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Molecular Events in Thrombosis

Molecular Events in Thrombosis. Nigel Mackman, Ph.D. The Scripps Research Institute La Jolla. Tissue Factor-Factor VIIa Initiated Coagulation. 5/05. Tissue Factor Pathway Inhibitor (TFPI). 4_05. Targets of New Anticoagulants. 5_3/04.

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Molecular Events in Thrombosis

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  1. Molecular Events in Thrombosis Nigel Mackman, Ph.D. The Scripps Research Institute La Jolla

  2. Tissue Factor-Factor VIIaInitiated Coagulation 5/05

  3. Tissue Factor Pathway Inhibitor (TFPI) 4_05

  4. Targets of New Anticoagulants 5_3/04

  5. Recombinant Factor VIIa (Novo Seven) as a Hemostatic Agent • Hemophilia patients with or without inhibitory antibodies • Patients with diffuse bleeding that are unresponsive to conventional therapies 6_3/04

  6. Lessons from KO Mice 6_3/04

  7. Phenotype of Mouse KOs Deficient in Clotting Factors and PARs Embryonic lethality (%) Perinatal lethality (%) Adult survival (%) KO 90 0 0 0 0 TF VII XI IX VIII 100 100 0 0 0 0 0 100 100 100 Extrinsic Pathway (MT) Intrinsic Pathway 50 10 100 100 0 0 V X Common Pathway Prothrombin 50 100 0 Fibrinogen PAR-4 0 0 75 0 25 100 0 50 PAR-4/Fibrinogen PAR-1 100 0 0 50

  8. Phenotype of Clotting Factor KOs and PAR KOs

  9. Phenotype of Anticoagulant KOs Embryonic lethality (%) Perinatal lethality (%) Adult survival (%) KO TFPI AT TM EPCR PC 100 100 100 100 0 0 0 0 0 0 0 0 0 0 100 (MT) Each of the three anticoagulants is required for regulation of the coagulation protease cascade

  10. Mouse Models of TF • Knock-out mice (mTF-/-)(1996) – embryonic lethal • Low TF mice (mTF-/- / hTF+)(1998) – 1% hTF expression • Cardiac specific TF mice – role of cardiomyocyte TF • Floxed TF mice – permits cell type-specific deletion • HCV mice (mTF-/-/HCV+)– wild-type levels of hTF 4/05

  11. Generation of Low TF Mice Complete deficiency of TF results in embryonic lethality Step 1: Make Transgenic Mice hTF promoter hTF minigene mTF+/+/hTF+ Step 2: Rescue mTF-/- Embryos X mTF+/- mTF+/-/hTF+ = Low TF Mice 1% expression mTF-/-/hTF+ 2/05

  12. Procoagulant Anticoagulant AT APC TFPI TF Hemostatic Balance

  13. Hemostatic Balance Procoagulant Anticoagulant Do low levels of TF rescue TFPI-/- embryos ?

  14. Genotype of Offspring at Wean Generated by Intercrossing TFPI+/-/mTF+/-/hTF+/- Mice Death due to no TF ( ) or no TFPI ( ) 32_3/04

  15. Conclusion TFPI plays an essential role in life by regulating TF:FVIIa activity 2/05

  16. Does TFPI deficiency rescue the hemostatic defects in low TF mice ?

  17. Tissue Specific Expression of TFPI in Mouse Tissues High Medium Low placenta heart lung uterus kidney testis brain liver spleen thymus skin

  18. TFPI Deficiency Prolongs the Survival of Low TF Mice 4/05

  19. TFPI Deficiency Rescues Some Hemostatic Defects in Low TF Mice Placenta Lung Heart Testis Low TF / TFPI+/+ Low TF / TFPI-/- Pedersen et al. Blood 2005

  20. Different thresholds of TF are required for hemostasis in different tissues

  21. Role of TF in Thrombosis

  22. Diseases Associated with Pathological Expression of TF and Life-Threatening Thrombosis • Atherosclerosis • Cancer • Sepsis 38_3/04

  23. Localization of TF in Normal and Atherosclerotic Vessels Normal Atherosclerotic Wilcox et al., Proc.Natl.Acad.Sci.USA 86:2839-2843, 1989 40_3/04

  24. Blood Borne Tissue Factor: Another View of Thrombosis Giesen et al., Proc.Natl.Acad.Sci.USA 96:2311-2315, 1999 42_3/04

  25. Controversy over Active Blood Borne TF in Healthy Individuals Nature Medicine (2004) • Against:Butenas and Mann“... there is no reliable data suggesting the presence of significant quantities of functional TF in the blood of healthy individuals.” • For:Bogdanov, Hathcock and Nemerson “It is abundantly clear that shear forces caused by laminar flow are involved in thrombogenesis.” and “Whole blood clotting times are performed using non flow conditions.”

  26. Contributions of Vessel Wall and Blood Borne TF to Thrombosis

  27. Two Models • Carotid artery thrombosis model (macrovasculature) • Laser injury thrombosis model (microvasculature)

  28. Experiment Analyze thrombosis in vivo in healthy mice with different levels of hematopoietic and non-hematopoietic cell-derived TF 50_3/04

  29. Low TF Mice Have Low Levels of Procoagulant Activity in the Vessel Wall (pM TF) Factor Xa Generation WT Low TF

  30. 6 wks Thrombosis experiments Bone marrow transplantation Lethal irradiation Donor Recipient Vessel wall Marrow Low TF WT Low TF BM:WT Normal Low TF WT WT BM:Low TF Normal 51_3/04

  31. Carotid Artery Thrombosis Model Rose Bengal Flow (ml/min) Time (min) Biarritz Slide 16_6-03

  32. Hematopoietic Cell-derived TF Does Not Contribute to Thrombosis Following Carotid Artery Injury Time (min) WT Low TF Low TFBM:WT WT BM:Low TF 7/04

  33. The vessel wall is the major source of TF in a carotid artery thrombosis model using healthy mice

  34. Real-time in vivo Imaging of Platelets, Tissue Factor and Fibrin During Thrombus Formation in the Mouse Falati et al. Nature Medicine 2002 45_3/04

  35. Bright Field Tissue factor Platelets Fibrin Merge In vivo Thrombus Formation Legend WT Mouse 47_3/04

  36. Bright Field Tissue factor Platelets Fibrin Merge Low hTF Mice Legend • Lack mTF gene • hTF mRNA and antigen ~1.0% of WT levels 48_3/04

  37. Hematopoietic Cell-derived TF Contributes to Microvascular Thrombosis Wild-type Fibrin : Platelet (%WT) Platelet TF Fibrin Low TF Fibrin : Platelet (%WT) Donor Low TF Recipient Wild-type Fibrin : Platelet (%WT) Donor Wild-type Recipient Low TF Fibrin : Platelet (%WT) 7/04

  38. Hematopoietic Cell Derived TF Contributes to Microvascular Thrombosis

  39. TF TF TF Role of TF in Thrombosis After Rupture of an Atherosclerotic Plaque

  40. Diseases Associated with Elevated Levels of TF-bearing Microparticles • Acute coronary syndromes • Sepsis • Diabetes • Sickle cell disease • Cancer

  41. TF Expression in Cancer • Expression of TF • Tumor cells • Host cells, i.e. monocytes & endothelial cells • Microparticles containing TF released by various cells • TF antigen levels correlated with disease progression, malignancy and metastasis

  42. high rate of DVT with catheter • Prophylaxis not provided prior to DVT • May increase TF PCA Increased Procoagulant State in Cancer Patients Tumor released factors e.g. cytokines & VEGF Host’s inflammatory response TF Expression Thombosis + Endogenous Exogenous Chemotherapy drugs Central venous catheter Surgery

  43. Hypothesis Blood borne TF is a marker of a pre-thrombotic state and may contribute to thrombosis in cancer patients

  44. Development of a New Functional Assay to Measure Levels of Blood Borne TF 5ml blood collected from healthy individuals into tubes containing sodium citrate (anti-coagulant) and corn trypsin inhibitor (FXII inhibitor) Whole blood + citrate + CTI 200 x g, 15min Platelet rich plasma (PRP) 20,000 x g, 15min Microparticle (MP) + platelet (P) pellet

  45. + LPS - LPS Levels of Blood Borne TF in Blood of Healthy Individuals with or without LPS Stimulation Ex Vivo Procoagulant activity n = 6 – – TF Antibody + +

  46. Cancer patients Healthy Procoagulant Activity Arbitrary Threshold – – + + TF Antibody Levels of Blood Borne TF in Cancer Patients

  47. Levels of blood borne TF may be a useful marker of a pre-thrombotic state in cancer patients

  48. Role of Monocyte TF and Endothelial Cell TF in LPS Induced Coagulation

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