1. INHERITED THROMBOPHILIA

2. Defects in physiologic anticoagulant pathways Increased production of procoagulant Antithrombin deficiency Protein C deficiency Protein S deficiency Factor V Leiden (APC resistance) Prothrombin G20210A gene mutation  Many other genes affect coagulation – the contribution of mutations in these genes to thrombotic risk is important but presently difficult to quantify

3. THE ANTITHROMBIN SYSTEM Antithrombin inhibits thrombin, Xa, IXa, XIa

4. E C TM THE PROTEIN C SYSTEM A negative feedback loop that degrades factors Va, VIIIa IIa P S APC Va Vi VIIIa VIIIi P C

5. Typically 30-60% of normal plasma activity of affected protein Genetically heterogeneous  Type 1: low antigen and activity  Type 2: normal antigen, low activity (missense mutations) Thrombotic risk varies from family to family Together account for approximately 10-15% of cases of inherited thrombophilia THROMBOPHILIA DUE TO DEFICIENCY OF ANTICOAGULANT PROTEIN Antithrombin, Protein C, Protein S

6. Missense mutation changes amino acid 506 of factor V from arginine to glycine Mutation is at preferred protein C cleavage site, slows inactivation of factor Va by protein C Factor Va procoagulant activity not affected  Not a “deficiency” Single mutation responsible for all cases  Usually diagnosed by DNA testing Very common  About 5% of US population heterozygous, 0.05% homozygous FACTOR V LEIDEN A highly prevalent inherited risk factor for thrombosis

7. FACTOR V LEIDEN Prevalence in different ethnic groups Lancet 1995;346:1133 Group Allele frequency Heterozygote frequency Homozygote frequency European4.4% 8.6% 0.2% Asia Minor0.61.20.004 African000 SE Asian000 Native American 000

8. Mutation in 3' untranslated (non-coding) part of prothrombin gene No effect on prothrombin structure or function Heterozygotes have 5-10% higher plasma levels of prothrombin Heterozygotes have 2-3 fold risk of venous thromboembolism About 1-2% of population heterozygous; 5- 7% of young patients with DVT/PE Diagnosis: DNA testing PROTHROMBIN G20210A GENE MUTATION

9. GENETIC RISK FACTORS FOR THROMBOSIS Phenotype Approximate prevalence in thrombophilia Number of genotypes Antithrombin deficiency5% or lessMany Protein C deficiencyabout 5%Many Protein S deficiency5% or lessMany Factor V Leiden40-50%One Prothrombin G20210A5-7%One

10. CLINICAL FEATURES OF INHERITED THROMBOPHILIA

11. INHERITED THROMBOPHILIA Clinical findings in homozygous state CONDITION CLINICAL FINDINGS IN HOMOZYGOTES Antithrombin III deficiencylethal? Protein C deficiencyneonatal purpural fulminans Protein S deficiencyneonatal purpural fulminans (? - rare) FVL Prothrombin mutation Premature thrombosis in many (most?) - some asymptomatic

12. HOMOZYGOUS PROTEIN C DEFICIENCY WITH NEONATAL PURPURA FULMINANS

13. Venous thromboembolism  No convincing evidence of increased risk of arterial thrombosis Onset often in 20s and 30s  Many carriers are asymptomatic throughout life About half of VTE episodes associated with other risk factors, half "idiopathic" Increased risk of pregnancy loss INHERITED THROMBOPHILIA Clinical Features in Heterozygotes

14. RISK OF VTE HIGHER WITH INHERITED ANTICOAGULANT PROTEIN DEFICIENCY THAN FVL, PROTHROMBIN MUTATION No defect AT PC PS PT FVL Blood 2009;113:5314 Thrombosis-free survival in relatives of patients with thrombophilia

15. INCREASED THROMBIN GENERATION IN HETEROZYGOUS FACTOR V LEIDEN Can such testing help predict risk of thrombosis? CONTROLS FVL

16. Genetic heterogeneity – mutation detection more difficult than for FVL, PT mutations Blood levels affected by other conditions, drugs  Heparin lowers antithrombin levels  Warfarin lowers protein C, protein S levels  Liver disease lowers all three  Pregnancy, inflammation, contraceptives decrease free protein S Some mutations affect protein activity, not antigen  Measurements of anticoagulant protein levels in unselected patients have low predictive value INHERITED THROMBOPHILIA Diagnosing anticoagulant protein deficiency

17. Likelihood that a gene mutation is present if measured protein activity is 50% of normal: –Antithrombin = 75% –Protein C =60% –Protein S = 25% Thromb Haemost 2012; 108: 247 Low blood level ≠ inherited deficiency

18. LOW PROTEIN S LEVEL IS A POOR PREDICTOR OF THROMBOSIS RISK IN THE GENERAL POPULATION The Leiden Thrombophilia Study Koster et al, Blood 1995;85:2756 Relative risk of thrombosis 95% CI Low total protein S (one measurement) 0.70.3-1.8 Low total protein S (two measurements) 0.80.2-3.0 Low free protein S 1.60.6-4.0 Low free and total protein S 1.71.0-4.7

19. Subjects: 122-member protein S-deficient family, 44 of whom carried Gly295-Val mutation Diagnosis of protein S deficiency established by DNA testing  Hazard ratio for thrombosis associated with carriage of protein S mutation was 11.5 (95% CI = 4.33-30.6) PROTEIN S DEFICIENCY IS A STRONG PREDICTOR OF THROMBOTIC RISK IN A FAMILY WITH A KNOWN MUTATION Simmonds et al, Ann Intern Med 1998;128:8

20. Family history predicts thrombotic risk just as well as laboratory testing for thrombophilia A case-control study Arch Intern Med 2009;169:610 EventOR for event with positive FH OR for event with positive test for thrombophilia Unprovoked VTE2.52.3 Provoked VTE16.421.2 Fam Hx VTEOdds ratio for thrombosis (95% CI) Negative1 (reference) Any relative2.2 (1.9-2.6) Relative < 502.7 (2.2-3.4) > 1 Relative3.9 (2.7-5.7)

21. The presence of thrombophilia does not predict thrombotic risk in the absence of a family hx of VTE Thromb Haemost 2011;106:646 Event in proband leading to diagnosis of thrombophilia Incidence of VTE in relatives per 1000 patient-yrs (95% CI) Carriers Non-carriers VTE1.6 (1.2-2.2) 0.5 (0.3-1.0) Arterial thrombosis0.5 (0.1-2.8) 0.8 (0.2-3.0) Obstetric complication0.6 (0.2-1.6) 0.0 (0-0.8) Asymptomatic0.3 (0.1-1.2) 0.0 (0-0.7)

22. RISK OF THROMBOSIS IN THROMBOPHILIA

23. EFFECT OF GENE DOSE Relative risk of thrombosis in heterozygous and homozygous factor V Leiden Rosendaal et al, Blood 1995;85:1504 GenotypeRelative Risk Normal1 Heterozygous7 Homozygous80

24. EFFECT OF GENE INTERACTIONS Co-inheritance of protein C deficiency and factor V Leiden within a family Koeleman et al, Blood 1994;84:1031 Gene Mutation Protein C and Factor V Thrombosis present (%) 16 (73) Thrombosis absent (%) 6 (27) Protein C5 (31)11 (69) Factor V2 (13)11 (87) None011 (100)

25. INTERACTION WITH ACQUIRED RISK FACTORS Oral contraceptive Vandenbroucke et al, Lancet 1994;344:1453 RISK FACTOR RELATIVE RISK OF THROMBOSIS Oral contraceptive4 Factor V Leiden8 Both35

26. INTERACTION WITH ACQUIRED RISK FACTORS Estrogen replacement Rosendaal, 2001 RISK FACTOR RELATIVE RISK OF THROMBOSIS Estrogen replacement3.5 Factor V Leiden4.6 Both11

27. FACTOR V LEIDEN INCREASES RISK OF VENOUS, BUT NOT ARTERIAL, THROMBOSIS Physicians' Health Study (15,000 subjects) Ridker et al, NEJM 1995;332:912 Type of thrombosis 0 2 4 6 8 10 12 % Heterozygotes None MIStroke MI or Stroke DVT or PE P = 0.9 P = 0.4 P = 0.7 P = 0.02

28. WHAT ARE THE IMPLICATIONS OF A POSITIVE TEST FOR THROMBOPHILIA IN AN ASYMPTOMATIC PERSON?

29. RELATIVE RISK OF VENOUS EVENTS IN RELATIVES OF PATIENTS WITH THROMBOPHILIA Vossen et al, J Thromb Haemost 2004;2:1526

30. THE ABSOLUTE RISK OF VENOUS EVENTS IN ASYMPTOMATIC RELATIVES OF THROMBOPHILIC PATIENTS IS LOW Vossen et al, J Thrombos Haemost 2005;3:459 Bleeding risk with long term anticoagulation estimated at 1-3%/year

31. INCIDENCE OF FIRST VTE EVENTS IN SPECIFIC RISK SITUATIONS IN THROMBOPHILIC INDIVIDUALS Vossen et al, J Thrombos Haemost 2005;3:459 Analysis restricted to individuals not given prophylaxis RISK SITUATION THROMBOPHILIC INDIVIDUALS CONTROLS Travel > 8h0% (0/504)0% (0/1244) Surgery or immobilization > 2 w 2% (3/176)0.04% (2/407) Plaster cast0% (0/33)0% (0/71) Cancer10% (1/10)6% (1/17) Pregnancy7% (2/28)0% (0/75)

32. SHOULD ORAL CONTRACEPTIVES ROUTINELY BE WITHHELD FROM WOMEN WITH FACTOR V LEIDEN? PREDICTED OUTCOMES WITH ALTERNATIVE CONTRACEPTIVE METHODS Oral contraceptive Levonorgesterol -IUD Copper IUDCondom 1 st VTE/100 pregancy-yr 0.550.25 VTE/100,000 pregnancy-yr 550250 Unintended pregnancies/ 100,000 p-y 200700140012,000 Additional cases of VTE 62040336 Total # VTE556270290586 Blood 2011;118:2055

33. MANAGEMENT OF ASYMPTOMATIC INDIVIDUALS WITH INHERITED THROMBOPHILIA Counseling/reassurance Prophylaxis in high-risk situations Carefully consider risk/benefit ratio and alternatives when prescribing oral contraceptives or HRT

34. IS THE MANAGEMENT OF PATIENTS WITH VTE AFFECTED BY THE RESULTS OF THROMBOPHILIA TESTING?

35. The presence of inherited thrombophilia does not usually affect treatment of patients with VTE Idiopathic VTE is a strong independent predictor of recurrence risk, and so is a potential indication for long-term anticoagulation The presence of inherited thrombophilia is not a good predictor of VTE recurrence risk and so should not be used as the basis for prolonging therapy

36. The risk of recurrent venous thromboembolism is higher in patients with idiopathic events Lancet 2003; 362: 523–26 Idiopathic VTE Postop VTE Other risk factor

37. The risk of recurrent VTE is not significantly affected by the presence of inherited thrombophilia Lancet 2003; 362: 523–26 Hazard ratio 1.50 (95% CI = 0.82-2.77) p=0.187

38. A diagnosis of thrombophilia does not affect overall survival after an episode of VTE Thromb Haemost 2013;1:79 Median followup time = 5 yrs

39. Warfarin-induced skin necrosis in a protein C-deficient patient Compound heterozygote for FVL and protein C deficiency Day 5 of warfarin treatment, on heparin Concomitant bilateral adrenal hemorrhagic infarction

40. WARFARIN LOWERS LEVELS OF PROTEIN C FASTER THAN LEVELS OF PROCOAGULANT VITAMIN K-DEPENDENT PROTEINS Protein C Prothrombin Transient hypercoagulability?

41. THROMBOPHILIA AND PREGNANCY

42. INCREASED RISK OF FETAL LOSS IN WOMEN WITH HERITABLE THROMBOPHILIA European Prospective Cohort on Thrombophilia (1384 women) Lancet 1996;348:913 CONDITION RR OF STILLBIRTH 95% CI RR OF MISCARRIAGE 95% CI ANTITHROMBIN DEFICIENCY 5.21.5-18.11.71.0-2.8 PROTEIN C DEFICIENCY 2.30.6-8.31.40.9-2.2 PROTEIN S DEFICIENCY 3.31.0-11.31.20.7-1.9 FACTOR V LEIDEN20.5-7.70.90.5-1.5 COMBINED DEFECTS14.32.4-86.00.80.2-3.6 ALL THROMBOPHILIA 3.6 1.4-9.41.270.94-1.71

43. LATE FETAL LOSS IN THROMBOPHILIA Due to factor V or prothrombin mutation NEJM 2000;343;1015 Mutation % of women with late fetal loss % of women with normal pregnancy RR (95% CI) Factor V or prothrombin 1663.3 (1.4-7.8) Factor V Leiden 733.2 (1.0-10.9) Prothrombin933.3 (1.1-10.3)

44. BUT…

45. There is no evidence that anticoagulant (LMWH) or antiplatelet (ASA) prophylaxis improves pregnancy outcomes in women with inherited thrombophilia

46. ANTICOAGULATION IN WOMEN WITH RECURRENT PREGNANCY LOSS 2012 ACCP CONSENSUS RECOMMENDATIONS For women with recurrent pregnancy loss and no evidence of antiphospholipid syndrome, whether or not they have inherited thrombophilia:  No antithrombotic therapy recommended CHEST 2012; 141:e691S

47. VTE PROPHYLAXIS DURING PREGNANCY 2012 ACCP CONSENSUS RECOMMENDATIONS Women homozygous for FVL or prothrombin mutation, no prior VTE – If positive FH: antepartum prophylaxis (LWMH) and postpartum prophylaxis x 6 weeks (warfarin or LMWH) – If no FH: antepartum “clinical vigilance” and postpartum prophylaxis x 6 weeks CHEST 2012; 141:e691S

48. VTE PROPHYLAXIS DURING PREGNANCY 2012 ACCP CONSENSUS RECOMMENDATIONS All other forms of thrombophilia, no prior VTE – If positive FH: antepartum “clinical vigilance” and postpartum prophylaxis x 6 weeks (LMWH, warfarin OK if not protein C or S deficient) – If no positive FH: clinical vigilance only CHEST 2012; 141:e691S

49. WHO TO TEST? Inherited thrombophilia is more likely if a patient with VTE  Is young  Has a family history of VTE  Had unprovoked VTE  Had warfarin-induced skin necrosis (protein C)  Test results rarely affect patient management!

50. WHEN TO TEST? FVL, prothrombin mutation: any time (not informative after liver transplantation) Antithrombin:  Not during acute thrombosis  Not during pregnancy or estrogen/OCP use  Off heparin/LMWH at least 2 weeks Protein C:  Off warfarin (preferable), or on stable warfarin dose at least 2 weeks  Preferably not during acute thrombosis Protein S:  As for protein C  Not during pregnancy, OCP use or acute inflammation Neonatal period, DIC, liver disease, asparaginase Rx can all cause acquired deficiency of AT, PC, PS  Testing should usually be done in the outpatient setting

51. ACQUIRED THROMBOPHILIA Antiphospholipid syndrome Hyperhomocysteinemia (may be inherited) Cancer Myeloproliferative disorders Nephrotic syndrome Pregnancy Oral contraceptive/estrogen Hyperviscosity

52. HOMOCYSTEINE

53. SEVERE homozygous cystathione beta-synthase deficiency (1:250,000) homozygous methylenetetrahydrofolate reductase deficiency MILD OR MODERATE heterozygous CBS deficiency (0.3-1.4% of population) thermolabile variant of MTHFR (5% of population) B12, folate or B6 deficiency Aging Chronic renal failure CAUSES OF HYPERHOMOCYSTEINEMIA

54. HIGHER HOMOCYSTEINE LEVELS ARE ASSOCIATED WITH VASCULAR RISK Meta-analysis Condition Increase in risk per 5 micromole increase in plasma HC (95% CI) Ischemic heart disease1.32 (1.19-1.45) Stroke1.59 (1.29-1.96) VTE1.60 (1.15-2.22) BMJ 2002;325:1202

55. BUT…

56. LOWERING HOMOCYSTEINE DOES NOT DECREASE VASCULAR RISK VISP trial (JAMA 2004): Moderate reduction in HC had no effect on vascular risk during 2 yr followup HOPE 2 trial (NEJM 2006): Vitamin supplements lowered HC levels but had no effect on vascular risk NORVIT trial (NEJM 2006): More aggressive vitamin supplementation associated with increased vascular risk VITRO study (Blood 2007): Lowering HC did not prevent recurrent VTE

57. ANTIPHOSPHOLIPID ANTIBODIES

58. Lupus anticoagulant Cardiolipin antibodies (IgG, IgM) Beta-2 glycoprotein I antibodies (IgG, IgM) Thrombotic risk associated with higher antibody levels, positive tests for more than one type of antibody

59. INCIDENCE OF ANTIPHOSPHOLIPID ANTIBODIES Love and Santoro, Ann Intern Med 1990 PATIENT GROUPANTIBODY TYPE APPROX INCIDENCE SLELAC30% SLEaCL40% Blood donorsaCL2% Healthy elderlyaCL52%

60. Thrombosis (arterial and venous) Recurrent fetal loss Hematologic abnormalities:  Immune thrombocytopenia  Immune hemolytic anemia CLINICAL CONDITIONS ASSOCIATED WITH ANTIPHOSPHOLIPID ANTIBODIES The “antiphospholipid syndrome”

61. Arthritis & Rheumatism 2002;46:1019-27

62. 1% or less of APL patients Generalized vasculopathy (?thrombotic or inflammatory) Livedo reticularis Multiple organ system involvement  Renal failure  Hypertension  ARDS  CNS Rapid progression; sudden death in some patients Treatment: anticoagulation, plasma exchange, ?immunosuppresion CATASTROPHIC ANTIPHOSPHOLIPID SYNDROME (Asherson, 1992)

63. In study of 22000 male physicians: aCL titer above 95th percentile associated with 5-fold increase in relative risk of DVT No significant increase in ischemic stroke risk ANTIPHOSPHOLIPID ANTIBODIES AND THROMBOSIS IN HEALTHY PEOPLE Physicians Health Study Ginsberg et al, Ann Intern Med 1992

64. IgG Anticardiolipin Antibodies and Risk of Recurrence or Death in Patients with VTE After Stopping Anticoagulation RecurrenceDeath Am J Med 1998; 104:332

65. Incidence of first thromboembolic events in asymptomatic “high-risk” individuals with antiphospholipid antibodies Blood 2011; 118:4714 “High risk” defined as having persistently positive tests for lupus anticoagulant, cardiolipin antibodies and ß2-glycoprotein I antibodies

66. Blood 2013;122:817-824 “Although a positive APLA test appears to predict an increased risk of recurrence in patients with a first VTE, the strength of this association is uncertain because the available evidence is of very low quality”

67. ANTIPHOSPHOLIPID SYNDROME CLINICAL CRITERIA 1.One or more documented episodes of arterial, venous, or small vessel thrombosis (other than superficial venous thrombosis) in any tissue or organ –Thrombosis must be confirmed by objective validated criteria –For histopathologic confirmation, thrombosis should be present without significant evidence of inflammation in the vessel wall 2.Pregnancy morbidity a.One or more unexplained deaths of a morphologically normal fetus at or beyond the 10th week of gestation, with normal fetal morphology documented by ultrasound or by direct examination of the fetus, or b.One or more premature births of a morphologically normal neonate before the 34th week of gestation because of: (i) eclampsia or severe pre-eclampsia defined according to standard definitions, or (ii) recognized features of placental insufficiency, or c.Three or more unexplained consecutive spontaneous abortions before the 10th week of gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded J Thromb Haemost 2006;4:295

68. ANTIPHOSPHOLIPID SYNDROME LABORATORY CRITERIA 1.Lupus anticoagulant (LAC) present in plasma, on two or more occasions at least 12 weeks apart, detected according to the guidelines of the International Society on Thrombosis and Haemostasis (Scientific Subcommittee on LACs/phospholipid- dependent antibodies) 2.Anticardiolipin antibody (aCL) of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e., > 40 GPL or MPL, or > the 99th percentile), on two or more occasions at least 12 weeks apart, measured by a standardized ELISA 3.Anti-ß2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (in titer >the 99th percentile), present on two or more occasions at least 12 weeks apart, measured by a standardized ELISA, according to recommended procedures  APL syndrome considered present if at least one of the clinical and one of the laboratory criteria are present J Thromb Haemost 2006;4:295

69. TREATMENT OF PATIENTS WITH ANTIPHOSPHOLIPID ANTIBODIES Asymptomatic: no treatment History of thrombosis: –Consider prolonged treatment in selected patients Recurrent or unprovoked thrombosis (arterial or venous) Persistently high antibody levels More than 1 APL antibody test positive –Most patients can be treated with standard anticoagulant regimen Two RCTs have shown inferior outcomes with high intensity warfarin treatment A few patients exhibit warfarin failure – consider long term LMWH treatment (no data yet on newer oral anticoagulants)

70. ANTIPHOSPHOLIPID ANTIBODIES AND FETAL LOSS Antiphospholipid antibodies associated with lower live birth rates in unselected “low-risk” pregnancies Live birth rates in untreated women with APL and at least one fetal loss have ranged from 10-85% in published studies Aspirin and heparin have been associated with higher live-birth rates in several studies, but most of these did not include a placebo-treated arm Arth Rheum 2004;50:1028

71. ANTIPHOSPHOLIPID ANTIBODIES AND FETAL LOSS Testing for APL should be restricted to women with at least three consecutive miscarriages Other causes of pregnancy loss (especially abnormal karyotypes) should be ruled out If criteria for obstetric APL syndrome met, treat with aspirin and/or LMWH during pregnancy and postpartum period Arth Rheum 2004;50:1028

72. ANTICOAGULATION IN WOMEN WITH APLA AND RECURRENT PREGNANCY LOSS 2012 ACCP CONSENSUS RECOMMENDATIONS Women who meet lab and clinical criteria for obstetric APLA:  Antepartum prophylactic or intermediate- dose LMWH plus low dose ASA CHEST 2012; 141:e691S