelets further increase at 1.8 T1 and T2 (ASA only) remaining significantly higher than those of MT SA patients. 1 month 6 months DAT ASA only DES-treated patients Medicallytreated SA patients

52 TF-positive platelets are significantly higher in DES patients TF positive platelets (%) ADP STIMULATED p = p = p = p = Upon ADP stimulation, levels of TF positive platelets at T0 are comparable to those in MT patients, despite DAT. Platelet responsiveness to ADP is higher at T1 and T2 (after clopidopgrel withdrawal) compared to T0 and to MT patients. 1 month 6 months DAT ASA only DES-treated patients Medicallytreated SA patients

53 DES patients were compliant and responsive to clopidogrel GpIIbIIIa positive platelets (%) CD62 positive platelets (%) ADP STIMULATED GpIIbIIIa P-Selectin p= p< p=0.007 p=0.002 p=0.001 p< p< p< p=0.021 p< DAT 1 month 6 months ASA only Medicallytreated SA patients DAT 1 month 6 months ASA only Medicallytreated SA patients DES-treated patients DES-treated patients

54 Platelet-associated TF and recurrence of revascularization Below the median Above the median Δ stimulation ADP/C p=0.014 Patients with the highest response to ADP in terms of platelet-associated TF expression during DAT had the highest risk to undergo a new PCI within four years.

55 F 1+2 plasma levels and recurrence of revascularization Below the median Above the median F 1+2 plasma levels Patients with F 1+2 plasma levels above the median during DAT had the highest risk to undergo a new PCI within four years.

56 Risk score and recurrence of revascularization Both below the median One of the two above the median Risk score: Plat-TF+ F 1+2 Both above the median The score was highly associated with adverse events (p= by log-rank test; HR 7.5, 95% C.I , p=0.001, for one-step score increase). The area under the ROC curve is 0.82 (95% C.I , p<0.0001), indicating a very good prognostic value. Brambilla M et al, submitted

57 Take home message In DES-treated patients, the evaluation, during DAT, of the traditional markers of platelet activation (GPIIbIIIa and P-selectin) would indicate a degree of platelet inhibition comparable to that of MT patients, whereas, in fact, they show a prothrombotic phenotype in terms of platelet-associated TF expression; platelet-associated TF as well as activated GPIIbIIIa expression increase after clopidogrel suspension; markers of coagulation activation (platelet-associated TF and F 1+2 ) predict the need for revascularization in a four years follow-up. Accurate determination of platelet activation status remains a challenge, but could provide a useful tool in identifying patients at risk for future cardio-vascular events.

58 Acknowledgments Elena Tremoli Daniela Boselli Marta Brambilla Paola Canzano Laura Facchinetti Franco Moro Laura Rossetti Fabrizio Veglia Antonio Bartorelli Piero Montorsi Giovanni Teruzzi Daniela Trabattoni GianCarlo Marenzi Monica De Metrio Mara Rubino Vincenzo Toschi Dept. of Pharmacological Sciences, University of Milan and Centro Cardiologico Monzino IRCCS, Milan Lab. of Cell Biology and Biochemistry of Atherothrombosis Centro Cardiologico Monzino IRCCS, Milan Unit of Biostatistics, Centro Cardiologico Monzino IRCCS, Milan Interventional Cardiology Units, Centro Cardiologico Monzino IRCCS, Milan Intensive Cardiac Care Unit, Centro Cardiologico Monzino IRCCS, Milan San Carlo Hospital, Milan