Thrombophilia Failure of the Inherent Anticoagulation Defense System

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Thrombophilia Failure of the Inherent
AnticoagulationDefense System

• Mervyn A. Sahud, M.D., A.B.I.M.-Hem.
• Medical Director, Coagulation, Quest Diagnostics
  Nichols Institute

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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

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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

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Deep Vein Thrombosis A Leg Up on Blood Clots
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Valve Cusp Thrombus (Autopsy Specimen)
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Thrombophilia
DVT, deep vein thrombosis. Rosendall FR. Lancet.
19993531167-1173. Anderson FA Jr, et al. Arch
Intern Med. 1991151933-938.
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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

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DVTWhats 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. 1999191026-1033.
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DVTWhats 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..
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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. 1998114(Suppl 5)531S-560S.
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Thrombophilia Is Often MultigenicMultiple risk
factors raises the risk of thrombosis
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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

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Clinical DVT Risk Factors

• Age gt60 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
patients age and surgery duration increases
risk also increases with presence of varicose
veins and obesity.
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Frequency of Biological Risk Factors
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Synergistic Effects of Thrombotic Risk Factors
aProthrombin 20210G?A mutation.
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Synergistic Effects of Thrombotic Risk Factors
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Synergistic Effects of Thrombotic Risk Factors
aProthrombin 20210G?A mutation.
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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.
2004424-438.
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Age of Thrombosis Onset in Patients with Common
Thrombotic Risk Factors
APCR, activated protein C resistance. May be
responsible for venous thrombosis in individuals
older than 55-60 years.
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Relative Frequency of Various Thrombosis Types
Salomon O, et al. Arterioscler Thromb Vasc Biol.
199919511-518.
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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

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Coagulation Cascade
Crowther MA, Kelton JG. Ann Intern Med.
2003138128-134.
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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.
2003138128-134.
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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

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Antithrombin Deficiency

• Genetic deficiency
• Rare
• lt1 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

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Antithrombin Deficiency

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

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AT
IIa

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

Heparin
? Inactive Complexes
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Therapeutic Monitoring with Antithrombin III
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Factors Affecting Test Results
OAC, oral anticoagulant.
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Factors Affecting Test Results
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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

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Protein C Deficiency

• Genetic deficiency
• Rare
• Heterozygotes
• lt1 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

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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 gt1.0 IU
• Direct thrombin inhibitors

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Functional Protein C Assay
2 patients missed by clot assay
8 patients missed by chromogenic assay
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Protein C Testing Algorithm
Normal adult reference range ?70
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Therapeutic Monitoring with Protein C
Normal protein C
Protein C deficient
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Protein S Deficiency

• Genetic deficiency
• Homozygous (rare)
• Purpura fulminans
• Heterozygous
• lt 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

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• Role of
• protein C S
• Inhibition of IIa formation

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Therapeutic Monitoring with Protein S
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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. 200313-9.
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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 Arg506? Gin Nucleotide
  CGA? CAA

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APC Resistance Genetic Mechanism

• Molecular function
• APC cleaves bond between arginine and glycine
• If Arg506 residue is mutated
• Bond not cleaved
• Factor Va not inactivated by APC
• Continued generation of thrombin and clot
  formation
• Thrombosis

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Acquired APC Resistance

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

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Incidence of APC Resistance in Normals
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Prothrombin (Factor II) 20210G?A Mutation
A, adenine G, guanine. Zoller B, Svensson PJ,
He X, et al. J Clin Invest. 1994942521-2524.
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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

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Factor VIII Excess
ODonnell J, et al. Thromb Haemost.
199777825-828.
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Factor VIII Excess vs Other Thrombophilia Markers
aHistory of thromobophilia.
ODonnell J, et al. Thromb Haemost.
199777825-828.
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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

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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

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Case Study 1 Laboratory Workup
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Case Study 2

• 37 y/o, G1P1 W/F attempting to get pregnant for
  the 2nd time
• 1st 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)

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Case Study 2 (contd)

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

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Case Study 2 (contd)

• 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

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Case Study 2 (contd)

• 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.

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Case Study 2 (contd)
Protein S deficiency unlikely
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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

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Case Study 3 (contd)
aTested prior to initiation of anticoagulant
therapy. bTested after initiation of coumadin
therapy INR 3.1.
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Case Study 3 Conclusion

• Long-term anticoagulation indicated

Schulman S, et al. N Engl J Med. 1997336393-398.
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Case Study 4

• Active 49 year-old white male experienced
  charley horse of right leg for gt6 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

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Case Study 4 (contd)

• 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

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Case Study 4 (contd) Lab Results at Admission
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Case Study 4 (contd) Lab Results 30 d Post
Discontinuation of Coumadin Therapy
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Case Study 4 Conclusions

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

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Case Study 4 Conclusions

• Elevated thrombin generation markers 14 days post
  coumadin therapy are turning point in this
  patients workup eventual diagnosis
• 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
• CT scan of chest reveals 3 cm mass, and
  bronchoscopic washing demonstrated malignant
  cells, ie, bronchogenic carcinoma

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Case Study 5

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

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Case Study 5 (contd)
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Case Study 5 (contd)

• Patient recovers, but D-dimer still elevated (241
  µg/mL)
• False-positive identified
• Demonstrated high heterophile antibody titer in
  patient serum
• Incubated serum w/ heterophile blocking antibody,
  then repeated D-dimer test
• Repeat D-dimer test normal

Medical Devices Agency 2002 Evaluation Report.
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Heterophile antibodies may contribute to
false-positive D-dimer results in various methods
Medical Devices Agency 2002 Evaluation Report.
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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

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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.
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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.
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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.
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Thrombophilia Quiz
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Thrombophilia Quiz

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

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Thrombophilia Quiz

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

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Thrombophilia Quiz

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

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Thrombophilia Quiz

• Of patients who experience venous thrombosis,
• deficiencies of protein C, protein S, and
• antithrombin are collectively present in
• Approximately 15 of patients
• Approximately 30 of patients
• Approximately 50 of patients
• Approximately 70 of patients

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Thrombophilia Quiz

• Patients with deficiency of protein C, protein S,
• or antithrombin usually present with
• Arterial thrombosis
• Venous thrombosis
• Recurrent arterial thromboembolic disease
• All of these

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Thrombophilia Quiz

• Which of the following statement(s) concerning
• methylene tetrahydrofolate reductase (MTHFR)
• is/are true?
• MTHFR and cystathionine ß synthase are the
  primary enzymes responsible for methionine
  metabolism.
• 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.
• All of these

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Thank You