Disorders of Fibrinogen
Ali Akalin
Resident in Pathology
UCHSC
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
(<150 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).
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
(Aα, Bβ, γ).
• 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)
D-Domain
E-domain
D-Domain
Aα: Red
Bβ: Blue
γ: Green
Structure
• Two major forms exist, separated from each other by ion
exchange chromatography: Fibrinogen 1 and 2.
• Fibrinogen 1: contains 2 γ chain (411 aa)
• Fibrinogen 2: contains one γ chain and one γ’ chain (427
aa), has a more anionic carboxyterminal sequence.
• Factor XIII (protransglutaminase, fibrinoligase) binds
specifically to γ’ chain of fibrinogen 2 (factor XIII is
carried by fibrinogen 2 in the plasma). Thrombin has been
shown to bind to the anionic γ’ extension of fibrin 2.
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.
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-α and B-β polypeptides, forming
fibrin monomer. Significant portion of abnormal fibrinogens
have mutations at this site.
• Fibrin polymerization: Initiated by complementary noncovalent binding of the D-domain (γ chain) of one molecule to
the central E-domain (A-α and B-β) of an adjacent fibrin
monomer (two molecule thick strand or protofibril).
Structure and Function
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 γ - γ, α-α, α- γ 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 crosslinking might presdispose to thromboemblic phenomena.
Structure and Function
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-α chain associated with
abnormal albumin binding.
• Defective fibrinolysis is a predisposition to thrombosis.
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
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 Aα, Bβ, or γ genes.
• Over 350 examples are reported (http://www.geht.org/databaseang/fibrinogen)
• Overall, ~ 55 % are silent, ~ 25 % manifests as bleeding
and ~ 20 % experience thrombosis with or without
bleeding.
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.
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
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.
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
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
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
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.
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).
Diagnosis
• Fibrinogen disorders are rare and diagnosis should be
considered after other causes of bleeding and/or
thrombosis have been ruled out.
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.
Prolonged TT and RT
• Prolonged TT
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Heparin
Heparin-like inhibitors
FDP
Hypofibrinogenemia
Excess fibrinogen
Hypoalbuminemia (<2 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
Causes of Prolonged or Shortened Thrombine Time
Diagnosis
• Confirmatory tests:
– Fibrinogen Activity–Antigen 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.
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.
Diagnosis
• Thrombine time 1:1 mixing study
– Indicated when the fibrinogen activity–antigen ratio is
within the reference range, yet, thrombin time is prolonged.
– Thrombine time is repeated on
• 1:1 mix of patient plasma and normal pooled plasma (Part 1)
• 1:1 mix of defibrinated patient plasma and normal pooled
plasma (part 2)
• The patient plasma is defibrinated by heating at 56°C for 10
minutes.
• The control for part 2 of the assay is a 1:1 mix of buffered
saline and pooled normal plasma.
• In acquired dysfibrinogenemia, the thrombin time 1:1 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.
Diagnosis
• Fibrinogen electrophoresis
– Based on the changes in molecular weight or isoelectric point of the
Aα, Bβ, or γ 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 (γ 375: Gly Arg), which causes a defect in high-affinity
calcium binding. In the presence of calcium, fibrinogen Osaka V
has a slower migrating γ chain compared to the normal γ chain on
1-dimensional electrophoresis. This difference in protein migration
rate allows detection of both the heterozygous and homozygous
states.
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, γglutamyltransferase, 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.
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).
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)
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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)
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)
College of American Pathologists
Consensus Conference XXXVI:
Diagnostic Issues in Thrombophilia,
Atlanta, Ga, November 9–11, 2001
Conclusions
Specific Recommendations
Cryofibrinogenemia
• Presence of abnormal cold-insoluble protein, composed of
fibrinogen, fibrin, fibronectin, in plasma (but not serum).
• Symptoms include cold sensitivity, Raynaud’s
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.
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 fibrinogenfibrin complexes.
• Other components of cryofibrinogen include α1antitrypsin and α2- 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
Diagnostic Criteria for
Cryofibrinogenemia
• Essential Criteria
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Compatible clinical presentation
Presence of cryofibrinogen in plasma
Absence of cryoglobulins
Absence secondary causes of cryofibrinogenemia (infection,
neoplasm)
• Supportive Criteria
– Elevation of serum α1-antitrypsin and α2- macroglobulin
– Angiogram with abrupt occlusion of small to medium sized
arteries.
– Typical skin biopsy findings (cryofibrinogen plugging vessels,
leukocytoclastic vasculitis, or dermal necrosis.
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.
Initial plasma with cryofibrinogen (left); initial serum
without cryoglobulins (middle); plasma after streptokinase
showing decreased cryofibrinogens (right).
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.
References
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Hayes, T. Dysfibrinogenemia and thrombosis. Arch Pathol Lab Med. 2002 Nov;126(11):138790.
Cunningham MT, Brandt JT, Laposata M, Olson JD. Laboratory diagnosis of
dysfibrinogenemia. Arch Pathol Lab Med. 2002 Apr;126(4):499-505.
Siebenlist K. R. http://academic.mu.edu/bisc/siebenlistk/research.html
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