Disorders of Fibrinogen

1. Disorders of Fibrinogen Ali Akalin Resident in Pathology UCHSC

2. 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).

3. 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)

4. D-Domain E-domain D-Domain Aα: Red Bβ: Blue γ: Green

5. 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.

6. 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.

7. 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 non-covalent 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).

8. Structure and Function

9. 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 cross-linking might presdispose to thromboemblic phenomena.

10. Structure and Function

12. 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.

13. 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 Classification

14. 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.

15. 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.

16. 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

18. 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.

19. 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

20. 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

21. 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

22. 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.

23. 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).

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

26. 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.

27. Prolonged TT 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 Prolonged TT and RT

28. Causes of Prolonged or Shortened Thrombine Time

29. 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.

30. 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.

31. 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.

32. 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.

33. 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, γ-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.

34. 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).

35. 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)

36. 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)

37. College of American Pathologists Consensus Conference XXXVI: Diagnostic Issues in Thrombophilia, Atlanta, Ga, November 9–11, 2001

38. Conclusions

39. Specific Recommendations

40. 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.

41. 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 α1- antitrypsin 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

44. 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 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.

45. 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.

46. Initial plasma with cryofibrinogen (left); initial serum without cryoglobulins (middle); plasma after streptokinase showing decreased cryofibrinogens (right).

47. 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.

48. References • Bérubé, C.http://www.uptodateonline.com/enterprise.asp?bhcp=1 • Pengh S.L., Schur P.H. http://www.uptodateonline.com/enterprise.asp?bhcp=1 • Hayes, T. Dysfibrinogenemia and thrombosis. Arch Pathol Lab Med. 2002 Nov;126(11):1387-90. • 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 • Amdo TD, Welker JA. An approach to the diagnosis and treatment of cryofibrinogenemia. Am J Med. 2004 Mar 1;116(5):332-7.