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האם ל Tissue Factor הנוצר במונוציטים יש חשיבות קלינית ? Has Monocyte Tissue Factor Any

האם ל Tissue Factor הנוצר במונוציטים יש חשיבות קלינית ? Has Monocyte Tissue Factor Any Clinical Significance ?. דר' אברהם קורנברג מכון המטולוגי מרכז רפואי אסף הרופא. האיגוד הישראלי להמטולוגיה ולעירויי דם כנס האביב מאי 2009.

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האם ל Tissue Factor הנוצר במונוציטים יש חשיבות קלינית ? Has Monocyte Tissue Factor Any

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  1. האם ל Tissue Factor הנוצר במונוציטים יש חשיבות קלינית ? Has Monocyte Tissue Factor Any Clinical Significance ? דר' אברהם קורנברג מכון המטולוגי מרכז רפואי אסף הרופא האיגוד הישראלי להמטולוגיה ולעירויי דם כנס האביב מאי 2009

  2. Coagulant Activity of Leukocytes: TF activity Niemetz J. Clin Invest 51,307-310 1972 The Role of Protein synthesis on the Generation of TF Activity by Leukocytes Pro Soc Exp Biol 139, 1276-8, 1972

  3. Mononuclear Cell Tissue Factor: Cell of Origin and Requirements for Activation Edwards RL, Rickles FR and Bobrove AM Blood 54, 350-370, 1979

  4. Tissue Factor Generation by human mononuckear cells: Effects of endotoxin… Rickles FR, Levine J, Hardin JA, Barr CF, Conrad Jr ME J Lab Clin Med 89, 792-803, 1977

  5. Lymphocyte Cooperation is Required for Amplification of Macrophage PCA Levy G , Edgington TS J Exp Med 151, 1232-1244 Entotoxin stimulated PCA Cells added To macrophages CT (sec) Units None 69 2.3 Lymphocytes 49 7.7 Neutrophils 69 2.2 RBC 71 2.1

  6. Granulocytes Do Not Express But Acquire Monocyte-Derived TF in Whole Blood: Evidence for a Direct Transfer Egorina EM, Sovershaev MA, Olsen JO, Osterud B Blood 111, 1208-1216, 2008

  7. C-Reactive protein Induces Human Peripheral Monocytes To Sythesize TF Ceramak J, Key NS, Bach RR, Balla J, Jacob HS, Vercellotti GM Blood 82, 513-520, 1993

  8. C5 Chemotactic Fragment Induce Leukocyte Production of TF Activity Muhlfelder TW, Niemetz J, Kreutzer D, Beebe D, Ward P, Rosenfeld S J Clin Invest 63, 147-150, 1979

  9. Tumor Necrosis factor Induces TF-Like Activity in Human Leukemia Cell Line U937 and Peripheral blood Monocytes Crookling PR, Greenberg CS, Weinberg JB Blood 72, 128-133, 1988

  10. Substances that induce TF in monocytes Immune complexes Lectins Cytoxines Lipoproteins Prostaglandins Viruses Vascular permeability factor Adenine nucleosides Ionomycin

  11. Activation of Blood Coagulation in Crohn’s Disease: Increased plasma Fibrinopeptide A Levels and Enhanced Generation of monocyte TF Activity Edwards RL, Levine JB, Green R, Duffy M, Mathews E, Brande W, Rickles Fr Gastroenterology 92, 329-337, 1987

  12. Abnormalities of Blood Coagulation in Patients with Cancer. Mononuclear TF generation Edwards RL, Rickles FR, Cronlund M J Lab Clin Med 98, 917-928, 1981

  13. Edwards RL et al. Cancer an MTF (3) Effect of warfarin on MTF in cancer MTF generation Stimulus Before warfarin During warfarin p PHA 205 171 0.3 PWM 180 139 0.06 PPD 138 108 0.2 SKSD 108 43 0.005 LPS 197 128 0.002 All cultures 165 106 <0.001

  14. Increased Production of Mononuclear PCA in Hodgkin’s disease Viero P, Cortellazzo S, Barbui t, Colucci M, Semeraro N Eur J Cancer Oncol 19, 1539-1543, 1983 Increased Sensitivity to thromboplastin in blood Monocytes from Pre- eclamptic Patients BOian P, Omsjo I, Maltau JM, Osterud B Br J Obst Gynaecol 92, 511-517, 1985 Generation of TF by Patient Monocytes:Correlation to Thromboembolic Complications (in Trauma) Miller CL, Graziano C, Lim RC, Chin M Thrombos Haemostas 46, 489-495, 1981 Septicemia Glomerulonephritis Allograft rejection SLE Sarcoidosis FMF

  15. Generation of PCR by Phorbol-Esters Induced Macrophages Derived from a Leukemic Promyelocytic Cell Line (HL-60) Kornberg A, Treves A, Rachmilewitz Ea, Fibach E Blood, 59, 1061-1066, 1982

  16. Kornberg A et al: Phorbol-esters, PCA and HL-60 cells (2) The effect of various compounds on PCA of HL-60 cells and NMNC . Recalcification time (sec) Compound HL-60NMC None 130 98 TPA 51 62 Phrbol 155 108 PHA 135 - Endotoxin 134 60

  17. Kornberg A et al : Porbol-ester, PCA and HL-60 cells (3) Effect of TPA-induced macrophages on clotting time of factor-deficient plasmas Recalcification time (sec) Normal F VII def F VIII def FIX def F X def Simplastin 53 148 5155162 TPA-induced Macrophages 511604850183

  18. Kornberg A et al: Phorbol-ester, PCA and HL-60 cells (4) Con-A inhibition of PCA . Recalcification time (sec) . Cells+Con ACells+ConA+alfaMM Con A Sec % inhibition Sec % protection 0 65 0 63 - 4 82 26 66 94 16 100 53 68 92 64 122 87 74 84 256 128 97 72 58

  19. Generation of PCA by Macrophage-like Cells Derived from Acute and Chronic Myeloid Leukemia cells in Response to Phorbol Esters Kornberg A, Treves A, Rachmilewitz EA, Fibach E Scan J Haematol 31, 102-108, 1983 Effect of TPA on PCA generation By cells . . PCA (units) . . Plasma . . CellsPts NoNormalF VIII defF VII defF X def AML 16 6.8 5.2 3.2 <1 AML+TPA 16 72.557.5 4<1 ALL 6 2.4 2.9 2.2 <1 ALL+TPA 6 4 2.7 2.1 <1 CML 5 3.3 4.5 4.1 <1 CML+TPA 5 75 65 3.2<1 NMC 9.3 NMC+TPA 45 Buffer <1 <1 <1 <1

  20. Kornberg A et al. Leukemoc cells, Phorbol-esters and PCA (2) • Con-A inhibition of PCA • Cells+ConA Cells+ConA+alfa MM • Con A Units % inhib Units % protection • 1 0 42 0 42 - • 16 16 60 35 70 • 64 7 83 23 45 • 0 65 0 65 - • 16 37 32 46 38 • 64 10 84 28 33 • 0 35 0 35 - • 16 10 69 28 74 • 64 6 80 16 35

  21. Modulation of the Maturation of Human leukemic Promyelocytres (HL-60) to Granulocytes or macrophages Fibach E, Peled T, Treves A, Kornberg A, Rachmilewitz EA Leuk Rsearch 6, 781-790, 1982

  22. In Vitro Generation of PCA by leukemic Promyelocytes in Response to Cytotoxic Drugs Fibach E, Treves A, Kornberg A, Rachmilewitz EA Am J Hematol 20, 257-265, 1985

  23. Fibach E et al. Cytotoxic drugs and PCA in HL-60 cells (2) Effect of cytotoxic agents on PCA generation of HL-60 cells . Drug Recalcification time Control 135 Act D 65 Ara C 108 Pur 62 Hyd 89 VCR 97 Dox 78 Ceru 90 Effect of Act D-treated HL-60 cells on CT of factor-deficient plasma Recalcification time Normal F VII def F VIII def FX def Untreated 144 >180 142 >180 Act D treated 59 101 59 >180

  24. Tuftsin Induces TF-Like Activity in Human Mononuclear Cells and in Monocytic Cell Lines Kornberg A, Catane R, Peller S, Kaufman S, Fridkin M Blood, 76, 814-819, 1990

  25. Kornberg A et al. Tuftsin and TF (2) Characterization of Tuftsin-induced M- PCA Cells Plasma Inhibitor PT-modified TF N - 53 MNC N - 69 MNC VII def - 143 MNC X def - 162 MNC VIII def - 60 MNC IX def - 68 MNC XI def - 64 MNC N Con-A 16 102 64 116 128 132 monoclonal anti-TF 2 55 10 118 40 136

  26. Kornberg A et al. Tuftsin and TF (3) Generation of PCA by various fractions of Tuftsin-treated MNC Cell Cell fraction PT-modified Intact Whole 69 Sonicated Supernatant 152 Pellet 60 Sonicated-tuftsin Pellet 146 Culture media 164

  27. Kornberg A et al. Tuftsin and TF (4) Effect of Inhibitors and T lymphocytes on PCA generation by Tuftsin Cells Cytarabine Act D PT-modified MNC - - 69 MNC 5 - 64 MNC - 10 134 T lymp depleted MNC - - 70

  28. Kornberg A et al. Tuftsin and PCA (5) Effect of Tuftsin analogs on MPCA generation PT-modified Analog 5 250 Tuftsin Thr-Lys-Pro-Arg 118 58 (Leu’)-tuftsin Leu-Lys-Pro-Arg 125 72 Nitro-tuftsin Lev-Lys-Pro-Arg-Nitro 154 162 Tuftsin+ (dis-Thr’)-tuftsin (Des-Thr’)-Lys-Pro-Arg 147 124

  29. Kornberg A et al. Tuftsin and PCA (6) Generation of PCA by Tuftsin-treated cell lines Modified PT 5 tuftsin 250 tuftsin Cell line MNC 120 69 Raji (B) 172 178 Hd-Mar (T) >200 188 REH (pre-B) >200 >180 U p37 (mono) 128 83 THP (mono) 125 86

  30. Induction of TF-Like Activity in Monocytes by Anti-Cardiolipin Antibodies Kornberg A, Blank M, Kaufman S, Shoenfeld Y The J Immunol 153, 1328-1332, 1994

  31. Kornberg a et al. ACLA and TF (3) Characterization of ACLA-induced PBMC PCA Inhibitor PCA (U) Plasma CMA CAL Normal - 2630 VIII def - 28 26 VII def 1.82.2 Normal Phospholipase 2.12.6 Normal ant-TF abs 40 4.4 6

  32. Kornberg A et al. ACLA and TF (4) Cell of origin of PCA induced by ACLA PCA (U) Cell - CAM CAL PBMN 2 2629 Monocytes 2 28 26 Lymphocytes 2 1 1 CLL 1 1 1 U937 9 (mono) 2 1414 FHP-1 (mono) 2 2224 Raji (B) 1 1 2 Hd-Mar (T) 2 2 2 HL-60 2 1 1

  33. Kornberg A et al. ACLA and PCA (5) Effect of protein synthesis inhibitors on ACLA-induced PCA Cytarabine Puromycin PCA (U) Cells CAM CAL PBMC 28 26 PBMC 102928 PBMC 10 5 5 T cell depleted PBMC 28 25

  34. Enhanced Capacity of Monocytes from Patients with Ischemic Heart Disease to Generate TF Kornberg A, Faibel H, Schlesinger Z Blood 88, (10) Supp , 177a, 1997 Patients n TF activity (U) TF antign Plasma F1+2 (nM) Endotoxin - + - + Normals 20 1.5 5 26 78 0.4 Stable AP 10 2 36 30 6001.6 Unstable AP 10 2 46 32 576 3 Acute MI 13 2 40 30 690 4.3

  35. Correlation between CRP, F1+2 and M-TF in Acute MI Kornberg A et al

  36. Enhanced Generation of Monocyte TF and Increased Plasma Prothrombin Fragment 1+2 Levels in Patients with Polycythemia Vera: Mechanis of Activation of Coagulation Kornberg A, Rahimi-Levene , Yona R, Mor A, Rachmilewitz EA Am J Hematol 56, 5-11, 1997 TF activity (U) Endotoxin p - + Patients No. . Normals 20 2 7 Apparent poly 20 2 9 Second poly 20 2 8 PV active 20 2 41 < 0.001 PV treated 20 2 40 < 0.001 PV spent 6 2 42 < 0.001 TF antigen (pg/ml) Endotoxin p - + Patients No. . Normals 20 53 364 Apparent poly 20 44 367 Second poly 20 41 362 PV active 20 51 1157 < 0.001 PV treated 20 50 1153 < 0.001 PV spent 6 47 1020 < 0.001

  37. Kornberg A et al. PV and TF (3) Cell of origin of TF in PV Cells n TF activity TF antigen p PV MNC 20 431158 < 0.001 PV monocytes 8 501210 <0.001 PV lymphocytes 8 2 46 Plasma F1+2 in polycythemia Patients n F1+2 (nM) p Normals 20 0.3 Apparent poly 15 0.2 Second poly 16 0.3 PV active 13 2.5 <0.001

  38. Kornberg A et al. PV, MTF and F1+2 (2) r = 0.77 r = 0.87

  39. Generation of Monocyte TF in Patients with Essential Thrombocytosis Kornberg A, Zahavi M, Rahimi-Levene N, Rapoport M

  40. Increased Expression of TF and RAGE in PBMC of Patients With Type 2 DM with Vascular Complications Bucks AE, Kornberg A, Zahavi M, Aharoni D, Zarfati C, Rapoport MJ Experimental Diab Res 5, 163-169, 2004 Expression of TF in DM TF activityTF protein Basal Stimulated Basal Stimulated Complications 18 56 33108 No complications 6 19 10 45 Expression of FT and RAGE mRNA in DM TF RNARAGE RNA Basal Stimulated Basal Stimulated Complications 0.58 0.790.20.2 No complications 0.21 0.46 0.04 0.13

  41. Buchs AE et al. DM and monocyte TF (2)

  42. Anti-Beta2-Glycoprotien I Antibodies and Anti-Endothelial Cell Antibodies Induce TF in Endothelial Cells Kornberg A, Renaudineau Y, Blank M, Youinou P, Shoenfeld Y IMAJ 2, 27-31 Supp, 2000

  43. ממצאים • מונוציטים מאנשים בריאים מיצרים TF לאחר הדגרה in vitro עם חומרים שונים • (אנדוטוקסין, CRP, TNF, משלים משופעל, קומפלכסים אימוניים ועוד) • ברוב המחלות עם שכיחות יתר של תופעות תרומבואמבוליות אשר בהן המונוציטים • מיצרים כמות גדולה של TF, התופעה הודגמה בעקר in vitroלאחר הדגרה עם • אנדוטוקסין או אחד החומרים הנ"ל • רמת החומרים הנ"ל עולה בדם בזמן זהום ותהליכים דלקתיים • היפותיזה • קיימת אפשרות שבמהלך האפיזודות של זהום או תהליכים דלקתיים החומרים • הנ"ל (שרמתם בדם עולה) משרים יצירת TF במונוציטים in vivo, אשר משפעל את • מערכת הקרישה וגורם להתפתחות קרישי דם. לכן יש לבדוק האם קיימת קורלציה בין • אפיזודות של זהום או תהליכים דלקתיים לבין התפתחות קרישי דם • יש לצרף את התופעה של יצירת TF במונוציטים לרשימת המצבים של קרישיות • יתר The data and the hypothesis merit further exploration and confirmation

  44. תודה על ההקשבה

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