Disorders of Fibrinogen

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Disorders of Fibrinogen

• Ali Akalin
• Resident in Pathology
• UCHSC

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Terms

• Dysfibrinogenemia fibrinogen with abnormal
  function.
• Hypofibrinogenemia Reduced amount of fibrinogen
  in the plasma.
• Hypodysfibrinogenemia inherited fibrinogens
  which are both functionally abnormal and reduced
  amounts in the plasma (lt150 mg/dL) as measured by
  immunologic methods.
• Afibrinogenemia absence of circulating
  fibrinogen in the plasma.
• Cryofibrinogenemia Fibrinogen in the plasma
  (but not serum) that precipitates on exposure to
  low temperatures (4 C).

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Structure

• 340 kD glycoprotein that circulates in plasma at
  a concentration of 200-400 mg/dL, with a half
  life of 4 days and a catobolic rate of 25 .
• Hexamer, consisting of three paired polypeptide
  chains (Aa, Bß, ?).
• Synthesized in hepatocytes under the control of
  three different genes located on chromosome 4q.
• Assembly takes place in the liver, carbohydrate
  side chains are added to the beta and gamma
  chains before it is secreted into plasma.
• It has a trinodular structure central E-domain
  (aminoterminal portions of the three
  polypeptides) and two D-domain (carboxyterminal
  portions)

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E-domain
D-Domain
D-Domain
Aa Red Bß Blue ? Green
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Structure

• Two major forms exist, separated from each other
  by ion exchange chromatography Fibrinogen 1 and
  2.
• Fibrinogen 1 contains 2 ? chain (411 aa)
• Fibrinogen 2 contains one ? chain and one ?
  chain (427 aa), has a more anionic
  carboxyterminal sequence.
• Factor XIII (protransglutaminase, fibrinoligase)
  binds specifically to ? chain of fibrinogen 2
  (factor XIII is carried by fibrinogen 2 in the
  plasma). Thrombin has been shown to bind to the
  anionic ? extension of fibrin 2.

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Functions

• Substrate for fibrin clot formation.
• Fibrin clot is a template for both thrombin
  binding and fibrinolytic system.
• Binds to platelets to support platelet
  aggregation.
• Has a role in wound healing.
• The balance between fibrin clot formation and
  fibrinolysis determines whether the clinical
  manifestations include bleeding, thrombosis,
  both, or neither.

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Functions

• Sites of important function Fibrinopeptide
  cleavage site (thrombin binding site), Factor
  XIIIa binding site, t-PA binding site, alpha-2
  antiplasmin binding site and platelet binding
  site.
• Fibrinopeptide cleavage Thrombin binds to
  fibrinogen and cleaves fibrinopeptides A (FPA)
  and B (FPB) from the aminoterminal portion of A-a
  and B-ß polypeptides, forming fibrin monomer.
  Significant portion of abnormal fibrinogens have
  mutations at this site.
• Fibrin polymerization Initiated by complementary
  non-covalent binding of the D-domain (? chain) of
  one molecule to the central E-domain (A-a and
  B-ß) of an adjacent fibrin monomer (two molecule
  thick strand or protofibril).

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Structure and Function
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Functions

• Fibrin polymerization Followed by longitudinal
  growth (D-D contact between adjacent fibrin
  monomers) and branching to form the final fibrin
  network. Mutations affecting this binding sites
  may delay fibrin polymerization and produce
  heterogenous clinical manifestations.
• Fibrin cross-linking Formation of covalent bonds
  between D domains of fibrin fibers by factor
  XIIIa, activated by thrombin. Involves
  interaction between ? - ?, a-a, a- ? chains.
• Cross-linking stabilizes the clot and makes it
  resistant to disruption. Defective cross-linking
  may be responsible for delayed wound healing,
  wound dehiscence. Increased cross-linking might
  presdispose to thromboemblic phenomena.

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Structure and Function
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Functions

• Fibrinolysis Fibrin has binding sites for
  plasminogen, t-PA, and alpha-2- plasmin
  inhibitor. Mutations at these sites may result in
  defective plasmin generation and reduced
  fibrinolysis. In addition, resistance to the
  action of plasmin can result from mutations in
  the C-terminus of the A-a chain associated with
  abnormal albumin binding.
• Defective fibrinolysis is a predisposition to
  thrombosis.

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Classification

• Quantitative Abnormalities
• Congenital
• Afibrinogenemia (uncommon, autosomal recessive)
• Hypofibrinogenemia
• Acquired
• Hypofibrinogenemia (consumptive coagulapathies,
  DIC)
• Hyperfibrinogenemia (inflammation, neoplasia)

• Qualitative abnormalities
• Congenital
• Dysfibrinogenemia
• Hypodysfibrinogenemia
• Acquired
• Liver disease
• Malignancies,
• Antifibrinogen antibodies

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Inherited dysfibrinogenemia

• Named after the city where the first patient was
  identified and evaluated. Roman numerals are
  added after the city name when there are several
  dysfibrinogens from the same city (eg, Carcas
  V).
• Result from the mutations in the coding region of
  the fibrinogen Aa, Bß, or ? genes.
• Over 350 examples are reported (http//www.geht.or
  g/databaseang/fibrinogen)
• Overall, 55 are silent, 25 manifests as
  bleeding and 20 experience thrombosis with
  or without bleeding.

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Inherited dysfibrinogenemia

• Thrombotic variants
• Estimated to represent 0.8 of patients with a
  history of venous thrombosis.
• Estimated that thrombosis is seen in 10-20 of
  patients with dysfibrinogenemia.
• Usually presents with venous thrombosis of lower
  extremities although arterial and/or venous
  thrombosis have also been reported.
• A highly convincing association between
  thrombophilia and and dysfibrinogenemia could be
  established for five families (Caracus V, Melun,
  Naples, Paris V, Vlissinger/Franckfurt IV). High
  rate of pregnancy-related complications
  (postpartum thrombosis, spontaneuos abortions)
  were seeen. Fibrinogen concentrations were
  normal or low.

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Inherited dysfibrinogenemia

• Suggested mechanisms for thrombosis
• Increased clot formation
• Defective thrombin binding by the abnormal
  fibrinogen, resulting in excess circulating
  thrombin that may stimulate platelet activiation.
• Impaired clot dissolution
• Resistance to lysis by plasmin
• Abnormal binding of tissue type plasminogen
  activator

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Inherited dysfibrinogenemia

• Bleeding variants
• clinical presentation is heterogenous, and may
  include epistaxis, menorrhagia, easy
  bruisability, soft tissue hemorrhage,
  postoperative bleeding, antepartum and postpartum
  bleeding, as well as hematomas and hemarthrosis.
  
• fibrinogen levels less than 50 to 100 mg/dL have
  a higher frequency of bleeding complications.
• Results from mutations impairing fibrinopeptide
  release or fibrin monomer polymerization.

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Inherited dysfibrinogenemia

• Other disease manifestations
• Hereditary renal amyloidosis
• Deposition of a mutant fibrinogen alpha chain in
  the kidney
• Autosomal dominant inheritance
• Presents with renal failure
• Hepatic storage disease
• Abnormal fibrinogen remain in the endoplamic
  reticulum in the hepatocytes
• Delayed wound healing and/or wound dehiscence

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Inherited afibrinogenemia

• A rare condition,
• Autosomal recessive inheritance
• The vast majority results from truncating
  mutations in the fibrinogen alpha chain
• (virtually) complete lack of circulating
  fibrinogen
• Bleeding manifestation range from mild to
  catastrophic
• Excessive bleeding and early miscarriages in
  pregnant women
• Fatal umbilical cord bleeding in the neonate

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Acquired dysfibrinogenemia

• Production of abnormal fibrinogen secondary to
• Liver disease such as cirrhosis, metastatic
  hepatocellualr carcinoma, acute and chronic
  hepatitis.
• Characterized by an inceased content of sialic
  acid residues (an increase in negative charge)
  and delayed fibrin polymerization.
• Removal of sialic acid from the abnormal
  fibrinogen normalizes the thrombin time and
  corrects the polymerization defect.
• Cleavage of A and B fibrinopeptides and
  cross-linking of fibrin by factor XIII are normal

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Acquired dysfibrinogenemia

• Whether the abnormal fibrinogen seen in liver
  disease is associated with an increased bleeding
  risk is unclear because most of these individuals
  also have associated other conditions such as
  decreased synthesis of coagulation factors,
  varices, thrombocytopenia, dysfunctional
  platelets.
• Other conditions and proposed mechanisms
• Autoantibodies inhibiting specific functions of
  fibrinogens, such as fibrinopeptide release,
  fibrin monomer polymerization, fibrin
  cross-linking SLE, ulcerative colitis, multiple
  myeloma, therapy with isoniazid, use of fibrin
  glue (sealant).
• Unknown mechanisms renal carcinoma,
  mithramycine, isotretinoin therapy, biliary
  obstruction, digital gangrene.
• Usually presents with bleeding, vary rarely
  thrombosis.

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Acquired hypofibrinogenemia

• Result from decreased hepatic synthesis and/or
  increased turnover of fibrinogen
• Causes include DIC, hepatic failure,
  decompensated cirrhosis, drugs that impair
  hepatic synthesis of fibrinogen (L-asparaginase,
  valproic acid), etc.
• Fibrinogen is an acute phase reactant, with
  levels increasing as part of the inflammatory
  response. Plasma fibrinogen level 200 mg/dl may
  represent a significant decrease in a patient
  whose baseline should be 800 mg/dl. (Sepsis,
  underlying malignancy, inflammation).

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Diagnosis

• Fibrinogen disorders are rare and diagnosis
  should be considered after other causes of
  bleeding and/or thrombosis have been ruled out.

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Diagnosis

• Fibrinogen disorders are rare and diagnosis
  should be considered after other causes of
  bleeding and/or thrombosis have been ruled out.
• Initial screening tests thrombin time (TT) and
  reptilase time (RT) fibrinogen activity and
  antigen.
• Afibrinogenemia Prolonged TT, RT, virtually
  absent fibrinogen antigen and activity (clottable
  antigen).
• Dysfibrinogenemia Prolonged TT, RT, normal or
  increased fibrinogen antigen, normal or decreased
  clottable fibrinogen (activity).
• Fibrinogen Oslo I and Valhalla show normal or
  shortened TT.

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Prolonged TT and RT

• Prolonged TT
• Heparin
• Heparin-like inhibitors
• FDP
• Hypofibrinogenemia
• Excess fibrinogen
• Hypoalbuminemia (lt2 g/dl)
• Paraproteins
• Excess protamine
• Primary systemic amyloidosis
• Acquired antibodies to bovien thrombin
• Acquired dysfibrinogenemia

• Prolonged RT
• Cleaves only fibrinopeptide A from fibrinogen
  molecule
• The same conditions except
• for heparin
• Not as sensitive as TT for
• detection of dysfibrinogenemia

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Causes of Prolonged or Shortened Thrombine Time
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Diagnosis

• Confirmatory tests
• Fibrinogen ActivityAntigen Ratio
• The Clauss method (Fibrinogen activity) measures
  the rate of clot formation after adding a high
  concentration of thrombin to citrated plasma.
• Prothrombine time-based method for fibrinogen
  activity (not validated)
• Fibrinogen antigen concenration can be determined
  by immunologic (ELISA, radial immunodiffusion),
  precipitation (heat, sulphite), thrombin clotting
  methods.
• FDP can cause falsely elevated fibrinogen antigen
  values when using sulphite precipitation,
  thrombin clotting, and some immunologic methods.
• Falsely decreased fibrinogen antigen values can
  occur with the heat precipitation method in the
  presence of fibrin degradation products,
  cryoglobulins, and high plasma viscosity.

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Preanalytic and analytic issues on fibrinogen
activity-antigen ratio

• Activity and antigen assays should be performed
  on the same sample because fibrinogen levels can
  fluctuate from day to day.
• Activity-antigen ratio should be interpreted
  against a method-specific reference range because
  fibrinogen antigen and activity levels are method
  dependent.
• These variables can be controlled if a single
  laboratory performs the activity and antigen
  assays on the same sample and then reports the
  ratio result along with a method-specific
  reference range.

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Diagnosis

• Thrombine time 11 mixing study
• Indicated when the fibrinogen activityantigen
  ratio is within the reference range, yet,
  thrombin time is prolonged.
• Thrombine time is repeated on
• 11 mix of patient plasma and normal pooled
  plasma (Part 1)
• 11 mix of defibrinated patient plasma and normal
  pooled plasma (part 2)
• The patient plasma is defibrinated by heating at
  56C for 10 minutes.
• The control for part 2 of the assay is a 11 mix
  of buffered saline and pooled normal plasma.
• In acquired dysfibrinogenemia, the thrombin time
  11 mix is prolonged in part 1(dysfibrinogen
  inhibits fibrin clot assembly of normal
  fibrinogen), and normal (corrected) in part 2
  (inhibitory dysfibrinogen has been removed by
  heat precipitation).
• The sensitivity, specificity, and predictive
  values of this test are unknown.

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Diagnosis

• Fibrinogen electrophoresis
• Based on the changes in molecular weight or
  isoelectric point of the Aa, Bß, or ? chain as a
  result of mutations
• One-dimensional electrophoresis separates
  polypeptides based on apparent molecular weight.
  Two-dimensional electrophoresis separates
  polypeptides based on apparent molecular weight
  in the first dimension and isoelectric point in
  the second dimension.
• These analyses can be performed either on
  purified fibrinogen or on plasma if the
  electrophoresis is followed by immunoblotting
  with fibrinogen-specific antibodies.
• An example in which electrophoresis has been used
  is fibrinogen Osaka V (? 375 Gly Arg), which
  causes a defect in high-affinity calcium binding.
  In the presence of calcium, fibrinogen Osaka V
  has a slower migrating ? chain compared to the
  normal ? chain on 1-dimensional electrophoresis.
  This difference in protein migration rate allows
  detection of both the heterozygous and homozygous
  states.

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Distinguishing Acquired and Inherited Forms

• Acquired dysfibrinogenemia is typically diagnosed
  by demonstrating
• abnormal laboratory tests of hepatocellular or
  cholestatic function (ie, aspartate
  aminotransferase, alanine aminotransferase,
  alkaline phosphatase, ?-glutamyltransferase,
  direct bilirubin) and
• normal thrombin time and/or reptilase time in
  family members.
• The diagnosis can be further substantiated by
  repeat testing after the condition resolves to
  show that fibrinogen function returns to normal.
• The possibility of an inherited defect should be
  considered if fibrinogen dysfunction persists
  after resolution of the hepatobiliary disease.

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Distinguishing Acquired and Inherited Forms

• Dysfibrinogenemia is most likely inherited, if
  liver function tests are normal.
• The inherited nature of the disease can be
  confirmed by demonstrating a similar abnormality
  in a family member, presentation during neonatal
  period or infancy, and detecting a protein
  abnormality
• by fibrinogen electrophoresis, or
• by identifying a mutation within 1 of the 3
  fibrinogen genes by molecular genetic analysis
• fibrinopeptide release (Fibrinopeptide A is
  tested by radioimmunoassay on human plasma after
  the contact in vitro with the biomaterial or
  artificial device or on the plasma from patients
  implanted with devices in contact with
  circulating blood).

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Treatment

• Most patients with dysfibrinogenemia is
  asymptomatic and do not require treatment.
• Patients with known history of previous bleeding
  should receive fibrinogen replacement therapy
  prior to surgery and during pregnancy (as early
  as 4-5 weeks) with the goal to increase the
  fibrinogen concentration to 50-100 mg/dl. (during
  labor, the target is 150-200 mg/dl)
• Cryoprecipitate
• Fresh frozen plasma
• Virally inactivated human fibrinogen
  concentrations (In Europe)
• Local treatment with antifibrinolytic agents
  (aminocaproic acid, tranexamic acid for oral or
  dental surgery)

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Treatment

• Patients with thrombotic complications should
  receive anticoagulation.
• The optimal duration of anticoagulation is
  unknown (the benefit of anticoagulation should be
  weighed against a potentially higher risk of
  bleeding).
• Patient education concerning thrombotic risk
  factors (surgery, pregnancy, oral contraceptives,
  immobilization)

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College of American Pathologists Consensus
Conference XXXVI Diagnostic Issues in
Thrombophilia, Atlanta, Ga, November 911, 2001
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Conclusions
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Specific Recommendations
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Cryofibrinogenemia

• Presence of abnormal cold-insoluble protein,
  composed of fibrinogen, fibrin, fibronectin, in
  plasma (but not serum).
• Symptoms include cold sensitivity, Raynauds
  phenomenon, purpura, urticaria, skin ulcerations,
  gangrene, arterial or venous thrombosis.
• Seen most frequently in autoimmune disorders,
  malignancy, infections, thrombotic disorders.
• May be accompanied by DIC.

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Pathogenesis of Cryofibrinogenemia

• Largely unknown.
• Major components of cryofibrinogen are
  fibrinogen, fibrin and fibronectin
  (cold-insoluble globulin)
• Fibronectin binds to fibrinogen and fibrin and
  acts as a nucleus for the cold-induced
  precipitation of fibrinogen-fibrin complexes.
• Other components of cryofibrinogen include a1-
  antitrypsin and a2- macroglobulin (both can
  inhibit plasmin activity and thereby contribute
  to thrombus formation.
• Fibronectin may also interact with circulating
  immunoglobulins or immune complexes.
• Molecular changes in rare familial forms of CF
  remain unknown

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Diagnostic Criteria for Cryofibrinogenemia

• Essential Criteria
• Compatible clinical presentation
• Presence of cryofibrinogen in plasma
• Absence of cryoglobulins
• Absence secondary causes of cryofibrinogenemia
  (infection, neoplasm)
• Supportive Criteria
• Elevation of serum a1-antitrypsin and a2-
  macroglobulin
• Angiogram with abrupt occlusion of small to
  medium sized arteries.
• Typical skin biopsy findings (cryofibrinogen
  plugging vessels, leukocytoclastic vasculitis, or
  dermal necrosis.

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Measurement of Cryofibrinogen

• Plasma should be collected at 37 C in a EDTA,
  citrate, or oxalate tube.
• Simultaneously, a serum sample should be prepared
  by collecting blood in a tube free of
  anticoagulant to rule out cryoglobulins.
• False negative collection below 37 C
  (autoabsorption of cryofibrinogens by the red
  blood cells)
• False positive Heparin tube, therapeutic heparin
  (complex with fibrinogen, fibrin and fibronectin)
  (heparin precipitable fraction)
• Plasma is placed in Winthrobe tube and
  refrigerated for 72 hours. Then, centrifuged and
  cryocrit is read as fraction.
• May be quantified by chromatography,
  immunodiffusion and/or electrophoresis.

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Initial plasma with cryofibrinogen (left)
initial serum without cryoglobulins (middle)
plasma after streptokinase showing decreased
cryofibrinogens (right).
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Treatment of Cryofibrinogenemia

• Avoidance of cold-exposure and other
  environmental triggers of symptoms
• Cessation of smoking and avoidance of
  sympathomimetic agents (diet pills,
  decongestants, caffeine)
• Anabolic steroid Stanozolol
• Fibrinolytic therapy, streptokinase,
  streptodornase, urokinase
• Immunosuppressive and cytotoxic agents
  prednisone, chlorambucil, azatioprine
• Plasmapheresis
• For secondary cryofibrinogenemia treatment of
  underlying disease.

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References

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  se.asp?bhcp1
• Pengh S.L., Schur P.H.
• http//www.uptodateonline.com/enterprise.asp?bhcp
  1
• Hayes, T. Dysfibrinogenemia and thrombosis. Arch
  Pathol Lab Med. 2002 Nov126(11)1387-90.
• Cunningham MT, Brandt JT, Laposata M, Olson JD.
  Laboratory diagnosis of dysfibrinogenemia. Arch
  Pathol Lab Med. 2002 Apr126(4)499-505.
• Siebenlist K. R. http//academic.mu.edu/bisc/siebe
  nlistk/research.html
• Amdo TD, Welker JA. An approach to the diagnosis
  and treatment of cryofibrinogenemia. Am J Med.
  2004 Mar 1116(5)332-7.