Moleküler Geneti̇k Kongresi̇ 2009

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Information about Moleküler Geneti̇k Kongresi̇ 2009

Published on November 11, 2016

Author: sercankuarktek

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1. THROMBOSIS AND THALASSEMIA Prof.Dr.Tansu Sipahi Ufuk University, Faculty of Medicine, Department of Pediatric Hematology 3rd InternationalCongress of Molecular Medicine, Istanbul, Turkey, May 5-8, 2009

2. Thalassemia is a congenital hemolytic disease caused by defective globin synthesis resulting in decreased quantity of globin chains. Worldwide, about 300 000 infants are born each year with a hb.pathy and an estimated 5% of the world’s population are carriers of a gene mutation for autosomal recessive conditions.

3. The severity of clinical course distinguishes this disease into two main subtypes: – Thalassemia major (TM) – Thalassemia intermedia (TI) Currently used therapeutic approaches such as regular blood transfusions and iron chelation have prolonged life expectancy in patients with thalassemia.

4. A consequence of this brings new complications Deep venous thrombosis (DVT) Portal venous thrombosis (PVT) Pulmonary embolism (PE) Cerebral thromboembolism (CTE) Postsplenectomy thrombosis

5. In recent years, thromboembolic events have been increasingly recognized. However, there are relatively few epidemiological data on the overall frequency of these complications.

6. Prevalence of thromboembolic episodes in β-thalassemia in previous studies Abbreviations: PTE, pulmonary thromboembolism; PVT, portal vein thrombosis; DIC, disseminated intravascular coagulation; CNS, central nervous system; STP, superficial thrombophlebitis. Taher et al. Blood Reviews 2008; 22: 283-292 Reference (year) # of patients with thromboembolism (%) Sites of thromboembolism Michaeli et al. (1992) 4 (TM) 4% Recurrent arterial occlusion, recurrent PTE, venous thrombosis and one fatal stroke Aessopos et al. (1997) 6 (3 TM, 3 TI) 1% Thrombotic strokes Moratelli et al. (1998) 26 (14 TM, 12 TI) 5.3% (overall), 3.3% (TM), 16.2% (TI) - Borgna Pignatti et al. (1998) 32 (27 TM, 5 TI) 3.27% (TM + TI) Thrombotic strokes-16, MT-1, PVT-2, DVT-6, intraatrial thrombosis-2, DIC in pregnancy-2, PTE-3 Akar et al. (1998) 17 homozygous β-thalassemia Nine had CNS involvement Senanayake and Lamabadusuriya (2001) 2 homozygous β-thalassemia Cerebral thrombosis (hemiparesis) Cappellini et al. (2000) 24 TI 29% PVT-9, DVT-3, STP-10, PTE-3, Priapism-1 Zalloua et al. (2003) 4 TI 8% Not specified Taher et al. (2006) 85 TI (3.9%), 61 TM (0.9%) Overall (1.65%) DVT 32%, stroke 18%, PVT 16%, PE 13% and STP 4.7%

7. Zurlo et al. (1989) A study concerning survival and causes of death in TM patients showed thromboembolism represented the primary cause of death in about 2.5% (4 of 159) of the transfusion dependent thalassemic patients. Zurlo MG. Lancet 1989; 2: 27-30

8. Pignatti et al. (1998) In a survey involving 9 Italian thalassemia centers, (735 patients), overall prevalence of TE episodes – Thalassemia major (n=683) → 2.3 % – Thalassemia intermedia (n=52) → 9.6 % Pignatti CB. Haematologica 2004; 89: 1187 Pignatti CB. Acta Haematol 1998; 99: 76-79 Pignatti et al. (2004) Seven Italian centers 1073 patients Primary cause of death 4.1% (thrombosis)

9. Causes of death for the entire population of patients All patients (N=1073) N % Heart Failure 133 60.2 Infection 15 6.8 Arrhytmia 15 6.8 Myocardial infarction 4 1.8 Cirrhosis 9 4.1 Thrombosis 9 4.1 Malignancy 8 3.6 Diabetes 7 3.2 Accident 4 1.8 Renal Failure 3 1.4 HIV/AIDS 3 1.4 Familial autoimmune disorder 2 0.9 Anorexia 1 0.5 Hemolytic anemia 1 0.5 Thrombocytopenia 1 0.5 Unknow 6 2.7 Total 221 Borgna-Pignatti et al. Haematologica 2004

10. Akar et al. (1998) The Turkish Thalassemia Study Group compile data from 11 Centers. Of the 519 homozygous β-thalassemia patients(442 TM, 77 TI). Seventeen (3.27%) had experienced thrombosis. Akar N. Acta Haematol 1998

11. Cappellini et al. (2000) 83 patients β-TI 29% of patients developed thromboembolic events (PE, DVT, PVT) (10 years follow-up) 9 of these patients recurrent VTEs All patients except one had undergone splenectomy Cappellini et al. Br J Haematol 2000

12. Taher et al. (2006) 8860 thalassemia patients 1.65% of patients had TE events Thromboembolic events occured 4.38 times more frequently in TI than TM, with more venous events occuring in TI and more arterial events occuring in TM OR:4.38 (%95 CI:3.14-6.10) Taher et al. Thromb Haemost 2006

13. Patient characteristics* *Not all patients responded to every question, so presented frequencies are based on responder numbers only. TI, thalassaemia intermedia; TM, thalassaemia major. TI (n=85) TM (n=61) All patients (n=146) Males % 42.4 (36/85) 51.7 (31/60) 46.2 (67/145) Females % 57.6 (49/85) 48.3 (29/60) 53.8 (78/145) Mean age at time of event, years 33.4 ± 14.9 25.1 ± 8.8 30.0 ± 13 Splenectomized % 94.0 (78/83) 91.8 (56/61) 93.0 (134/144) Haemoglobin <9 g/dl, % 67.5 (52/77) 43.3 (26/60) 56.9 (78/137) Regularly transfused % 33.3 (28/84) 93.3 (56/60) 58.3 (84/144) Recurrent thrombosis % 35.6 (26/73) 25.0 (13/52) 31.2 (39/125) Pulm. hypertension % 27.3 (15/55) 19.5 (8/41) 24.0 (23/96) Aspirin % 51.0 (26/51) 52.5 (21/40) 51.6 (47/91) Hydroxyurea % 35.3 (18/51) 4.7 (2/43) 21.3 (20/94) Taher et al. Thromb Haemost 2006

14. Thromboembolic events in TI and TM (%) 12 8 19 12 39 9 66 30 0 11 8 23 28 48 0 20 40 60 80 Others STP PVT PE DVT Stroke Venous Typeofevent Thromboembolic events (%) TI (n=85) TM (n=61) Taher A et al. Thromb Haemost 2006; 96: 488-91

15. Cerebral TE events, stroke syndrome, and neurological findings were also found in patients with beta thalassemia. The incidence of stroke 2% to 20%. Manfre L. AJR 1999 Pignatti . Acta Hemotologica 1998 Asymptomatic brain damage on MRI has also been reported on patients with TI as a frequent occurence affecting 37% of patients.

16. Autopsy series in patients with TM and TI describe the presence of Pulmonary embolism, arterial occlusion, thrombi in small and large pulmonary vessels. Chronic hypoxia and lung injuries due to infections Excessive iron deposition Right ventricular dysfunction Increased platelet activation Microembolization in the lungs Singer ST et al. Am J Hematol 2006

17. As a result of multiple recent clinical studies and laboratory data, thalassemia has been referred to as a “hypercoagulable state”. Several factors are implicated in the etiopathogenesis of the hypercoagulable state; namely inherent abnormality in the red cells, cardiac dysfunction, liver dysfunction, hypothyroidism, diabetes and post splenectomy thrombocytosis.

18. Summary of the pathogenesis of hypercoagulability in thalassemia Pathogenesis Description of the problem Reference Platelet activation Increased platelet aggregation, increased expression of activation markers, presence of platelet morphologic abnormalities Winichagoon et al., Del Principe et al., Ruf et al., Bunyaratvej et al., Eldor et al. Pathology and alteration in red blood cells Formation and precipitation of hemichromes, formation of reactive oxygen species, expression of negatively charged phospholipids which facilitate thrombin generation, enhanced cohensiveness and aggreability Kuypers and de Jong, Borenstain-Ben Yashar et al., Helley et al., Chen et al. Endothelial cell and peripheral blood activation Expression of endothelial adhesion molecules and tissue factor on endothelial cells, formation of microparticles, monocyte and granulocyte activation Carlos and Harlan, Pattanapanyasat et al. (2004), Pattanapanyasat et al. (2007), Wiener et al., Deo et al. Splenectomy High platelet counts following splenectomy, platelet hyperactivity Eldor and Rachmilewitz, Atichartakarn et al. Thrombophilic DNA mutations and acquired changes in coagulation factors and inhibitors High prevalence for both the factor V Leiden, decreased levels of antithrombin III, protein C and protein S. Anti-phospholipid antibodies Zalloua et al., Eldor et al., Cappellini et al., Iolascon et al., Giordano et al., Sharma et al., Kashef et al.

19. PATHOGENESIS Factors contributing to the hypercoagulable state in thalassemia Hypercoagulable State Iron overload Hyperviscosity Endothelial damage Monocyte activation Release of microparticles Genetic mutations Factor V Leiden Prothrombin G20210 MTHFR C677T (hyperhomocysteinemia) ? Others Cardiac dysfunction Liver dysfunction Hormonal deficiencies Antiphospholipid antibodies ? Intravascular hemolysis Decreased levels of Protein C Protein S Antithrombin III Platelets Increased platelet aggregation State of oxidative stress (ROS) Expression of activation markers (CD62P and CD63) Taher et al. Blood Reviews 2008; 22: 283-292 Red Blood Cells Abnormal erythroid cells Source of procoagulant phospholipids Enhanced cohesiveness

20. PATHOGENESIS Factors contributing to the hypercoagulable state in thalassemia Hypercoagulable State Red Blood Cells Abnormal erythroid cells Source of procoagulant phospholipids Enhanced cohesiveness Iron overload Hyperviscosity Endothelial damage Monocyte activation Release of microparticles Genetic mutations Factor V Leiden Prothrombin G20210 MTHFR C677T (hyperhomocysteinemia) ? Others Cardiac dysfunction Liver dysfunction Hormonal deficiencies Antiphospholipid antibodies ? Intravascular hemolysis Decreased levels of Protein C Protein S Antithrombin III Platelets Increased platelet aggregation State of oxidative stress (ROS) Expression of activation markers (CD62P and CD63) Taher et al. Blood Reviews 2008; 22: 283-292

21. Under normal conditions, the choline-containing phospholipids, phosphatidylcholine(PC) and sphingomyelin(SM) are mainly present in the outer monolayer of the plasma membrane, where as phosphatidylserine (PS) is exclusively and phosphatidylethanolamine (PE) is mainly found in the inner monolayer. Kuypers, ASH, Hematology, 2007 Red Blood Cells

22. In normal RBC, maintenance of membrane phospholipid asymmetry appears to be provided by the action of an ATP-dependent aminophospholipid translocase ( flipase), that transports PS and PE from the outer to the inner membrane surface. Red Blood Cells

23. Phospholipid Organisation of Red Blood Cells Kuypers, ASH, Hematology, 2007 Red Blood Cells

24. The Hypercoagulable State in Thalassemia Eldor A. Blood 2002; Kuypers&de Jong 2004; Rund&Rachmilewitz 2005 Endothelial activation Thrombus formation Endothelial perturbation Low plasma protein C and protein S RBC adhesion and aggregation Fibrin Platelets Fibrinogen Activation of granulocytes and monocytes RBC Tissue factor Prothrombinase complex Red Blood Cells

25. Ataga et al.2007 Pathophysiology of hypercoagulable state in thalassemia

26. Rund et al.2005

27. Atichartakran et al. Annexin V labelling of RBCs using flow cytometry These findings indicated that splenectomized patients with Hb E/β- thalassemia were in a chronic hypercoagulable state. increased numbers of circulating PS-exposed RBCs Atichartakran et al. 2002 Red Blood Cells

28. Several studies have demonstrated that RBCs from thalassemia patients also show enhanced cohesiveness and aggregability. RBCs may also act as activated platelets and enhance conversion of prothrombin → thrombin Enhanced adherence of the abnormal erythrocytes to endothelial cells is also described. Hovav T. Br J Haematol 1999 Red Blood Cells

29. PATHOGENESIS Factors contributing to the hypercoagulable state in thalassemia Hypercoagulable State Red Blood Cells Abnormal erythroid cells Source of procoagulant phospholipids Enhanced cohesiveness Iron overload Hyperviscosity Endothelial damage Monocyte activation Release of microparticles Genetic mutations Factor V Leiden Prothrombin G20210 MTHFR C677T (hyperhomocysteinemia) ? Others Cardiac dysfunction Liver dysfunction Hormonal deficiencies Antiphospholipid antibodies ? Intravascular hemolysis Decreased levels of Protein C Protein S Antithrombin III Platelets Increased platelet aggregation State of oxidative stress (ROS) Expression of activation markers (CD62P and CD63) Taher et al. Blood Reviews 2008; 22: 283-292

30. Platelet abnormalities Increased platelet aggregation Chronic platelet activation Increased circulating platelet aggregates Shortened platelet survival Enhanced excretion of urinary metabolites of thromboxane A2 (TxA2) and prostacyclin Expression the activation markers (CD62P and CD63)(flow cytometry) Platelets

31. Eldor et al. A significant increase (4-10 fold) in the urinary excretion of the stable hydrolysis products of TxA2, and PGI2 in beta-thalassemia patients compared to controls. The platelet life span is shortened – 107 ± 36 hour in splenectomized patients – 248 ± 51 hour in splenectomized controls Eldor A et al. Am J Hematol 1989 Platelets

32. Goldschmidt et al. Platelet adhesion under flow condition, the primary event in thrombus formation, is increased in thalassemic patients as compared to healthy controls. Thalassemic red blood cells promote platelet adhesion under flow. Goldschmidt et al. Thromb Haemost 2008 Platelets

33. PATHOGENESIS Factors contributing to the hypercoagulable state in thalassemia Hypercoagulable State Red Blood Cells Abnormal erythroid cells Source of procoagulant phospholipids Enhanced cohesiveness Iron overload NO deficiency Endothelial damage Monocyte activation Release of microparticles Genetic mutations Factor V Leiden Prothrombin G20210 MTHFR C677T (hyperhomocysteinemia) ? Others Cardiac dysfunction Liver dysfunction Hormonal deficiencies Antiphospholipid antibodies ? Intravascular hemolysis Decreased levels of Protein C Protein S Antithrombin III Platelets Increased platelet aggregation State of oxidative stress (ROS) Expression of activation markers (CD62P and CD63) Taher et al. Blood Reviews 2008; 22: 283-292

34. Endothelial cells, monocytes and granulocyte activation Elevated levels of endothelial adhesion proteins – Vascular cell adhesion molecule-1 (VCAM-1) – Intercellular adhesion molecule-1 (ICAM-1) – E-selectin (ELAM-1) – Von Willebrand factor (vWF) – Thrombomodulin Endothelial injury and activation Butthep P. Thromb Hemost 1995, Hovav T. 1999 Monocyte, Endothelial Cells

35. Adherence of red blood cells (RBC) from β-thalassaemia major (TM) and intermedia (TI) patients to endothelial cells (EC) RBC of the indicated composition were placed on confluent cultured bovine aortic endothelial cells for 45 min then washed five times with PBS, and the number of RBC remaining adhered to the EC were counted as described in Methods. Each datum is mean ±SD for six experiments (with blood samples taken from six patients). To examine the effect of the dilution of pathological RBC with normal RBC ones. TI-RBC were mixed with 30% of normal RBC because the dilution of TM-RBC by the routine transfusion of normal blood is usually about 30%. RBC composition Adherence (no. of RBC/103 EC) Normal 32 ± 7 TM prior to transfusion 342 ± 43 TM after transfusion 321 ± 37 TI 820 ± 54 TI (70%) with normal (30%) 813 ± 66 Hovav T. Br J Haematol 1999; 106: 178-181 Monocyte, Endothelial Cells

36. Microparticles are small vesicles formed by the membrane, which is derived from RBCs, platelets, EC and monocytes following activation. The number of RBC vesicles are especially marked following splenectomy in patients with beta-thalassemia/hemoglobin E. Hugel B et al. Physiology 2005 Recently, in a study it was shown that these particles are also derived from activated platelets. Pattanapanyasat K et al. Cytometry B Clin Cytom 2004 Pattanapanyasat K et al. Br J Haematol 2007 Monocyte, Endothelial Cells

37. Monocyte activation may play an important role in endothelial activation – High serum levels of monocyte colony- stimulating factor Granulocytes activation – Elevated granulocyte phagocytic function – Endothelial damage - Removal of leukocytes from transfused blood resulted in improved pulmonary function tests. Deo SS et al. Indian J Pediatr 1994 Kivity S et al. Pediatr Pulmonol 1999 Monocyte, Endothelial Cells

38. Intravascular hemolysis reduces nitric oxide bioactivity Kato GJ et al. Blood Reviews 2007 NO deficiency

39. Intravascular hemolysis releases Hb, Arginase, LDH Hb inactivates NO, generating MetHb and inert nitrat. Plasma Arginase deplete NO production. Decrease of NO is associated with Pulmonary. Decrease of NO is associated with Pulmonary hypertension, priapism, leg ulceration and nonhemorrhagic stroke NO deficiency

40. HEMOLYSIS NITRIC OXIDE Endothelial Cell Dysfunction Proinflammatory Effects Proliferation DECREASED NITRIC OXIDE SIGNALLING VIA cGMP Impaired Regulation of Smooth Muscle Tone Platelet Activation Local Vasoconstriction Intravascular Thrombosis Smooth Muscle Dystonias Vascular Constriction GI Contractions Pulmonary and Systemic Hypertension Dysphagia Erectile Dysfunction Abdominal Pain Cappellini MD et al. Am Soc Hematol 2007; adapted from Rother et al. NO deficiency Consequences of nitric oxide depletion during intravascular hemolysis

41. Role of splenectomy Summary of studies on the prevalence of thrombosis in splenectomized patients Authors No. of patients splenectomized Prevalence of thrombosis Type of thrombosis Underling disease Chaffanjon et al. 60 6.7% PVT (1 symtomatic, 3 asymptomatic) Myeloproliferative disorder Loring et al. 123 9.8% PVT (3 symtomatic, 9 asymptomatic) Hematologic diseases (myelofibrosis, MDS, lymphoma, leukemia) Valeri et al. 12 8.3% PVT ITP Van’t Riet et al. 563 1.6% PVT 10% autoimmune hemolytic anemia and myeloproliferative syndrome Tefferi et al. 223 7% TE Myelofibrosis with myeloid metaplasia Hassn et al. 50 10% PVT ? Cappellini et al. 83 29% TE Thalassemia intermedia Delaitre et al. 275 10% TE Hematologic diseases Akpek et al. 26 12% VT Myelofibrosis with myeloid metaplasia Winslow et al. 101 8% PVT 74% hematologic disease (myeloproliferative disorder) Fujita et al. 321 1.5% PVT Hemolytic anemia thalassemia and myelofibrosis Ikeda et al. 22 (LS) 21 (OS) 55% (LS) 33% symp- tomatic 19% (OS) Portal vein, mesenteric veins, splenic vein ? Cappellini MD. Ann NY Acad Sci 2005 Splenectomy

42. The development of these complications has been attributed to the presence of high platelet counts following splenectomy and/or to increased number of abnormal RBC. Eldor A. Blood 2002 A multicenter study (56 tertiary referral centers,8860 patients),146 (1.65%)TE events. The highest prevalence of thrombotic events was observed in splenectomized patients. Taher A. Thromb Haemost 2006 Splenectomy Taher et al. Eldor A.

43. The most significant changes occurred in the severe splenectomized group who have a higher risk for thrombosis than comparable patients with intact spleen. Tripatara A et al. Thrombosis Res 2007 Test Normal (mean±SD) Severe non-splenectomized (mean±SD) Severe splenectomized (mean±SD) TAFI (%) 115±17 108±22 95±12a,b Factor V (%) 114±15 108±18 94±12a,b Factor VII (%) 98±13 94±14 88±9 Factor VIII (%) 98±14 91±43 71±17a,b Factor IX (%) 97±36 98±39 77±29 Factor XI (%) 86±19 81±37 78±37 Prothrombin (%) 86±12 87±14 86±13 Fibrinogen (mg/dl) 261±40 220±42a 218±38a Protein C (%) 94±21 58±10a 63±14a Protein S (%) 92±35 68±19a 53±25a Tripatara et al. The alterations in the activity of coagulation and fibrinolytic proteins in normal individuals and in severe non-splenectomized and severe splenectomized β-thalassemia/Hb E patients For comparison purposes, values for NS group were prothrombin=87±13%; fibrinogen=189±47%; protein C=76±21%; protein S=59±20%. a p<0.05, in comparison with normal control. b p<0.05, in comparison with severe non-splenectomized. Splenectomy

44. Thrombocytosis The increased number of circulating abnormal RBC Activation of platelets Activation of the coagulation system may contribute to the development of TE phenomena in patients with βt Splenectomy

45. Alterations in markers of coagulation activation The marker of thrombin generation – Prothrombin fragment 1.2 (F 1.2) – Fibrinopeptide A The marker of increased fibrinolysis – Thrombin – antithrombin complexes – D-dimer

46. Thrombin generation by red cells and erythroid cells of patients with thalassemia intermedia and major **P < 0.001 compared with patients with thalassaemia intermedia who were non-splenectomized and compared with patients with thalassaemia major and healthy individuals. No. of individuals Generated thrombin (nmol/min/ml) Thalassaemia intermedia Non-splenectomized 5 22.6±3.4 Splenectomized 9 42.9±16.8* Thalassaemia major Non-splenectomized 3 29.7±1.3 Splenectomized 3 23.0±11.1 Healthy individuals Non-splenectomized 11 28.6±3.6 Splenectomized 8 26.3±4.4 Cappellini MD et al. Br J Haematol 2000; 111: 467-473

47. PATHOGENESIS Factors contributing to the hypercoagulable state in thalassemia Hypercoagulable State Red Blood Cells Abnormal erythroid cells Source of procoagulant phospholipids Enhanced cohesiveness Iron overload Hyperviscosity Endothelial damage Monocyte activation Release of microparticles Genetic mutations Factor V Leiden Prothrombin G20210 MTHFR C677T (hyperhomocysteinemia) ? Others Cardiac dysfunction Liver dysfunction Hormonal deficiencies Antiphospholipid antibodies ? Intravascular hemolysis Decreased levels of Protein C Protein S Antithrombin III Platelets Increased platelet aggregation State of oxidative stress (ROS) Expression of activation markers (CD62P and CD63) Taher et al. Blood Reviews 2008; 22: 283-292

48. Decreased levels of anticoagulant proteins: Protein C and Protein S levels ↓ Cappellini et al. Br J Haematol 2000 Protein C was low in 26.2% of patients Protein S was low in 28.6% of patients AT III was low in 46.8% of patients Naithani R et al. Hematology 2006 Anticoagulant Proteins

49. Nature anticoagulant proteins Parameter No No. of patient with abnormal values Percentage Thrombocytopenia 54 18 33.3 Prolonged PT 54 22 40.7 Prolonged aPTT 54 25 46.3 Protein C (70%↓) 42 11 26.2 Protein S (70%↓) 42 12 28.6 AT III (80%↓) 47 22 46.8 Pr C + Pr S 42 1 2.4 Pr C + AT III 42 6 14.3 Pr S + AT III 42 5 11.9 Pr C + Pr S + AT III 42 3 7.1 Naithani R et al. Hematology 2006 Anticoagulant Proteins Naithani et al.

50. Anticoagulant proteins and markers of coagulation and fibrinolysis activation in patients with thalassemia intermedia and major Values are expressed as mean ± 1SD. *P < 0.05 compared with healthy individuals. **P < 0.001 compared with healthy individuals. No. of individuals Protein C (IU/ml) Antithrombin (IU/ml) Fibrinopeptide (nmol/l) Prothrombin fragment 1+2 (nmol/l) D-dimer (ng/ml) Thalassaemia intermedia Non-splenectomized 10 0.54±0.10** 0.81±0.11** 0.8±0.3 0.7±0.3 36±26 Splenectomized 20 0.52±0.14** 0.80±0.10** 1.3±0** 1.2±0.7* 121±74** Thalassaemia major Non-splenectomized 8 - 0.96±0.13 0.7±0.1 1.1±0.3 17±9.3 Splenectomized 24 - 0.92±0.10** 1.1±0.7 1.0±0.2 34.5±24 Healthy individuals 30 0.87±0.11 0.98±0.07 0.8±0.3 0.9±0.3 27±16 Cappellini MD et al. Br J Haematol 2000; 111: 467-473 Anticoagulant Proteins

51. The most significant changes occurred in the severe splenectomized group who have a higher risk for thrombosis than comparable patients with intact spleen. Tripatara A et al. Thrombosis Res 2007 Test Normal (mean±SD) Severe non-splenectomized (mean±SD) Severe splenectomized (mean±SD) TAFI (%) 115±17 108±22 95±12a,b Factor V (%) 114±15 108±18 94±12a,b Factor VII (%) 98±13 94±14 88±9 Factor VIII (%) 98±14 91±43 71±17a,b Factor IX (%) 97±36 98±39 77±29 Factor XI (%) 86±19 81±37 78±37 Prothrombin (%) 86±12 87±14 86±13 Fibrinogen (mg/dl) 261±40 220±42a 218±38a Protein C (%) 94±21 58±10a 63±14a Protein S (%) 92±35 68±19a 53±25a Tripatara et al. The alterations in the activity of coagulation and fibrinolytic proteins in normal individuals and in severe non-splenectomized and severe splenectomized β-thalassemia/Hb E patients For comparison purposes, values for NS group were prothrombin=87±13%; fibrinogen=189±47%; protein C=76±21%; protein S=59±20%. a p<0.05, in comparison with normal control. b p<0.05, in comparison with severe non-splenectomized. Anticoagulant Proteins

52. Thalassemia Major Patients with Abnormal Values of Hematological Parameters Parameter No Number of patients with abnormal values (n) Percentage (%) Protein C deficiency 34 10 29.4% Protein S deficiency 34 13 38.2% AT III deficiency 34 1 2.9% Protein C and protein S deficiency 34 6 17.6% Protein C and AT III deficiency 34 1 2.9% Protein S and AT III deficiency 34 1 2.9% Protein C, protein S and AT III deficiency 34 1 2.9% Elevated FVIII level 24 2 8.3% Elevated FIX level 22 0 0 Sipahi T et al. Clin Appl Thromb Hemost 2008 Anticoagulant Proteins Sipahi et al.

53. Protein C and Protein S levels ↓ AT III = N Protein C, protein S, TAFI, fibrinogen, Factor V and VIII in the splenectomized groups were statistically lower than those in control group. Eldor A et al. 1999 Tripatara A et al. Thrombosis Res 2007 Anticoagulant Proteins

54. The presence of anti-phospholipid antibodies (aPL) reported a high prevalence (34%) Kashef et al. reported ACA in 42.7% Antiphospholipid ab Giardano at al. 1998 Kashef et al.2008

55. Hemostatic parameters in thalassemia Assay β-TM β-TI Platelet Life span Aggregation Urinary TXA2 Circulating platelet aggregates CD62, CD63 PF3 PF4, β-TG Short Impaired High Present High High High Short Impaired High Present High High High Vascular endothelium Thrombomodulin ICAM-1 VCAM-1 VWF E-selectin Urinary PGI2 High High High High High High High High High High High High RBC Annexin V binding Thrombin generation Increased Increased Increased Increased Coagulation factors Factor II Factors V, VII, X Low Normal Low Normal Coagulation inhibitors Protein C (antigen, activity) Protein S (free) ATIII HCII Low Low Normal Low Low Low Low Thrombin generation TAT F1,2 FPA D-dimer High Normal High High High High Eldor A and Rachmilewitz EA. Blood 2002; 99(1): 36-43

56. PATHOGENESIS Factors contributing to the hypercoagulable state in thalassemia Hypercoagulable State Red Blood Cells Abnormal erythroid cells Source of procoagulant phospholipids Enhanced cohesiveness Iron overload Hyperviscosity Endothelial damage Monocyte activation Release of microparticles Genetic mutations Factor V Leiden Prothrombin G20210 MTHFR C677T (hyperhomocysteinemia) ? Others Cardiac dysfunction Liver dysfunction Hormonal deficiencies Antiphospholipid antibodies ? Intravascular hemolysis Decreased levels of Protein C Protein S Antithrombin III Platelets Increased platelet aggregation State of oxidative stress (ROS) Expression of activation markers (CD62P and CD63) Taher et al. Blood Reviews 2008; 22: 283-292

57. Thrombophilic DNA mutations Genetic basis for the hypercoagulable state in thalassemia patients is not clear. Eldor et al. (1999) No increased prevalence of FVL, PT20210 A, MTHFR C 677 T mutations. Finkelstein et al. (2004) (5/23) had spesific mutations Finkelstein et al. Pediatr Hematol Oncol 2004 Zalloua et al. (2003) 14% were heterozygous for FVL Genetic Mutations

58. FVL (heterozygous) 8/48 (17%) FVL (homozygous) 1/48 (2%) PT20210A mutation 1/48 (2%) (heterozygous) Normal population (in Turkiye) FVL 10.4 % PT 20210A 2.7 % Sipahi T et al. Clin Appl Thromb Hemost 2008 Genetic Mutations Sipahi et al.

59. Distribution of PT20210 G-A Polymorphism in Patients and Controls Genotype n (%) G/G (%) GA (%) p OR Control 70 (100) 68 (97) 2 (3) 1 1.38 (0.1217-15.697 ) Patients 48 (100) 47 (98) 1 (2) Sipahi T et al. Clin Appl Thromb Hemost 2008 Genotype n (%) G/G (%) GA (%) AA (%) p OR Control 70 (100) 60 (86) 10 (14) 0 (0) 0.795 0.81 (0.29-2.24) Patients 48 (100) 39 (87) 8 (17) 1 (2) Distribution of FV1691 G-A Polymorphism in Patients and Controls Genetic Mutations Sipahi et al.

60. Distribution of MTHFR 677C-T Polymorphism in Patients and Controls Genotype n (%) C/C (%) CT (%) T/T (%) p OR Control 70 (100) 37 (52.9) 29 (41.4) 4 (5.7) 0.24 0.59 (0.28-1.28) Patient 48 (100) 19 (40) 25 (52) 4 (8) 0.44 0.51 (0.12-2.28) Sipahi T et al. Clin Appl Thromb Hemost 2008 Distribution of Elevated sEPCR Levels in Patients and Control EPCR level n (%) Normal (%) Elevated (%) p OR Control 61 (100) 46 (75.4) 12 (24.6) 0.34 1.68 (0.62-4.55) Patient 43 (100) 38 (88.4) 5 (11.6) Genetic Mutations Sipahi et al.

61. Result of our study was, significant decrements of protein C and protein S and slight increased prevalence of congenital thrombophilic mutations when compared to controls. Although five of the patients had high sEPCR levels, no significant change was found at sEPCR values between patients and controls. Sipahi T et al. Clin Appl Thromb Hemost 2008 Genetic Mutations Sipahi et al.

62. Other Factors Other pathogenetic mechanisms: Congestive heart failure Cardiac dysfunction Liver dysfunction Hormonal deficiencies

63. Prevention of thromboembolism The TE events in the majority of patients are usually manifested in the second or third decade of life Post transf. Hb should not exceed 15g Effective iron chelation should be initiated after 10-20 transfusions when serum ferritin exceeds 1000 µg/L Low dose aspirin can be given in splenectomized patients with platelet counts above 800 000/dl

64. The management of TE events: – LMWH (enoxaparin, dalteparin, nadroparin) 7 days Dose: 1 mg/kg twice a day sc – Warfarin (long term) Prophylaxis for TE in thalassemia – Antiplatelet agents – Hydroxyurea – Aspirin – Antioxidants Prevention of thromboembolism

65. Even if thromboembolic complications could be explained by hypercoagulable state found in thalassemia major patients; following the first thrombotic event, should be investigated for congenital thrombophilia. When they are exposed to thrombotic risk factors such as immobilisation, surgery and delivery, prophylactic antithrombotic agents may be recommended. In Conclusion

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