1. Thrombophilia Failure of the Inherent Anticoagulation Defense System Mervyn A. Sahud, M.D., A.B.I.M.-Hem. Medical Director, Coagulation, Quest Diagnostics Nichols Institute

2. 2 Outline Thrombophilia (impact on DVT and PE) Risk factors Coagulation cascade Thrombophilia testing –Antithrombin –Protein C –Protein S –Activated protein C resistance (APCR) –Prothrombin (factor II) mutation –Lupus anticoagulant –Factor VIII excess Case studies Recommended work-ups

3. 3 Thrombophilia (impact on DVT and PE) Risk factors Coagulation cascade Thrombophilia testing –Antithrombin –Protein C –Protein S –Activated protein C resistance (APCR) –Prothrombin (factor II) mutation –Lupus anticoagulant –Factor VIII excess Case studies Recommended work-ups Outline

4. 4 Deep Vein Thrombosis — A Leg Up on Blood Clots

5. 5 Valve Cusp Thrombus (Autopsy Specimen)

6. 6 DVT only DVT & PE Die from PE 2 million Americans affected with DVT each year 600,000 of these develop pulmonary embolism (PE) 60,000 of those with DVT and PE die each year Thrombophilia DVT, deep vein thrombosis. Rosendall FR. Lancet. 1999:353:1167-1173. Anderson FA Jr, et al. Arch Intern Med. 1991;151:933-938.

7. 7 5% - 8% of population affected by genetic defects leading to thrombosis predisposition 25% suffer chronic swelling, skin ulceration, and impaired mobility secondary to “venous hypertension” Thrombophilia

8. 8 DVT…What’s New? 50% (soon to be 100%) of “unprovoked” DVT cases associated with hereditary thrombophilia 60% of DVT cases in pregnant women associated with factor V Leiden mutation (A1698G) DVT often associated with multiple genetic and acquired risk factors DVT responds to prolonged anticoagulant therapy (eg, low molecular weight heparin) Bucciarelli P, et al. Arterioscler Thromb Vasc Biol. 1999;19:1026-1033.

9. 9 DVT…What’s New? New anticoagulants available for non-responsive patients and those who experience side-effects from standard anticoagulants Family members request DVT screening prior to being placed in a “high-risk” situation Elevated D-dimer level often indicates thrombosis Normal D-dimer level has strong NPV NPV, negative predictive value..

10. 10 What Else is New? Pulmonary embolism –4 million patients present to U.S. emergency departments with shortness of breath each year –Shortness of breath = heart failure or pulmonary embolism (PE) –60% of patients who die in the hospital have PE –PE diagnosis missed in 70% of hospital cases –10% of patients with acute PE die within first 60 minutes Clagett GP. Chest. 1998;114(Suppl 5):531S-560S.

11. 11 Thrombophilia Is Often Multigenic Multiple risk factors raises the risk of thrombosis

12. 12 Thrombophilia (Impact on DVT and PE) Risk factors Coagulation cascade Thrombophilia testing –Antithrombin –Protein C –Protein S –Activated protein C resistance (APCR) –Prothrombin (factor II) mutation –Lupus anticoagulant –Factor VIII excess Case studies Recommended work-ups Outline

13. 13 Clinical DVT Risk Factors Age >60 y Extensive surgery* Marked immobility, pre- or postoperative Major orthopedic surgery (eg, hip, knee) Fracture of pelvis, femur, or tibia *Risk of postoperative thrombosis increases as patient’s age and surgery duration increases; risk also increases with presence of varicose veins and obesity. Surgery for malignant disease Postoperative sepsis Major medical illness –Heart failure –Inflammatory bowel disease –Sepsis –Myocardial infarction –Stroke

14. 14 Frequency of Biological Risk Factors Factor VIII excess (30%) APCR, Factor V mutation (20%) Prothrombin (factor II) mutation (15%) Hyperhomocysteinemia (10%) Protein C (10%) Protein S (10%) Unknown (5%) Antithrombin (4%) LA (3%) Other (<1%)

15. 15 Synergistic Effects of Thrombotic Risk Factors Factor V LeidenFactor II a Thrombotic Risk Normal 1.0 AbnormalNormal2.8 NormalAbnormal2.4 Abnormal 22.8 a Prothrombin 20210G  A mutation.

16. 16 Synergistic Effects of Thrombotic Risk Factors Factor V LeidenMTHFRThrombotic Risk Normal 1.0 AbnormalNormal2.8 NormalAbnormal1.8 Abnormal 16.5

17. 17 Synergistic Effects of Thrombotic Risk Factors Factor V Leiden Factor II a MTHFRThrombotic Risk Normal 1.0 AbnormalNormal 2.8 Normal Abnormal1.8 NormalAbnormalNormal2.4 Abnormal 56.5 a Prothrombin 20210G  A mutation.

18. 18 Risk of Recurrent Venous Thromboembolism Risk of recurrent venous thromboembolism (VTE) in individuals with a thrombophilic defect relative to risk in those with a first episode of VTE and no thrombophilic defect. Weitz J, et al. Hematology Am Soc Hematol Edu Program. 2004;424-438.

19. 19 Age of Thrombosis Onset in Patients with Common Thrombotic Risk Factors Risk Factor Age of Thrombosis Onset HomozygoteHeterozygote Protein C deficiencyBirth26 years Protein S deficiencyBirth31 years APCR*28 years63 years AntithrombinEmbryonic death33 years APCR, activated protein C resistance. *May be responsible for venous thrombosis in individuals older than 55-60 years.

20. 20 Relative Frequency of Various Thrombosis Types Type of Thrombosis Patient with APCRPatient with Protein C or S Deficiency DVT and/or PE82%76% Abdominal6%11% Stroke/MI4%6% Other8%7% Total100% Salomon O, et al. Arterioscler Thromb Vasc Biol. 1999;19:511-518.

21. 21 Thrombophilia (impact on DVT and PE) Risk factors Coagulation cascade Thrombophilia testing –Antithrombin –Protein C –Protein S –Activated protein C resistance (APCR) –Prothrombin (factor II) mutation –Lupus anticoagulant –Factor VIII excess Case studies Recommended work-ups Outline

22. 22 Coagulation Cascade Crowther MA, Kelton JG. Ann Intern Med. 2003;138:128-134.

23. 23 Coagulation Cascade Factor VIIa binds to tissue factor (TF) at sites of vascular injury, causing a cascade that ultimately leads to factor IIa (thrombin) generation. Thrombin activates platelets; converts fibrinogen to fibrin; leads to generation of additional thrombin through an autocatalytic loop; converts factor XIII to factor XIIIa, which stabilizes the thrombus; inhibits fibrinolysis; and acts as an anticoagulant by activating protein C. The principal inhibitors of coagulation are activated protein C (which, in concert with protein S, inactivates factors Va and VIIIa), antithrombin (which forms an inactive covalent complex with thrombin and factors Xa, IXa, and XIa), and tissue factor pathway inhibitor (which bonds with and inactivates the tissue factor-factor VIIa complex). Crowther MA, Kelton JG. Ann Intern Med. 2003;138:128-134.

24. 24 Thrombophilia (impact on DVT and PE) Risk factors Coagulation cascade Thrombophilia testing –Antithrombin –Protein C –Protein S –Activated protein C resistance (APCR) –Prothrombin (factor II) mutation –Lupus anticoagulant –Factor VIII excess Case studies Recommended work-ups Outline

25. 25 How to test? When to test?What to test? Why test? Whom to test? Thrombophilia Testing

26. 26 Antithrombin Deficiency Genetic deficiency Rare –<1% of general population –~1% - 8% of those with VTE Heterozygotes –VTE by ~30 years of age –5- to 50-fold increased risk of VTE –High risk of VTE during pregnancy and postpartum

27. 27 Antithrombin Deficiency Acquired deficiency Causes –Liver disease –Malnutrition –Inflammatory bowel disease –Prematurity –DIC –Transfusion reactions –Chemotherapy –Heparin therapy

28. 28 AT IIa AT  Inactive Complexes IIa AT Xa AT IXa AT XIa AT XIIa AT Heparin Role of antithrombin Direct inhibitor of IIa Inhibits other factors leading to indirect inhibition of IIa formation

29. 29 Therapeutic Monitoring with Antithrombin III Normal antithrombin III Antithrombin III deficient

30. 30 Factors Affecting Test Results AntithrombinProtein C and S HeparinLong-term therapy  results False  in results from clot-based assays (>1.0 IU/mL) OAC or vitamin K deficiency No effectVitamin K dependent proteins; OAC will  levels FVIIINo effect High levels will falsely  results from clot-based assays Circulating inhibitors No effect May falsely  results from clot- based assays OAC, oral anticoagulant.

31. 31 Factors Affecting Test Results AntithrombinProtein CProtein S Pregnancy  No effect  Oral contraceptives  or no change No effect  Hormone replacement therapy  or no change No effect  Liver disease, DIC, active thrombosis, recent surgery 

32. 32 Protein C Deficiency – Genetic Mechanism Autosomal inheritance Complete penetrance (dominant mutation) –Heterozygotes –Primary symptom: venous thrombosis Mild or no penetrance (recessive mutation) –Homozygotes: symptomatic (purpura fulminans) –Heterozygotes: asymptomatic

33. 33 Protein C Deficiency Genetic deficiency –Rare –Heterozygotes <1% of population ~1-11% of patients with VTE 30% will have an event during lifetime ~50% experience VTE by age 40 Acquired deficiencies –Warfarin, vitamin K deficiency, liver disease, DIC, renal insufficiency

34. 34 Which Protein C Functional Assay? Chromogenic Pro –Fewer interfering substances Con –Measures cleavage of synthetic substrate –Rare mutations not detected by this method Clotting Pro –Measures cleavage of natural substrates (factor Va or VIIIa) Con –Many interfering substances  High factor VIII levels  Lupus anticoagulants  Heparin >1.0 IU  Direct thrombin inhibitors

35. 35 Functional Protein C Assay 2 patients missed by clot assay 8 patients missed by chromogenic assay

36. 36 Normal adult reference range:  70% Protein C Testing Algorithm No further testing Protein C Clot Assay Decreased (<70%) Borderline (70% - 75%) Normal or  Protein C Antigen Repeat Clot Assay Decreased (<70%) Increased (  70%) Borderline (70% - 75%) Chromogenic Assay

37. 37 Therapeutic Monitoring with Protein C Normal protein C Protein C deficient

38. 38 Protein S Deficiency Genetic deficiency Homozygous (rare) –Purpura fulminans Heterozygous –< 1% of general population –~1% to 3% of VTE population – ~50% experience first VTE by 45 years of age Acquired deficiency Pregnancy and oral contraceptive/HRT use Inflammation and acute thrombosis

39. 39 Protein C fPS PS TM IIa PC APC Va or VIIIa VIIIai or Vai Free PS C4b-BP IIa fPS Inactive Cofactors Role of protein C & S Inhibition of IIa formation

40. 40 Therapeutic Monitoring with Protein S Normal protein S Protein S deficient

41. 41 APC Resistance in Patient with Thrombosis APC, when added to plasma from a middle- aged man with recurrent thrombosis, did not result in a prolongation of the clotting time (APC resistance). In contrast, when added to normal plasma (control), APC prolongs the clotting time in a dose- dependent manner. Dahlback B. J Thromb Haemost. 2003;1:3-9.

42. 42 APC Resistance – Genetic Mechanism Molecular cause Mutation of APC cleavage site within Factor Va Mutation site Amino acid #506 (arginine) mutates to a glutamine Amino acid: Arg 506 → Gin Nucleotide: CGA→ CAA

43. 43 APC Resistance – Genetic Mechanism Molecular function –APC cleaves bond between arginine and glycine –If Arg 506 residue is mutated  Bond not cleaved  Factor Va not inactivated by APC  Continued generation of thrombin and clot formation  Thrombosis

44. 44 Acquired APC Resistance Associated with: Increased plasma levels of FVIIIa Presence of antiphospholipid antibodies 3 rd generation oral contraceptives, relative to 2 nd generation oral contraceptives Pregnancy

45. 45 Incidence of APC Resistance in “Normals” EthnicityPercent (%) Caucasian4 African American0.8 Asian American0.6 Hispanic/Latino2.3 Native American0.2

46. 46 Prothrombin (Factor II) 20210G  A Mutation A, adenine; G, guanine. Zoller B, Svensson PJ, He X, et al. J Clin Invest. 1994;94:2521-2524.

47. 47 Lupus Anticoagulant Screening Screening tests –LA sensitive aPTT reagent –Kaolin clotting time (KCT) –Dilute russell viper venom time (dRVVT) –Dilute prothrombin time (dPT) Minimum of 2 screening tests

48. 48 Factor VIII Excess O’Donnell J, et al. Thromb Haemost. 1997;77:825-828. Factor VIII LevelRelative Risk >150% (>1.5 IU/mL)3.0 >200% (>2.0 IU/mL)5.5

49. 49 Factor VIII Excess vs Other Thrombophilia Markers Abnormality Frequency (%) No History a With History a APCR14-2020-50 Prothrombin mutation4-810-20 Hyperhomocysteinemia5-10NA Protein C deficiency Protein S deficiency Antithrombin deficiency APCR/factor VIII mutation 1-4 1-3 25 3-12 1-6 NA O’Donnell J, et al. Thromb Haemost. 1997;77:825-828. a History of thromobophilia.

50. 50 Thrombophilia (impact on DVT and PE) Risk factors Coagulation cascade Thrombophilia testing –Antithrombin –Protein C –Protein S –Activated protein C resistance (APCR) –Prothrombin (factor II) mutation –Lupus anticoagulant –Factor VIII excess Case studies Recommended work-ups Outline

51. 51 Case Study #1 61 year-old white male with multiple myeloma (in remission) Receiving no treatment Flies to Singapore for vacation Experiences discomfort in right leg, no swelling, no rubor 2 days later, sudden dyspnea, left pleuritic chest pain Hospitalized with DVT/PE Returns home 2 weeks later on coumadin

52. 52 Case Study #1: Laboratory Workup TestResult Hemoglobin12.4 g/dL Platelet count 177,000/  L WBC 3,100/  L INR2.2 D-dimer <0.79  g/mL FEU APCR1.3 ratio Antithrombin87% Protein C<41% Protein S22% Factor V LeidenHomozygous Follow-up duplex ultrasound Residual thrombus in iliac system and IVC

53. 53 Case Study #2 37 y/o, G1P1 W/F attempting to get pregnant for the 2 nd time 1 st childbirth at age 21 y without incident Family history of protein S deficiency –Mother on long-term coumadin –Sister had DVT/PE while on birth control Lab results at 12 weeks –Protein S 47% (normal, 60-120%) –Protein C 141% (normal, 60-130%) –Antithrombin III 78% (normal, 60-120%)

54. 54 Case Study #2 (cont’d) 2 weeks later, concern raised about need for prophylactic anticoagulant Lab results –C4 binding protein212 mg/L (normal, 75-140) –Total protein S81% –Free protein S53% –Functional protein S43%

55. 55 Case Study #2 (cont’d) Physician discusses drug therapy –Coumadin –Unfractionated heparin –Low molecular weight heparin –Arixtra  (fondaparinux) Decision made to use graded elastic stockings and aspirin Lab results at 20 weeks –Protein S33% –Protein C 162%

56. 56 Case Study #2 (cont’d) Lab results at 36 weeks –Protein S 21% Enoxaparin initiated: 40 mg s.c. daily Delivery of baby uneventful Lab results 4 weeks postpartum –Protein S 31% Patient told she probably has hereditary protein S deficiency. Restarts enoxaparin for 4 weeks, daily 40 ng s.c.

57. 57 Case Study #2 (cont’d) Months postpartumProtein S (%) 374 681 1280 Protein S deficiency unlikely

58. 58 Case Study #3 39 year-old, physically active, white male bicyclist develops rapidly progressive drawing pain in right calf after 40-mile event History of DVT in left lower extremity after laparoscopic knee surgery at age 21 Mother on coumadin Father died mysteriously of unknown causes at age 51 years Sister has had recurrent miscarriages

59. 59 Case Study #3 (cont’d) TestResult PT a 10.4 sec. (normal, 10 – 12) aPTT a 33.0 sec. (normal, <36 sec.) Factor V LeidenR506Q heterozygous Prothrombin geneG20210A heterozygous MTHFRHeterozygous Lupus anticoagulant screenNegative Protein C b 31% (normal, 62 – 124) Protein S b 19% (normal, 58 – 112) Antithrombin III b 77% (normal, 62 – 114) MRI, vena cavaThrombus to vena cava; duplicate cava; old thrombosis noted a Tested prior to initiation of anticoagulant therapy. b Tested after initiation of coumadin therapy; INR 3.1.

60. 60 Case Study #3 Conclusion Long-term anticoagulation indicated Schulman S, et al. N Engl J Med. 1997;336:393-398.

61. 61 Case Study #4 Active 49 year-old white male experienced “charley horse” of right leg for >6 days Pain, swelling, and redness progressed to lower thigh area 16-year history of smoking Episode of superficial phlebitis following IV insertion during routine colonoscopy

62. 62 Case Study #4 (cont’d) Thrombophilic workup negative for –Factor V G1691A –Prothrombin G20210A –Lupus anticoagulant –Protein C and S –Antithrombin Ultrasound duplex assessment demonstrated DVT involving both lower extremities Patient hospitalized for IV heparin therapy followed by coumadin therapy

63. 63 Case Study #4 (cont’d) Lab Results at Admission TestResult Platelet count 515,000/  L (normal, 147,000 – 390,000) Prothrombin time9.9 secs (normal, 10.3 – 12.7) aPTT20.7 secs (normal, 23.1 – 34.2) Fibrinogen584 mg/dL (normal, 200 – 400) D-dimer4000-8000 ng/mL (normal, <250)

64. 64 TestResult D-dimer500 – 1000 ng/mL (normal, <250) Thrombin-antithrombin 11.3  g/L (normal, <4.0) Soluble tissue factor (CD142) 1114 pg/mL (normal, <250) Prothrombin fragment 1.2 7.6 nM/L (normal, 0.32 – 2.45) Case Study #4 (cont’d) Lab Results 30 d Post Discontinuation of Coumadin Therapy

65. 65 Case Study #4 Conclusions 1. Finding of unprovoked DVT and negative thrombophilic workup requires closer look for possible malignancy even without referable symptoms 2. Development of superficial thrombophlebitis after brief IV insertion and subsequent development of unprovoked bilateral DVT is significant 3. High level of fibrinogen, elevated platelet counts, and shortened prothrombin time and aPTT are suggestive of hypercoagulability

66. 66 Case Study #4 Conclusions 4. Elevated thrombin generation markers 14 days post coumadin therapy are turning point in this patient’s workup & eventual diagnosis 5. High level of thrombin generation markers and presence of soluble tissue factor suggest antithrombin or Xa inhibitors (eg, low molecular weight heparins) would be more appropriate therapy 6. CT scan of chest reveals 3 cm mass, and bronchoscopic washing demonstrated malignant cells, ie, bronchogenic carcinoma

67. 67 Case Study #5 19 year-old ballet dancer strains left calf and ankle Recently recovered from infectious mononucleosis Negative ultrasound duplex exam

68. 68 TestResult D-dimer497 µg/mL (normal, <0.79) Fibrin monomer+/- PT11.4 sec (normal, 12) aPTT31.7 sec (normal, <34) HAMANegative Rheumatoid factorNegative Fibrinogen232 mg % ANANegative Repeat ultrasound duplex (post 7 d) No DVT Case Study #5 (cont’d)

69. 69 Medical Devices Agency 2002 Evaluation Report. Patient recovers, but D-dimer still elevated (241 µg/mL) False-positive identified 1.Demonstrated high heterophile antibody titer in patient serum 2.Incubated serum w/ heterophile blocking antibody, then repeated D-dimer test 3.Repeat D-dimer test normal Case Study #5 (cont’d)

70. 70 Heterophile antibodies may contribute to false- positive D-dimer results in various methods Medical Devices Agency 2002 Evaluation Report. FEU, fibrinogen equivalent units; cut-off values are either those recommended by the manufacturer or 95% VTE sensitivity.

71. 71 Thrombophilia (impact on DVT and PE) Risk factors Coagulation cascade Thrombophilia testing –Antithrombin –Protein C –Protein S –Activated protein C resistance (APCR) –Prothrombin (factor II) mutation –Lupus anticoagulant –Factor VIII excess Case studies Recommended work-ups Outline

72. 72 Hereditary Thrombophilia Evaluation Who Age 18-45 years Family history of HTD No other cause of HTD When Asymptomatic Usually not receiving therapy, but may be on stable oral anticoagulant therapy How Work up in stages Most common factors first HTD, hereditary thrombophilia evaluation.

73. 73 Cost-Effective Hypercoagulation Workup Below Age 45 APCR Protein C Protein S Antithrombin If APCR is abnormal or borderline normal, perform factor V Leiden mutation analysis. If aPTT is abnormal, complete LA workup. If homocysteine is abnormal, perform MTHFR mutation analysis. Prothrombin mutation Homocysteine LA (aPTT) and ACA/APS

74. 74 Cost-Effective Hypercoagulation Workup Over Age 45 APCR Homocysteine Prothrombin mutation LA (aPTT) and ACA/APS If APCR is abnormal or borderline normal, perform factor V Leiden mutation analysis. If aPTT is abnormal, complete LA workup. If homocysteine is abnormal, perform MTHFR mutation analysis.

75. Thrombophilia Quiz

76. 76 Thrombophilia Quiz Which of the following statement(s) concerning activated protein C resistance is/are true? a.Activated protein C resistance is the most commonly inherited thrombophilic condition. b.Factor V Leiden mutation is reported in 3% to 7% of the Caucasian population. c.Approximately 20% to 60% of patients with venous thromboembolism have the Factor V Leiden mutation. d.All of these

77. 77 Thrombophilia Quiz Which of the following statements concerning prothrombin mutation are true? a.A mutated 20210 allele is a risk factor for venous and arterial thrombosis. b.The increased thrombotic risk in patients with a mutated 20210 allele appears to be caused by excessive activation of the coagulation pathway. c.In patients with a history of venous thrombosis, there is a 9- to 11-fold increase in the presence of the mutant 20210 allele. d.All of these

78. 78 Thrombophilia Quiz All of the following statements concerning antithrombin are true except: a.Patients with homozygous antithrombin deficiency usually present in the neonatal period with purpura fulminans. b.Antithrombin is a major regulator of coagulation and inhibits activated Factor II, IX, X, XI, and XII. c.Thrombosis is recurrent in approximately 60% of antithrombin deficient patients. d.There are multiple amino acid substitutions responsible for congenital deficiencies of antithrombin.

79. 79 Thrombophilia Quiz Of patients who experience venous thrombosis, deficiencies of protein C, protein S, and antithrombin are collectively present in: a.Approximately 15% of patients b.Approximately 30% of patients c.Approximately 50% of patients d.Approximately 70% of patients

80. 80 Thrombophilia Quiz Patients with deficiency of protein C, protein S, or antithrombin usually present with: a.Arterial thrombosis b.Venous thrombosis c.Recurrent arterial thromboembolic disease d.All of these

81. 81 Thrombophilia Quiz Which of the following statement(s) concerning methylene tetrahydrofolate reductase (MTHFR) is/are true? a.MTHFR and cystathionine β synthase are the primary enzymes responsible for methionine metabolism. b.At least 7 mutations of MTHFR have been reported to increase plasma homocysteine levels. c.It is estimated that up to ½ of the North American population is heterozygous for a MTHFR mutation. d.All of these

82. 82 Thank You