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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

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 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

Deep Vein Thrombosis

— A Leg Up on Blood Clots

Valve Cusp Thrombus

(Autopsy Specimen)

Thrombophilia

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 DVT, deep vein thrombosis.

Rosendall FR.

Lancet

. 1999:353:1167-1173.

Anderson FA Jr, et al.

Arch Intern Med

. 1991;151:933-938.

Thrombophilia

• 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” 7

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) 8 Bucciarelli P, et al.

Arterioscler Thromb Vasc Biol

. 1999;19:1026-1033.

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 9 NPV, negative predictive value..

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.

Thrombophilia Is Often Multigenic

Multiple risk factors raises the risk of thrombosis

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 12

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 • Surgery for malignant disease • Postoperative sepsis • Major medical illness – Heart failure – Inflammatory bowel disease – Sepsis – Myocardial infarction – Stroke *Risk of postoperative thrombosis increases as patient’s age and surgery duration increases; risk also increases with presence of varicose veins and obesity.

Frequency of Biological Risk Factors

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

Synergistic Effects of Thrombotic Risk Factors

Factor V Leiden Normal Factor II a Normal Thrombotic Risk 1.0

Abnormal Normal Normal Abnormal Abnormal Abnormal a Prothrombin 20210G  A mutation.

2.8

2.4

22.8

Synergistic Effects of Thrombotic Risk Factors

Factor V Leiden Normal MTHFR Normal Thrombotic Risk 1.0

Abnormal Normal Abnormal Normal Abnormal Abnormal 2.8

1.8

16.5

Synergistic Effects of Thrombotic Risk Factors

Factor V Leiden Normal Abnormal Normal Factor II Normal Normal Normal a Normal Abnormal Abnormal Abnormal a Prothrombin 20210G  A mutation.

MTHFR Normal Normal Abnormal Normal Abnormal Thrombotic Risk 1.0

2.8

1.8

2.4

56.5

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.

Age of Thrombosis Onset in Patients with Common Thrombotic Risk Factors

Risk Factor Protein C deficiency Protein S deficiency Age of Thrombosis Onset Homozygote Heterozygote Birth Birth 26 years 31 years APCR* 28 years 63 years Antithrombin Embryonic death 33 years APCR, activated protein C resistance.

*May be responsible for venous thrombosis in individuals older than 55-60 years.

Relative Frequency of Various Thrombosis Types

Type of Thrombosis DVT and/or PE Patient with APCR 82% Patient with Protein C or S Deficiency 76% Abdominal Stroke/MI Other Total 6% 4% 8% 100% 11% 6% 7% 100% 20 Salomon O, et al.

Arterioscler Thromb Vasc Biol

. 1999;19:511-518.

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 21

Coagulation Cascade

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

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.

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 24

Whom to test?

How to test?

Thrombophilia Testing

Why test?

When to test?

What to test?

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 26

Antithrombin Deficiency

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

AT Heparin IIa AT IIa

Role of antithrombin • Direct inhibitor of IIa • Inhibits other factors leading to indirect inhibition of IIa formation

IIa Xa AT AT IXa XIa AT AT XIIa AT



Inactive Complexes

Therapeutic Monitoring with Antithrombin III

29 Normal antithrombin III Antithrombin III deficient

Factors Affecting Test Results

Heparin OAC or vitamin K deficiency No effect FVIII Circulating inhibitors OAC, oral anticoagulant.

Antithrombin Long-term therapy  results Protein C and S False  in results from clot-based assays (>1.0 IU/mL) No effect No effect Vitamin K dependent proteins; OAC will  levels High levels will falsely  from clot-based assays results May falsely  results from clot based assays 30

Factors Affecting Test Results

Pregnancy Oral contraceptives Hormone replacement therapy Liver disease, DIC, active thrombosis, recent surgery Antithrombin   or no change Protein C No effect No effect  or no change No effect Protein S       31

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 32

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 33

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 34

Functional Protein C Assay

2 patients missed by clot assay 80 70 60 50 40 30 20 10 0 0 20 8 patients missed by chromogenic assay 40 60 80 100

Chromogenic Assay

120 140 160 35

Protein C Testing Algorithm

Protein C Clot Assay Decreased (<70%) Protein C Antigen Borderline (70% - 75%) Repeat Clot Assay Normal or testing



No further Decreased (<70%) Borderline (70% - 75%) Increased (



70%) Chromogenic Assay

Normal adult reference range:  70% 36

Therapeutic Monitoring with Protein C

37 Normal protein C Protein C deficient

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 38

Role of protein C & S • Inhibition of IIa formation

Free PS PS IIa APC TM IIa TM Protein C PC Va or VIIIa

VIIIai or Vai Inactive Cofactors TM

Therapeutic Monitoring with Protein S

40 Normal protein S Protein S deficient

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.

41 Dahlback B.

J Thromb Haemost

. 2003;1:3-9.

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 42

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 43

Acquired APC Resistance

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

Incidence of APC Resistance in “Normals”

Ethnicity Caucasian African American Asian American Hispanic/Latino Native American Percent (%) 4 0.8

0.6

2.3

0.2

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.

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 47

Factor VIII Excess

Factor VIII Level >150% (>1.5 IU/mL) >200% (>2.0 IU/mL) Relative Risk 3.0

5.5

O’Donnell J, et al.

Thromb Haemost

. 1997;77:825-828.

Factor VIII Excess vs Other Thrombophilia Markers

Abnormality APCR Prothrombin mutation Hyperhomocysteinemia Protein C deficiency Protein S deficiency Antithrombin deficiency APCR/factor VIII mutation a History of thromobophilia.

Frequency (%) No History a With History a 14-20 20-50 4-8 5-10 1-4 1-4 1-3 25 10-20 NA 3-12 3-12 1-6 NA O’Donnell J, et al.

Thromb Haemost

. 1997;77:825-828.

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 50

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 51

Case Study #1: Laboratory Workup

Test Hemoglobin Platelet count WBC INR D-dimer APCR Antithrombin Protein C Protein S

Factor V Leiden

Follow-up duplex ultrasound Result 12.4 g/dL 177,000/  L 3,100/  L 2.2

<0.79  g/mL FEU 1.3 ratio 87% <41% 22%

Homozygous

Residual thrombus in iliac system and IVC 52

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%) 53

Case Study #2

(cont’d)

• 2 weeks later, concern raised about need for prophylactic anticoagulant • Lab results – C4 binding protein 212 mg/L (normal, 75-140) – Total protein S – Free protein S 81% 53% – Functional protein S 43% 54

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 S 33% – Protein C 162% 55

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.

Case Study #2

(cont’d)

Months postpartum 3 6 12 Protein S (%) 74 81 80

Protein S deficiency unlikely

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 58

Case Study #3

(cont’d)

Test PT a aPTT a Factor V Leiden Prothrombin gene MTHFR Lupus anticoagulant screen Protein C b Protein S b Antithrombin III b MRI, vena cava Result 10.4 sec. (normal, 10 – 12) 33.0 sec. (normal, <36 sec.) R506Q heterozygous G20210A heterozygous Heterozygous Negative 31% (normal, 62 – 124) 19% (normal, 58 – 112) 77% (normal, 62 – 114) Thrombus 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.

Case Study #3 Conclusion Long-term anticoagulation indicated

Schulman S, et al.

N Engl J Med

. 1997;336:393-398.

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 61

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 62

Case Study #4

(cont’d)

Lab Results at Admission

Test Platelet count Prothrombin time 9.9 secs (normal, 10.3 – 12.7) aPTT Fibrinogen Result 515,000/  L (normal, 147,000 – 390,000) 20.7 secs (normal, 23.1 – 34.2) 584 mg/dL (normal, 200 – 400) D-dimer 4000-8000 ng/mL (normal, <250) 63

Case Study #4

(cont’d)

Lab Results 30 d Post Discontinuation of Coumadin Therapy

Test D-dimer Result 500 – 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) 64

Case Study #4 Conclusions

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 65

Case Study #4 Conclusions

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

Case Study #5

• 19 year-old ballet dancer strains left calf and ankle • Recently recovered from infectious mononucleosis • Negative ultrasound duplex exam 67

Case Study #5

(cont’d)

Test D-dimer Fibrin monomer PT aPTT HAMA Rheumatoid factor Fibrinogen ANA Repeat ultrasound duplex (post 7 d) Result 497 µg/mL (normal, <0.79) +/ 11.4 sec (normal, 12) 31.7 sec (normal, <34) Negative Negative 232 mg % Negative No DVT 68

Case Study #5

(cont’d)

• 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 Medical Devices Agency 2002 Evaluation Report.

Heterophile antibodies may contribute to false positive D-dimer results in various methods

FEU, fibrinogen equivalent units; cut-off values are either those recommended by the manufacturer or 95% VTE sensitivity. Medical Devices Agency 2002 Evaluation Report.

Outline

• Thrombophilia (

impact on DVT and PE

Recommended work-ups

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 72 HTD, hereditary thrombophilia evaluation.

Cost-Effective Hypercoagulation Workup

Below Age 45

• APCR • Protein C • Protein S • Antithrombin • Prothrombin mutation • Homocysteine • 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.

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.

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 76

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 77

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.

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 79

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 80

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.

It is estimated that up to ½ of the North American population is heterozygous for a MTHFR mutation.

d. All of these 81