ANTITHROMBIN DEFICIENCY

PROTEIN C DEFICIENCY

PROTEIN S DEFICIENCY

FACTOR V LEIDEN (ACTIVATED PROTEIN C RESISTANCE)

PROTHROMBIN GENE MUTATION

HYPERHOMOCYSTEINEMIA

ANTIPHOSPHOLIPID ANTIBODIES

LABORATORY TESTING FOR THROMBOPHILIA

MANAGEMENT OF PATIENTS WITH THROMBOPHILIA

Link to thrombophilia PowerPoint presentation

References



Antithrombin deficiency ("Antithrombin III")
    1. Pathophysiology: deficiency of natural anticoagulant protein
      1. Antithrombin inhibits thrombin, Xa, IXa, XIa
    2. Thrombotic risk unknown, varies according to molecular defect
    3. Venous >> arterial thrombosis
    4. Inheritance: Autosomal dominant
      1. Homozygotes (with no plasma antithrombin) unknown (embryonic lethal?)
    5. Frequency: less than 5% of young patients with DVT/PE
    6. Molecular basis: genetically heterogeneous
      1. Type I: decreased production (gene deletion, nonsense mutation, etc)
      2. Type II: production of dysfunctional protein (missense mutation)
        1. Not all variants carry same thrombotic risk
    7. Laboratory diagnosis
      1. Approximately 50% of normal level of plasma antithrombin
      2. Most laboratories use functional (activity) assay
      3. Heparin therapy often decreases plasma antithrombin; order test at least a week after stopping heparin.

Protein C deficiency
    1. Pathophysiology: deficiency of natural anticoagulant protein
      1. Protein C inactivates factors Va, VIIIa
      2. Protein C is vitamin K dependent (levels fall with warfarin Rx)
    2. Thrombotic risk unknown, may vary according to molecular defect
    3. Venous >> arterial thrombosis
      1. Increased risk of warfarin-induced skin necrosis, due to further decrease in plasma protein C induced by warfarin (analogous to neonatal purpura fulminans)
    4. Inheritance: Autosomal dominant
      1. Homozygotes (completely lacking protein C) have neonatal purpura fulminans
    5. Frequency: up to 0.2% of population; about 5% of young patients with DVT/PE
    6. Molecular basis: genetically heterogeneous
      1. Type I: decreased production (gene deletion, nonsense mutation, etc)
      2. Type II: production of dysfunctional protein (missense mutation)
        1. Not known if all variants carry same thrombotic risk
    7. Laboratory diagnosis
      1. Approximately 50% of normal level of plasma protein C
      2. Assays
        1. Protein C antigen: low in type I deficiency, not in type II
        2. Protein C activity by chromogenic assay: low in type I, most type II
        3. Protein C activity by clotting assay: low in type I and all type II
        4. a and b, but not c, can be done in patients taking warfarin
      3. Warfarin therapy causes decreased protein C activity and antigen
        1. Preferable to measure protein C before giving warfarin
        2. If patient already on warfarin, can compare protein C level to another vitamin K-dependent protein (not reliable if warfarin dose changed within prior week)

Protein S deficiency
    1. Pathophysiology: deficiency of natural anticoagulant protein
      1. Protein S is cofactor for protein C, which inactivates Va, VIIIa
      2. Protein S is vitamin K-dependent (levels fall with warfarin Rx)
      3. Protein S exists in both bound and free forms in plasma; only free form is active
    2. Thrombotic risk unknown, may vary according to molecular defect
    3. Venous>>arterial thrombosis
    4. Inheritance: autosomal dominant
      1. Homozygotes (no plasma protein S) may have neonatal purpura fulminans
    5. Frequency in general population unknown: about 5% of young adults with DVT/PE
    6. Molecular basis: genetically heterogeneous
      1. Type I: decreased synthesis (deletions, nonsense mutations, etc), total plasma protein S antigen 50% of normal
      2. Type II: missense mutation of protein S or binding protein causing increased binding, decreased free protein S with normal total protein S antigen
      3. Type III: dysfunctional protein (missense mutations), normal antigen with decreased protein S activity
      4. The Heerlen mutation (Ser460Pro) is present in a majority of patients in whom a mutation can be identified
    7. Laboratory diagnosis
      1. Protein S antigen (total protein)
      2. Free protein S antigen
      3. Protein S activity (difficult assay, false positive result with APC resistance)
      4. Molecular testing for Heerlen mutation
      5. All assays affected by warfarin therapy (lower levels of protein S)
      6. Acquired variation of protein S in pregnancy, etc may complicate diagnosis; repeat testing recommended to exclude acquired deficiency

Factor V Leiden (Activated protein C resistance)
    1. Pathophysiology and molecular basis: missense mutation in factor V at activated protein C (APC) cleavage site, mutant protein less susceptible to cleavage/inactivation by APC
    2. Thrombotic risk:
      1. Heterozygotes have up to 7-fold increased lifetime risk of DVT/PE
      2. Homozygotes have approximately 40-50 fold increased lifetime risk of DVT/PE
      3. Not clearly associated with increased risk of recurrence in patients with single prior DVT/PE
    3. Venous>>arterial thrombosis
    4. Inheritance: autosomal dominant
      1. Homozygotes have further increased risk, but some homozygotes healthy
    5. Frequency: about 5% of those of European descent are heterozygous; about 40% of young patients with DVT/PE
    6. Molecular basis: genetically homogenous - single mutation responsible for almost all known cases
    7. APC resistance not due to factor V Leiden ("acquired APC resistance") can be detected in some patients, though not with APC resistance assay used here. Clinical significance not well understood.
    8. Laboratory diagnosis
      1. Functional assay (aPTT ratio without/with APC); current generation assay has high sensitivity and specificity
      2. DNA assay: indicated if borderline result of functional assay, or to confirm homozygote status.  Some laboratories offer dual PCR assay for factor V Leiden and Prothrombin G20210A mutation.  No need to do both functional and DNA assay in most cases.

Prothrombin G20210A gene mutation
    1. Pathophysiology and molecular basis: mutation in 3'-untranslated region of prothrombin gene associated with higher levels of plasma prothrombin
      1. No alteration of prothrombin structure or function
    2. Thrombotic risk up to 3-fold higher in heterozygotes; risk for homozygotes unknown
    3. Venous > arterial thrombosis
    4. Inheritance: autosomal dominant
    5. Frequency: about 1-2% of population heterozygous; 5-7% of young patients with DVT/PE
    6. Molecular basis: genetically homogeneous (single mutation)
    7. Laboratory diagnosis: DNA assay

Hyperhomocysteinemia
    1. Pathophysiology: elevated blood level of homocysteine causes endothelial damage
      1. Homocysteine is intermediary in methionine, cysteine metabolism (see figure)
      2. Inherited deficiencies of enzymes involved in its metabolism can cause elevated blood levels
        1. Cystathione -synthase (CBS)
        2. Methylenetetrahydrofolate reductase (MTHFR)
      3. Deficiencies of B-12, folate, pyridoxine (all involved in homocysteine metabolism) cause elevated level
      4. Homocysteine levels rise with age, after menopause, in renal failure, other acquired disorders
    2. Thrombotic risk varies with homocysteine level
      1. Homozygous CBS deficiency (homocystinuria) associated with childhood thrombosis and marked elevations of homocysteine
      2. Heterozygous CBS deficiency, MTHFR deficiency, acquired forms generally have lower risk
    3. Venous arterial thrombosis
    4. Frequency: heterozygous CBS deficiency in about 0.3% of population; thermolabile variant of MTHFR in up to 15% (not all have elevated serum homocysteine); about 10% of individuals with thrombotic disorders have elevated serum homocysteine
    5. Diagnosis
      1. Serum homocysteine level
        1. Methionine loading may enhance sensitivity, but impractical for screening
      2. DNA analysis for common MTHFR mutation [NOTE: 5-16% of the population are homozygous for this mutation; in the absence of hyperhomocysteinemia, it has not been shown to confer an increased risk of thrombosis]
    6. Folate/pyridoxine/B-12 administration can normalize homocysteine level, but to date no prospective trial has shown that this decreases thrombotic risk

Antiphospholipid antibodies: lupus anticoagulant (LAC) and anticardiolipin antibodies (ACL)
    1. Pathophysiology: mechanism(s) of thrombosis uncertain; possible explanations include
      1. Interference with natural anticoagulant function
        1. Protein C activation or function
        2. ß2-glycoprotein I (anticoagulant function uncertain)
        3. Annexin V (placental anticoagulant)
      2. Platelet activation
      3. Endothelial injury
    2. Thrombotic risk variable
      1. Many patients with these antibodies are asymptomatic
      2. Some have recurrent/lifethreatening thrombotic episodes
      3. History of prior thrombosis increases risk (high risk of recurrent DVT)
      4. Risk may be associated with antibodies to Beta2-glycoprotein I-phospholipid complex
      5. Retrospective analyses suggest some patients benefit from long-term anticoagulation, but thus far no prospective trials justify indefinite anticoagulation after a single thrombotic episode
    3. Clinical manifestations
      1. Venous or arterial thrombosis
        1. Individual patients tend to have one or the other, less often both
      2. Recurrent pregnancy loss
      3. Neurologic symptoms - TIAs, migraine-like headaches, retinal vascular occlusion
      4. Ulcerative skin lesions on legs/feet (arterial occlusion)
      5. Cardiac valve dysfunction ("Libman-Sacks endocarditis")
      6. Adrenal insufficiency (thrombotic infarction?)
    4. Frequency
      1. Incidence rises with age
        1. About 2% of healthy blood donors have ACL
        2. Up to 50% of healthy elderly have ACL
        3. 30-40% of patients with SLE have either ACL or LAC
      2. Association with certain infections (benign?), incl HIV
      3. Drug-induced (benign?): chlorpromazine, quinidine, procaineamide
    5. Diagnosis
      1. Lupus anticoagulant (LAC) assay
        1. Interference with phospholipid-dependent coagulation reactions in vitro causes long aPTT (no evidence that in vivo procoagulant reactions affected)
          1. Heparin interferes with the assay
          2. Must be distinguished from "true" clotting factor deficiencies or inhibitors
        2. Variable effect on clotting times - screening by several methods recommended (aPTT, dilute Russell viper venom time, kaolin clotting time, etc)
        3. Some antibodies also bind prothrombin/phospholipid complex, prolong PT/INR
          1. this may make monitoring warfarin therapy more difficult
      2. Anticardiolipin antibodies (ACL)
        1. IgG and possibly IgM clinically significant; significance of IgA ACL not proven
        2. Immunologic assay on serum, not affected by anticoagulant treatment
      3. "False positive" VDRL (less useful for screening)
      4. Anti-ß2-glycoprotein I antibody? (not widely available, but may correlate with thrombotic tendency)

Testing for hypercoagulable states

Testing is indicated if the results will affect the patient's treatment, or if knowledge of an inherited disorder might benefit the patient's relatives.  If a decision has already been made to keep patient on lifelong anticoagulation, diagnosing a hypercoagulable state is unlikely to change treatment, but may be useful if there are living first-degree relatives who might benefit from knowing of an inherited condition

    1. Indications (not absolute):
      1. Young (<50) patient with venous thrombosis
      2. Patient < 50 with arterial thrombosis (?)
      3. Venous thrombosis and no other identifiable risk factors
      4. Family history of venous thromboembolism or hypercoagulable state
      5. Recurrent venous thromboembolism, particularly DVTs in multiple sites
      6. Warfarin-induced skin necrosis
    2. What tests to do?
      1. If patient is not on any anticoagulants (in approximate order of priority):
          1. Factor V Leiden DNA test (no need to do functional test for APC resistance in most cases)
          2. Prothrombin gene mutation DNA test (may be combined with factor V Leiden DNA test)
          3. Lupus anticoagulant screen
          4. Anticardiolipin antibody test
          5. Protein C activity or antigen (antigen if patient on warfarin, or known family hx of type I protein C deficiency)
          6. Antithrombin activity
          7. Serum homocysteine
          8. Protein S:  Free protein S antigen  (total protein S optional)
      2. If patient is on heparin, wait to order antithrombin activity and lupus anticoagulant screen until off heparin at least 1-2 weeks
      3. If patient is taking warfarin but not heparin, can do all of above tests but remember that proteins C and S will be lower because of warfarin therapy
        1. Some protein C activity assays extremely sensitive to warfarin effect, unusable in patients on warfarin
        2. Total protein S preferable to free protein S in warfarin-treated patients
        3. Can correct for effect of warfarin by comparing levels of protein C and S to level of another vitamin K-dependent protein (usually factor X) if warfarin dose is stable
    3. Interpretation of results of tests
      1. APC resistance assay usually expressed as ratio (aPTT without APC to aPTT with APC). Current generation assay has high sensitivity and specificity for factor V Leiden mutation, but will not detect APC resistance due to other causes (so-called "acquired APC resistance").
        1. Ratio 1.4 - 1.9 usually indicates heterozygosity for factor V Leiden mutation
        2. Ratio 1.3 or less usually indicates homozygosity
        3. Consider DNA assay for borderline results or to confirm homozygosity.  This assay highly sensitive/specific.
      2. Antithrombin activity < 60% in patient who has not recently received heparin suggests antithrombin deficiency. (Lower values more suggestive)
        1. consider family study to confirm suspected deficiency
      3. Protein C activity or antigen < 60% in patient not on warfarin suggests protein C deficiency (lower values more suggestive)
        1. If activity << antigen, suggests "Type II" deficiency (dysfunctional protein)
        2. If patient on warfarin, protein C:factor X ratio 0.5 or less suggests protein C deficiency
        3. Consider family study to confirm suspected deficiency
      4. Protein S (total protein) < 60%, and/or free protein S < 40-50%, (patient not taking warfarin) suggest protein S deficiency (lower values more suggestive)
        1. Strongly consider family study to confirm suspected deficiency
        2. If family testing not possible, repeat measurements of total and free Protein S recommended to rule out transient acquired protein S deficiency
        3. Consider genetic testing for Heerlen mutation
        4. If patient on warfarin, total protein S:factor X ratio 0.5 or less suggests protein S deficiency
      5. Prothrombin gene mutation DNA test highly sensitive and specific for heterozygous/homozygous states
      6. If lupus anticoagulant test or anticardiolipin antibody result borderline or equivocal, consider repeating the test in 3-6 months. "Benign" antiphospholipid antibodies more likely to be transient.
    4. Diagnostic pitfalls
      1. Many conditions cause acquired deficiencies of proteins C and S, antithrombin; clinical implications of acquired deficiency unclear/different from inherited deficiency
        1. Liver disease, consumptive coagulopathy lower all three proteins
        2. Heparin treatment lowers antithrombin
        3. Warfarin treatment lowers proteins C, S
        4. Pregnancy/oral contraceptives lower protein S
        5. Children < 1 year old have lower protein C, S levels
      2. If inherited deficiency of one of these proteins is suspected, confirmation by family study (parents, siblings, children) is recommended whenever possible; this is particularly important if a condition that could cause acquired deficiency is present

Management of patients with hypercoagulable states
    1. Patients without symptoms or history of thrombosis
      1. Prophylactic anticoagulation not warranted in most instances
      2. Education about signs/symptoms of thromboembolism, need for prompt medical attention, etc.
      3. DVT prophylaxis in high risk situations (surgery, immobilization, etc)
      4. High index of suspicion for thrombotic events
      5. Counselling and discussion of risks and alternatives before prescribing oral contraceptives or hormone replacement therapy.  Strongly consider alternative therapies in patients with multiple risk factors or family histories of thromboembolism.
    2. Patients with history of thrombosis
      1. Factors favoring long-term anticoagulation
        1. History of recurrent thrombosis
        2. History of unprovoked ("idiopathic") venous thromboembolism
        3. History of pulmonary embolus (recurrence more likely to be fatal)
        4. Comorbidity due to prior thrombosis or other conditions - less ability to tolerate recurrence
        5. Antiphospholipid syndrome?
        6. Presence of multiple genetic risk factors, or additional acquired risk factors for recurrence
          1. Relative risk due to each risk factor may be multiplicative - e.g., APC resistance (7x) + oral contraceptive (5x) = 35 x risk if both factors present
        7. Persistently elevated D-dimer level after stopping anticoagulation?  (associated with higher risk of recurrence)
      2. Factors weighing against long-term anticoagulation
        1. Thrombosis associated with reversible risk factor
        2. Single thrombotic episode
        3. "Lower-risk" form of thrombophilia (heterozygous factor V Leiden, prothrombin gene mutation)
        4. Uncertainty of diagnosis of hypercoagulable state (e.g., borderline result, inability to confirm inherited condition via family study)
        5. Increased risk of anticoagulation (history of bleeding, poor compliance with meds, etc)
    3. Warfarin is drug of choice for long-term anticoagulation
      1. Target INR 2-3 appropriate for most patients
      2. Lower target (around 1.5) may be adequate in patients at high risk of bleeding
    4. Pregnant women with history of thrombosis:
      1. Begin adjusted dose heparin (target aPTT 1.5 x baseline) or low molecular weight heparin (e.g., enoxaparin 30 mg b.i.d.) in first or second trimester
      2. Switch to warfarin after deliver (target INR 2-3), continue x 6 weeks
    5. Pregnant women with history of fetal loss and antiphospholipid antibodies:
      1. No convincing evidence that immunosuppressive Rx beneficial, may be harmful
      2. No convincing evidence that aspirin beneficial
      3. Heparin or low molecular weight heparin treatment during pregnancy may be beneficial