1 Comment


Williams Hematology




Conversion and Assembly


Thrombin Binding to Fibrin(Ogen)
Afibrinogenemia and Hypofibrinogenemia

Definition, History, and Clinical Features

Laboratory Features

Therapy, Course, and Prognosis

Definition and History

Etiology, Pathogenesis, Clinical and Laboratory Features
Chapter References

There are two general overlapping classes of hereditary fibrinogen disorders, afibrinogenemia and dysfibrinogenemia. Afibrinogenemia is inherited as an autosomal recessive trait and is associated with a variable degree of bleeding ranging from minimal to catastrophic. The underlying mechanism for this condition is not well established but is probably due to failure of hepatic fibrinogen synthesis and/or secretion. A related form of this disorder is manifested as hypofibrinogenemia associated with hepatic storage disease, in which liver secretion of a mutant fibrinogen is impaired. Hereditary dysfibrinogenemia is characterized by the biosynthesis of a structurally abnormal fibrinogen molecule that exhibits altered functional properties. Although most dysfibrinogenemics are asymptomatic, the disorder is commonly associated with bleeding, thrombophilia, or with fibrinogen-related organ pathology such as amyloidosis. Dysfibrinogenemic disorders can be grouped into functionally distinguishable subcategories that are exemplified in each of the phases of the fibrinogen-fibrin conversion: (1) Abnormal fibrinopeptide release, or a defective EA or EB site; (2) defects in D domains involving the Da site, Db site, D:D site, gXL site, or the aC domain. Defects in these regions are usually associated with distinctive functional abnormalities, and bleeding or thrombophilia are found in almost all categories. Some aC domain defects are associated with hereditary renal amyloidosis, in which an abnormal fragment of the Aa chain is deposited in the kidneys. Hypodysfibrinogenemia is a subcategory characterized by low circulating levels of abnormal fibrinogen levels, a category that overlaps with hypofibrinogenemia manifested as hepatic storage disease. The pathophysiological basis for hypodysfibrinogenemia includes hypercatabolism, reduced synthesis, or impaired secretion, but in most cases neither the genetic basis nor the structural abnormality is known.

Acronyms and abbreviations that appear in this chapter include: ADP, adenosine 5′ diphosphate; HRA, hereditary renal amyloidosis.

Fibrinogen is a 340-kD plasma protein that circulates at a concentration of 1.5 to 3.5 mg/ml. Each fibrinogen molecule is about 45 nm in length and comprises a symmetrical disulfide-bridged dimer consisting of two outer D domains and a central E domain that are joined through coiled-coil regions1,2 (Fig. 124-1). Electron microscopy has confirmed its tridomainal structure and additionally shows a twofold axis of symmetry perpendicular to the long axis.3,4 and 5 Each half-molecule consists of two sets of three different polypeptide chains, Aa, Bb, and g, that are joined in their amino terminal regions by disulfide bridges to form the E domain.1,6,7 and 8 The Aa chain consists of 610, the Bb chain of 461, and the major form of the g chain, gA, 411 residues1. A minor g chain variant termed g’, amounting to about 8 percent of the total,9 is comprised of 427 residues and has a unique acidic amino acid sequence10 after position 407. Carbohydrate side chain groups are attached to the Bb and g chains and are linked through N-acetylglucosamine to Asp52 of each g chain, and Asp364 of each Bb chain.11

FIGURE 124-1 Diagram of fibrinogen and fibrin showing the major domains and the intermolecular association sites that participate in fibrin polymerization and cross-linking.
Enzymatic conversion of fibrinogen to fibrin by thrombin results in release of fibrinopeptides A (FPA) and B (FPB) and exposure of EA and EB polymerization sites, respectively. Structural features within the D domains of this model include two separate self-association sites, gXL and D:D. Other constitutive association sites are Da, which interacts with an available EA site in fibrin (i.e., the fibrin D:E interaction), and Db, which interacts with an EB site in fibrin. gXL overlaps the factor XIIIa cross-linking site in the C-terminal region of gA or g’ chains, which are known g chain variants. Their C-terminal sequences (nonidentical residues in bold) and the residues used by XIIIa for cross-linking are shown. Only gA chains contain the platelet fibrinogen receptor aIIbb3 binding sequence. D:D site interaction promotes end-to-end alignment of fibrin or fibrinogen molecules in assembling fibrils. The Aa chain cleavage site that produces plasma fraction I-9 fibrinogen is indicated. The aC domain in the C-terminal region of the Aa chain emerges from the D domain and is shown dissociating from its noncovalent association with the fibrin E domain as a result of thrombin cleavage of FPB. The two locations of thrombin binding in fibrin and tPA binding at fibrin-specific epitopes in fibrin are also shown, as is the endothelial cell binding site (b15-42) that becomes exposed by cleavage of FPB. (Reproduced with permission from ref. 2.)

Fibrinogen is synthesized by hepatocytes12,13 and chain synthesis is under the coordinated control of three separate genes located on chromosome 4.14,15,16 and 17 Subsequent to hepatic assembly of the constituent polypeptide chains and the addition of carbohydrate side chains, the mature molecule is secreted into the circulation, where it manifests a half-life of 4 days and a fractional catabolic rate of 25 percent per day.18,19 In addition to the fibrinogen in plasma, blood contains an intracellular fibrinogen pool that is stored within platelet a-granules. Both megakaryocytes and platelets are capable of internalizing plasma fibrinogen via the fibrinogen glycoprotein(GP)IIb/IIIa (aIIbb3) receptor20,21 and 22 (see Chap. 111). This absorptive process is specifically dependent upon GPIIb/IIIa binding to the C-terminal platelet recognition sequence that is present on gA chains, but absent from gchains. Thus, internalized platelet fibrinogen molecules contain only gA chains.22,23,24 and 25 This phenomenon is especially interesting in relation to the abnormal g chains in fibrinogen Paris I (gParis I), which are not found in platelets,26 a subject that is discussed more fully in a later section.
Fibrin(ogen) participates in numerous physiological processes, most of which are listed below. Fibrinogen when converted by thrombin into fibrin, forms an insoluble fibrin clot; it supports platelet aggregation, and it binds to vascular endothelial and other cells. Fibrinogen also binds to plasma or tissue matrix proteins such as fibronectin, to peptide growth factors, and it serves as a carrier protein for factor XIII. Fibrin provides a template for the assembly and activation of the fibrinolytic system, has binding sites for tissue matrix components such as glycosaminoglycans, and possesses nonsubstrate thrombin binding sites. Both fibrinogen and fibrin serve as substrates for factor XIIIa and other transglutaminases, which catalyze covalent cross-linking.
Conversion of fibrinogen into fibrin has three distinct phases: (1) enzymatic cleavage by thrombin to produce fibrin; (2) fibrin self-assembly to an organized polymeric structure; (3) covalent cross-linking of fibrin by factor XIIIa. In the first phase of conversion to fibrin, cleavage of fibrinogen at Aa16R-17G* and later Bb14R-15G results in release of two fibrinopeptides A (FPA) and two fibrinopeptides B (FPB),27,28 and 29 and exposure of EA and EB polymerization sites, respectively. One portion of the EA site is located at the amino terminal end of the fibrin a chain comprising the amino terminal Aa17-20 GPRV sequence.30,31 Another portion of this site is in the amino terminal region of the fibrinogen Bb chain, specifically in the b15-42 sequence.32,33,34,35 and 36 The EA site in fibrin interacts with a constitutive complementary association site, Da, in the D domain of another molecule (the so-called D:E interaction) to initiate and accelerate the fibrin assembly process.29,37,38 and 39 This site is situated in a segment of the D domain encompassed by g337-379.40,41 and 42
The initial assembly of a fibrin clot involves Da:EA associations that result in formation of double-stranded fibrils in which fibrin molecules become aligned in an end-to-middle domain staggered overlapping arrangement43,44,45 and 46 (Fig. 124-2). Subsequently fibrils undergo branching and lateral fibril associations that result in fiber networks.47,48 Two types of fibril branching occur, the first of which, called a tetramolecular junction, consists of two fibrils that converge to form a four-stranded fibril.47,49 The second, termed a trimolecular junction, consists of three fibrils which converge at a junction comprised of three D:E interacting molecules to form a symmetrical three-armed structure. Progressive lateral fibril associations result in formation of thick fiber bundles and large fiber branches.

FIGURE 124-2 Schematic diagram of fibrin assembly and cross-linking.
Fibrin assembly begins after FPA release with noncovalent D:E interactions between the EA and Da sites (dotted lines) to form end-to-middle staggered overlapping double-stranded fibrils (upper). Fibrils also branch and undergo lateral associations to form wider fibrils and fibers [Inset, Critical point dried fibril matrix containing trimolecular (arrows) and tetramolecular (arrowheads) junctions; bar, 100 nm]. After cleavage of FPB (lower), aC domains become available for self-association with other aC domains, thereby promoting lateral fibril association and fiber assembly [Inset, negatively contrasted fibrin fiber showing 22.5 nm periodicity; bar, 100 nm]. Activation of factor XIII to XIIIa by thrombin results in introduction of e-(g-glutamyl) lysine cross-links among C-terminal gXL sites (thick lines between D domains) mainly between the strands of each fibril to form g dimers. g trimers and g tetramers form by interfibril g chain cross-linking. (Reproduced with permission from ref. 2.)

FPB (Bb1-14) release occurs more slowly than release of FPA27,28 and 29 and exposes an independent polymerization site, EB,50,51 beginning with the b15-18 sequence, GHRP.30,31 FPB cleavage is accelerated by fibrin polymerization whereas fibrinopeptide cleavage at the Aa site is independent of fibrin polymerization, per se.52,53 and 54 EB is utilized through interactions with a complementary binding pocket, Db, located in the b chain segment of the D domain encompassed by b397-432.55,56 The interaction is evidently not required for lateral fibril and fiber association but contributes to this process through cooperative interactions resulting from alignment of D domains in the fibrin polymer.50,51
In addition to the Da:EA site interactions that guide fibrin assembly, there are other distinct D domain self-association sites termed ‘gXL’ and ‘D:D’, respectively,57,58 and 59 that contribute to this process. Interactions between D:D sites, which are situated at the distal ends of D domains, promote end-to-end alignment of fibrin molecules in assembling polymers.57,58 The interface for this site lies between g275R and g300S,42 but other nearby g chain residues contribute to the site, as evidenced by impaired D:D interactions in dysfibrinogenemic molecules such as fibrinogen Kurashiki I (gG268E).60
The so-called gXL assembly site is situated in the C-terminal region of each g chain. It contains the factor XIIIa crosslinking sequence between g398/399 Gln and g406 Lys and overlaps the platelet fibrinogen receptor aIIbb3 binding site at g400-411 of gA chains.61,62 The g’ variant chain also contains this cross-linking peptide sequence and therefore undergoes factor-XIIIa-mediated cross-linking normally.63 g’ chains, however, lack a functional aIIbb3 platelet binding site and instead they contain an acidic C-terminal sequence beginning at g’408 and ending at g’427.10 By binding to factor XIII B subunits via its unique g’ sequence, fibrinogen serves as a carrier protein for factor XIII in plasma.64 Fibrin g’ chains also bind to thrombin with high affinity.65
Still another association site that plays a role in fibrin assembly and other functions is located in the C-terminal region of the Aa chain and is commonly referred to as the ‘aC’ domain. This segment of the molecule originates in the D domain at residue 111 and ends at residue 610.1 Fibrin formed from circulating fibrinogen “catabolite” molecules (e.g., fraction I-9) lacking a C-terminal portion of the aC domain manifests a prolonged thrombin time and reduced turbidity development and produces thinner fibrin fibers.66,67 In fibrinogen molecules, the aC domains tend to be noncovalently tethered at the E domain4,68,69 but become dissociated from it as a result of FPB cleavage.68,69 This event evidently makes the aC domain available for noncovalent interaction with other aC domains, a process that promotes lateral fibril associations and fibrin network assembly. A considerable number of symptomatic dysfibrinogenemias are associated with irregularities in this region of the molecule.
Besides its role in mediating fibrin polymerization, the b chain of fibrin participates in cell-matrix interactions. Exposure of the b15-42 sequence by release of FPB promotes platelet spreading,70 fibroblast proliferation,71 endothelial cell spreading, proliferation and capillary tube formation,71,72 and 73 and release of von Willebrand factor.74,75 Endothelial cell binding is a heparin- or proteolglycan-dependent process,76,77 that correlates with exposure of a heparin-binding domain in b15-42.78
In the presence of factor XIIIa, fibrin undergoes intermolecular covalent cross-linking by virtue of forming e-(g-glutamyl)lysine isopeptide bonds.79,80 The resulting cross-linked clot exhibits almost perfect elastic behavior and becomes more rigid and resistant to deformation.81 Cross-linking of g chains first involves formation of g dimers,82 which occur as reciprocal bridges between lysine at position 406 of one g chain and glutamine at position 398 or 399 of another.83,84 and 85 Slower cross-linking among a chains creates a chain oligomers and polymers.86 Cross-linking also occurs between a chains and g chains,47,87 and other plasma proteins, notably a2-plasmin inhibitor and fibronectin, become cross-linked to a chains.88,89,90 and 91 Oligomeric forms of crosslinked g chains also occur, namely g trimers and g tetramers.47,87 They form more slowly than g dimers or even a polymers,47,92 and contribute to development of resistance to fibrinolysis.92 Because there is a single donor lysine residue at g406,83,85 trimeric and tetrameric cross-linked structures form by utilization or reutilization of that same lysine 406 residue. Whereas g dimers usually lie in a transverse position between the strands of each fibril,57,59,93,94 and 95 trimeric and tetrameric structures form through interfibrillar interactions subsequent to intrafibrillar g chain dimerization (see Fig. 124-2).
Thrombin binds to its substrate, fibrinogen, at N-terminal sites on Aa and Bb chains through an anion-binding fibrinogen recognition site in thrombin termed exosite I.96,97 Substrate binding leads to proteolytic cleavage and release of FPA and FPB, respectively. The binding site for FPA cleavage is contained within residues 1 to 51 of the N-terminal Aa chain, whereas the binding site for FPB cleavage is at least in part in the N-terminal Bb chain.52,65,98 Other evidence for a thrombin binding sequence in the Bb chain comes from studies of two dysfibrinogenemic Bb chain mutant molecules, New York I (des Bb9-72)32,99 and Naples (BbA68T),100,101 both of which showed impaired thrombin binding. These dysfibrinogenemias are discussed in subsequent sections.
There are two classes of nonsubstrate thrombin binding sites in fibrin, one of high affinity and the other of low affinity,65,102 and each class has a distinct location.65 The low-affinity binding sites are in the fibrin E domain and represent, at least in part, a residual aspect of fibrinogen substrate recognition. The a27-50 sequence in fibrin contributes to the low-affinity site.103,104 The high-affinity site is located exclusively on g’ chains of fibrin(ogen)65 and probably competes for thrombin binding at thrombin exosite-dependent sites in other proteins or cells (e.g., the platelet/endothelial cell thrombin receptor) or even at fibrinogen substrate sites.
Congenital afibrinogenemia is a rare disorder that has been described in more than 150 families.105,106 In the classical disorder the disease is inherited as an autosomal recessive trait. Consanguinity is common. Typically, low levels of plasma fibrinogen in both parents supports a recessive inheritance pattern. Clinical expression in terms of hemmorhagic manifestations ranges widely from minimal to catastrophic.105,106 Umbilical cord bleeding may be the first manifestation, and it can be the cause of death in newborns. Later in life, such problems as gum bleeding, epistaxis, menorrhagia, gastrointestinal hemorrhage, muscle hemorrhage, spontaneous abortions, and hemarthrosis can present, but the leading cause of hemorrhagic death is intracranial bleeding. Classical afibrinogenemia appears to be due to a biosynthetic disorder rather than a consumptive defect, since injection of autologous fibrinogen into affected individuals exhibits normal plasma survival.107,108 and 109 A recent report on a Swiss family with congenital afibrinogenemia corroborates that idea109a in that homozygous deletion of the majority of the fibrinogen Aaa chain gene results in absence of the capacity to synthesize functional fibrinogen.
There is an hereditary form of hypofibrinogenemia that is associated with hepatic storage disease,110,111,112 and 113 and including that disorder in this section rather than under the category of hypodysfibrinogenemia is somewhat arbitrary. The condition may not be very rare, and evidence of abnormal fibrinogen storage in the form of PAS-negative inclusion bodies in the cisternae of the endoplasmic reticulum was found in 9 of 700 liver biopsies.113 This type of abnormal hepatic storage of fibrinogen fulfills the criteria for an endoplasmic reticulum storage disease such as occurs in a1-antitrypsin deficiency in which an abnormal a1-antitrypsin variant is retained within the endoplasmic reticulum of the liver instead of being secreted into the circulation.114,115 and 116
The circulating fibrinogen level, measured by a functional assay, in the kindred reported by Callea and coworkers112 was low in the propositus (0.2–0.4 mg/ml), in two of her four sons, in her sister, two of her sister’s daughters, and in four of her sister’s grandchildren (0.4–0.85 mg/ml). Immunoreactive plasma fibrinogen in the propositus was higher (1.0 mg/ml) suggesting that the circulating fibrinogen was dysfunctional. George and coworkers117 recently showed by DNA sequencing that there is a heterozygous mutation in the g chain (gG284R) of fibrinogen Brescia that evidently impairs its secretion by the liver. None of the mutant form of fibrinogen Brescia is found in plasma, but the secreted fibrinogen is hypersialated, suggesting an explanation for the presence of dysfunctional plasma fibrinogen.
All tests based upon the appearance of a fibrin clot are abnormal in afibrinogenemic and hypofibrinogenemic subjects. These include the whole blood clotting time, partial thromboplastin time, prothrombin time, and thrombin and reptilase times. These abnormalities are correctable by the addition of normal plasma or purified fibrinogen. The diagnosis is established by immunological measurements of fibrinogen concentration.118,119 The platelet fibrinogen content in most cases is negligible.118 Related coagulation abnormalities include a prolonged bleeding time and abnormal platelet aggregation, especially with ADP. These abnormalities can be corrected by infusion of plasma or fibrinogen concentrates.119,120 and 121
Patients with afibrinogenemia or hypofibrinogenemia may require replacement therapy to control bleeding episodes or in preparation for surgery. Many patients receive fibrinogen replacement in the form of cryoprecipitate, a plasma-derived fibrinogen concentrate that contains approximately 300 mg fibrinogen per unit. An afibrinogenemic adult with a plasma volume of 3 liters will require 12 units of cryoprecipitate to increase the fibrinogen level to an adequate hemostatic level of 1 mg/ml.122 Since the fractional catabolic rate of fibrinogen is 25 percent per day, patients should receive one-third of the initial loading dose daily for as long as fibrinogen level elevation is desired. Cryoprecipitate is also used during pregnancy to prevent spontaneous abortion and to assist in carrying pregnancies to term.123 Development of antifibrinogen antibodies has been reported,124 and some patients undergoing replacement therapy have experienced thrombosis.125,126 and 127
Inherited dysfibrinogenemia is characterized by the biosynthesis of a structurally abnormal fibrinogen molecule that exhibits altered functional properties. More than 260 cases of congenital dysfibrinogenemia were reported by 1994,128 and now that number is in excess of 300. Approximately 25 percent of the reported cases of dysfibrinogenemia have had a history of bleeding, and in about 20 percent of the families there has been a tendency toward thrombosis (thrombophilia),128 the remainder being clinically asymptomatic in these regards. In some families with dysfibrinogenemia there is associated fibrinogen-related organ pathology such as amyloidosis or hepatic storage disease.
The abnormality in blood is most commonly but not always discovered by a prolonged thrombin-mediated clotting time. With some exceptions, the various mutants described carry the name of the city of origin of the patient. The main focus for discussion of dysfibrinogenemia in this chapter is placed on abnormalities for which a structure/function correlation can be made, whose dysfunction can be assigned to a particular domain or region in the molecule, or which exhibit interesting or unique clinical or pathophysiological features. Some dysfibrinogens are discussed under more than one subtopic.
Fibrinogen abnormalities are usually reflected in one or more phases of the fibrinogen-fibrin conversion and fibrin assembly process including: (1) impaired release of fibrinopeptides, (2) defects in fibrin polymerization, (3) defective cross-linking by factor XIIIa. Other important abnormalities involve other aspects of fibrin(ogen) function or metabolism such as defective intrahepatic assembly, storage and/or secretion, catabolism, abnormal deposition in tissues, defective assembly of the fibrinolytic system, abnormal interactions with platelets, endothelial cells, or calcium binding.
Fibrinogen Detroit was the first abnormal fibrinogen in which a specific amino acid substitution was identified (AaR19S)129; this mutation is at the fibrin EA polymerization site (i.e., GPRV) and results in impaired fibrin polymerization and a bleeding tendency. Amino acid substitutions at this site in other kindreds are associated with bleeding in some (Munich I, AaR19N128; Mannheim I, AaR19G128) and with thrombosis in others (Aarhus, AaR19G128; Kumamoto, AaR19G130). As pointed out in the section on thrombophilia, there may be coexisiting as yet undetermined genetic or environmental risk factors in some patients that account for such seemingly paradoxical clinical manifestations. Substitution of Aa18 Pro with Leu (Kyoto II)131 is also associated with a bleeding tendency due to a defective EA site. In fibrinogen Canterbury (AaV20D),132 the EA polymerization site is missing as well as the entire FPA sequence (i.e., Aa1 to 19). This arises because the AaV20D mutation creates a cleavage site at R19 (RGPRD) for the intracellular enzyme furin, and the mutant Aa chain is cleaved at that site prior to secretion to create a sequence beginning with Aa20D. A mild bleeding tendency exists with this situation. Fibrinogen Nijmegen (BbR44C)133 exhibits abnormal polymerization (consistent with an abnormal EA polymerization site) plus abnormal tPA-induced plasminogen activation and binding134 but does not exhibit impaired fibrinopeptide release like fibrinogen Naples (BbA68T).100,101,135
Thrombin binds to fibrinogen and cleaves each Aa chain at 16R/17G to release FPA. The most common mutation is at Aa16, with Arg replacement leading to delayed (R®H) or absent (R®C) FPA release. Most of these subjects, especially those who are heterozygous, have no bleeding tendencies. Some patients with a bleeding tendency are homozygous for the defect (Metz136; Giessen I137; Bicêtre I128) or have other demonstrable abnormalities, such as an abnormal von Willebrand factor138 or impaired cross-linking.139 Substitution of V for G at Aa17 (Bremen I140) results in delayed FPA release as well as a modest impairment of fibrin monomer polymerization, indicating that there is also a defect at the EA polymerization site resulting from the change of GPRV to VPRV. This defect is associated with a bleeding tendency and delayed wound healing.
Dysfibrinogenemias with a structural alteration in Aa 7-12 segment of the FPA sequence (Lille, Mitaka II, Rouen I128) exhibit defective substrate binding with thrombin and impaired FPA release. There is a bleeding tendency only with Mitaka II. Delayed FPA release is also associated with dysfibrinogens manifesting a structural defect in the N-terminal region of the Bb chain, including fibrinogen New York I (Bb9-72 deletion32), fibrinogen Naples (BbA68T100,101,135), and dysfibrinogenemias involving substitutions at the FPB cleavage site, BbR14C (Table 124-1) or BbG15C (Ise128; Fukuoka II141).


FPB release results in exposure of the EB polymerization site,50,51 but does not occur in mutant Bb chains with Cys substitutions at the FPB cleavage site, BbR14C (Table 124-1) and in fibrinogen New York I, which exhibits deletion of Bb9-72, corresponding to exon 2 of the Bb chain gene.32 Impaired FPB release also occurs in dysfibrinogenemias having a mutation at the FPB cleavage site BbG15C (Ise128; Fukuoka II141) and in fibrinogen Naples I (BbA68T100,101,135). Fibrinogen Nijmegen (BbR44C133) does not exhibit impaired fibrinopeptide release but rather abnormal polymerization (consistent with an abnormal EA polymerization site). Further details on Bb chain defective dysfibrinogenemic families exhibiting thromboembolic disorders, such as fibrinogens New York I, Naples I, and Nijmegen, are contained in the section on thrombophilia.
Fibrin assembly initially involves Da:EA site interactions that drive the formation of staggered overlapping end-to-middle molecular associations resulting in double-stranded fibrils. In addition, there are two other distinct association sites in each D domain, termed ‘gXL’ and ‘D:D’, respectively. In the following sections we consider each of these sites in terms of known congenital abnormalities of fibrinogen. These structural changes have been reviewed.142,143
Da Site D domain mutations are associated with defective fibrin polymerization, and many are located in a region of the Da site, covering a rather large stretch of the g chain. These include fibrinogen Matsumoto I (gD364H144) and fibrinogen Melun I (gD364V145); the latter is associated with thrombophilia. Other dysfibrinogenemias that induce functional changes at the Da site include: Kyoto III (gD330Y146), Milano I (gD330V147), Nagoya I (gQ329R148), Osaka V (gR375G149), and Bern I (gN337K150). Fibrinogen Milano VII (gS358C-albumin) molecules, which are disulfide-linked to albumin151 displayed a marked polymerization defect. It is not clear how this abnormality relates to abnormal polymerization, since the location itself does not appear to contribute to Da site function, and removal of albumin did not normalize the defect. None of these last-mentioned dysfibrinogens have thrombotic or bleeding manifestations.
Db Site To date, no dysfibrinogenemias have been described involving this region of the D domain. The substitution in fibrinogen Pontoise (BbA335T152) results in a new glycosylation site in the b chain portion of its D domain, and the molecule exhibits defective polymerization. The defect may be related to a steric or a charge effect on the polymerization process, but it is not likely to be directly related to an abnormality in the GHRP binding pocket, per se.
D:D Site The interface for the D:D site lies between g275R and g300S, with g280T contacting g275R at the D:D interface.42 The nearby g chain residues also contribute to the site, as evidenced by functional impairment of D:D interactions in molecules such as fibrinogen Kurashiki I (gG268E60,153).
Fibrinogen Tokyo II58,154 is one of many reported clinically asymptomatic gR275C-substituted dysfibrinogens and was the first to be characterized in terms of its D:D site defect. Several others have Arg substituted by His and one by Ser (Table 124-1). Tokyo II fibrin displayed normal D:E associations and normal g chain cross-linking. Factor XIIIa-cross-linked fibrinogen polymers were defective in that otherwise linear double-stranded fibrils were disorganized due to failure of normal end-to-end molecular associations. For the same reason, Tokyo II fibrin showed increased fiber branching.58 The same type of abnormal fibrin branching has been shown for fibrinogen Haifa I (gR275H155), a case in which thrombophilia was prominent. Fibrinogen Banks Penninsula (gY280C156) which is associated with mild bleeding, shows the same polymerization defect as described above for Tokyo II fibrinogen, because the Cys substitution at g280 disrupts the normal D:D contact with g275R.
Fibrinogen Baltimore I (gG292V128) may represent another example of defective D:D site function, although its characteristics are not identical to those of fibrinogen Tokyo II. The Baltimore I patient had a history of recurrent thrombosis, pulmonary embolism, and mild bleeding.157 Electron microscopy of fibrin networks showed thinner, relatively more branched fibers. Cross-linking of g chains was evidently normal, as seems to be characteristic of purely D:D defective molecules, but a-polymer formation was also delayed, though correctable. There are other g chain mutations that may also cause abnormal D:D site function. These include mutations at g308 N to I (Baltimore III158) or N to K (Kyoto I159; Bicêtre II160; Matsumoto II161), and glycosylation at g308N due to a gM310T mutation (Asahi I162).
gXL Site The gXL association site contains the factor XIIIa cross-linking sequence and overlaps the platelet fibrinogen receptor aIIbb3 binding site. There are no reported changes specifically involving this region of the molecule, although markedly impaired to absent g chain cross-linking at this site has been documented in fibrinogen Asahi I162 and in fibrinogen Paris I (see below).163 Fibrinogen Asahi I has a carbohydrate group incorporated at g308N that may sterically interfere with cross-linking at the gXL site, although it does not interfere with factor-XIIIa-mediated incorporation of amine donors at g398Q. A dysfibrinogen with the same g310 Met to Thr substitution as Asahi I (Frankfurt VII164) has been reported to display abnormal ADP-induced platelet aggregation, probably by sterically hindering the C-terminal platelet-binding sequence at the gXL site. Fibrinogen Vlissingen (gdel 319N,320D165) also displayed abnormal platelet aggregation,164 evidently resulting from disruption of its calcium binding site. These molecules are also discussed in the section on thrombophilia.
The fibrinogen Paris I abnormality is characterized by markedly impaired fibrin polymerization and clot retraction166 but is not associated with either clinical bleeding or thrombophilia. The propositus, however, displayed surgical wound dehiscence. The defect (g350/insert/gG351S) involves a point mutation in intron 8 that results in insertion of a 15 amino acid sequence after g350 and substitution of S for G at g351.167 The gParis I chains have a normal C-terminal sequence beyond g351,167,168 yet despite this, the gXL site does not participate in factor-XIIIa-mediated g chain cross-linking nor can amine donors be incorporated into gParis I chains.163 Furthermore, gParis I chains manifest defective ADP-induced platelet aggregation,168 and gParis I-containing molecules are not incorporated into platelets.26 This suggests that marked conformational changes have occurred in the Paris I D domain that make the C-terminal gA chain sequence unavailable for any function attributable to the gXL site.
Fibrinogen Marburg169 is a homozygous hypodysfibrinogenemia lacking amino acid residues Aa461-610. This abnormality is covered in the section on thrombophilia, as is another Aa chain truncation mutant, fibrinogen Milano III.170,171 Fibrinogen Dusart (AaR554C-albu-min172,173,174,175,176 and 177) is also covered in that section. Fibrinogen Otago217 a hypodysfibrinogenemic Aa chain truncation mutant, is discusssed in the section on hypodysfibrinogenemia.
Fibrinogen Caracas II (AaS434N-glycosylated178) is characterized by impaired fibrin gelation that is related to N-glycosolation. The fibrin ultrastructure shows thinner, less well-ordered fibers.179 The carbohydrate group may interfere with fibrin polymerization in the same way as the bulky albumin group does in the case of fibrinogen Dusart, or possibly the problem may be related to repulsive forces generated by its negative charge. In contrast to several other thrombophilic dysfibrinogenemias that are located in this region of the molecule, this dysfibrinogenemia is asymptomatic.
Fibrinogen Lima (AaR141S,Aa139N-glycosylation180) is a homozygous dysfibrinogenemia discovered in a family without a history of bleeding or thrombosis. The glycosolation site is located in the proximal portion of the D domain and evidently does not interefere with D:E or D:D interactions, nor with fibrin-dependent plasminogen activation by tPA. It most likely interferes with lateral fibril associations due to the bulky carbohydrate group or to the repulsive negative charge on the carbohydrate group.
Amyloidosis Hereditary amyloidosis is an autosomal dominant trait characterized by progressive extracellular deposition of an amyloid protein in various organs. Hereditary forms of amyloidosis are associated with mutants of several plasma proteins: transerythretin, gelsolin, apolipoprotein A1, lysozyme, and, most recently, fibrinogen.181 Transerythretin-associated amyloidoses, the most common form, have peripheral neuropathy and cardiomyopathy as major clinical manifestations, but patients have also developed gastrointestinal dysfunction, nephropathy, sexual impotence, vitreous opacification, and cerebral hemorrhage. In amyloidosis related to apolipoprotein A1, lysozyme, and fibrinogen, renal amyloid deposition has been the major finding. In 1993, amyloid fibrils obtained from the kidney of a patient with hereditary renal amyloidosis (HRA) were found to contain an extractable fragment of the fibrinogen Aa chain covering residues 499 through 580 and containing an Aa R554L mutation.182 The propositus in this family developed nephrotic syndrome and azotemia at the age of 36, requiring cadaveric renal transplantation at the age of 40. A second renal transplantation for the same condition was done at the age of 50. After death from septicemia, an autopsy revealed the same amyloid deposition in the transplanted kidney as had been found in the original and first transplanted kidneys. A sibling had died at the age of 28 with nephrotic syndrome, and the propositus’ son died at the age of 24 with azotemia and renal amyloidosis. It is important to note that substitution of Cys for Arg at position 554 in the Aa chain, namely fibrinogens Dusart and Chapel Hill III (AaR554C), does not result in amyloidosis such as occurs in the R554L mutation but instead causes a different functional impairment and clinical picture (see “Thrombophilia”).
Other mutations in the Aa chain gene in families with HRA have been reported. Four kindreds were described with an AaE526V mutation with HRA manifested clinically in the fifth to seventh decades of life.183,184 In one study184 plasma fibrinogen levels were found to be within the normal range as were the thrombin times, and the distribution of normal and mutant Aa chains was equal, indicating a heterozygous defect.
Another kindred with clinical recognition of HRA in the fourth to fifth decade of life and low plasma fibrinogen levels has been described.185 DNA sequence analysis showed a nucleotide deletion causing a frame shift after position Aa524 that produced an abnormal sequence and Aa chain termination after position 547. The abnormal peptide sequence was found in amyloid deposits in the kidneys but was not present in plasma fibrinogen. A similar frame shift mutation in the Aa chain gene in another kindred with the onset of kidney disease as early as the second decade resulted in renal deposition of a 49-residue hybrid peptide whose N-terminal 23 amino acids were identical to Aa499 to 521.186 The remaining 26 C-terminal amino acids in the peptide resulting from a frame shift at codon 522 had a unique sequence that terminated at position 547.
Therapy and Course Renal transplantation is at best a temporizing solution to the problem of renal failure in fibrinogen-related HRA, since amyloid deposits accumulate in transplanted kidneys, just as in the original kidneys, causing eventual destruction of the allograft.182,186 Liver transplantation would seem to be preferable to renal transplantation, but has not yet been attempted to our knowledge.
Inherited hypodysfibrinogenemia includes cases of dysfibrinogenemia in which plasma fibrinogen levels are less than 1.5 mg/ml, as measured immunochemically or by other physical methods. Clottable protein measurements are not a reliable indicator of fibrinogen concentration, since functionally abnormal molecules are not always incorporated into a clot. Eighteen cases of hypodysfibrinogenemia have been described.128,217,218 In the first described case, fibrinogen Parma,187 the patient had a severe bleeding disorder that was corrected by infusing normal fibrinogen, which itself seemed to exhibit normal plasma survival. Fibrinogens Philadelphia I188 and Bethesda III189 possess the same properties, including normal fibrinopeptide release, defective fibrin polymerization, increased catabolic rate of the autologous protein, but normal survival of infused fibrinogen. These findings suggest that hypercatabolism is caused by an intrinsic molecular defect of the mutant fibrinogen that accounts for the hypofibrinogenemia. In the case of fibrinogen Chapel Hill I,190 homologous and autologous fibrinogen turnover were normal or nearly normal, suggesting a different mechanism for the hypofibrinogenemia than in Philadelphia I or Bethesda III. In fibrinogen Baltimore II,191 FPB release was delayed, and autologous fibrinogen synthesis was reduced, but homologous and autologous catabolism were normal. A better understanding of the cause for this abnormality awaits details on the exact structural anomaly.
Fibrinogen Frankfurt I (gA357T) is a heterozygously transmitted dysfibrinogen associated with hypofibrinogenemia, bleeding, impaired fibrin polymerization, and decreased support of platelet aggregation.217 Fibrinogen Otago, a homozygous hypodysfibrinogenemia (AaR268Q/frameshift/271Pstop), is associated with mild bleeding, multiple spontaneous first trimester abortions, and abnormal scar formation,218 a behavior consistent with that of patients with afibrinogenemia. Cryoprecipitate infusion during one pregnancy in the propositus resulted in a normal birth. The data suggest that there is decreased hepatic assembly and/or secretion of molecules with truncated Aa chains. Fibrinogens Marburg I169 and Malmöe I193 are discussed in the section on thrombophilia.
Management of these conditions with plasma or fibrinogen concentrates is generally the same as that described for afibrinogenemia.
Dysfibrinogenemia associated with thrombosis occurs in about 20 percent of dysfibrinogenemic families.128,194 Among such families, there has been a high incidence of thrombotic problems related to pregnancy, in particular spontaneous abortion and post partum thromboembolism. Most of the thrombophilic familes that have been identified to date are listed in Table 124-1, and those with defined or potential mechanisms for the thrombophilic condition are discussed below.
Fibrinogens Nijmegen and Ijmuiden Fibrinogen Nijmegen is associated with abnormal tPA-induced plasminogen activation but normal fibrinopeptide release.133,134 Abnormal high-molecular-weight fibrinogen complexes and albumin-linked fibrinogen molecules were also found. Fibrinogen Ijmuiden was similar to Nijmegen in these respects, and in addition showed abnormal FPB release.133 The polymerization abnormality that both fibrinogens show may cause abnormal tPA-mediated plasminogen activation, which in turn may contribute to the thrombophilia.
Fibrinogen Cedar Rapids Of the numerous dysfibrinogenemic families which are characterized by an amino acid substitution at position 275R of the normal g chain, either R to C, R to H, or R to S (Table 124-1), all but five are asymptomatic. Of the families reporting thrombophilia, there are two with gR275C, Bologna I194 and Cedar Rapids195 and three with gR275H substitutions. The gR275H Haifa I patient196 presented at the age of 30 with arterial occlusions, Barcelona III with venous thrombosis,197 and Bergamo II with pulmonary embolism associated with pregnancy.198 Clearly there is no simple direct linkage between this type of structural abnormality and thromboembolism, and other contributory conditions must be sought.
Several genetic risk factors are important in the pathogenesis of venous thromboembolism, and these include abnormalities of protein C, protein S, antithrombin III, plasminogen, prothrombin, fibrinogen, plasma homocysteine levels, and most recently, factor V.199,200 The aforementioned defect was manifested as resistance to activated protein C due to a mutation in the factor V gene resulting in substitution of Gln for Arg at position 506, commonly referred to as factor V Leiden.201,202,203 and 204
Fibrinogen Cedar Rapids is a heterozygous dysfibrinogenemia in which thromboembolic disease was associated with pregnancy in three second-generation family members.195 Each affected member of the family was heterozygous for the factor V Leiden defect, whereas the parents and their siblings manifested either the factor V Leiden defect (paternal) or fibrinogen Cedar Rapids (maternal) and were asymptomatic. These observations suggest that coexpression of factor V Leiden and fibrinogen Cedar Rapids is associated with thrombophilia. Another possible example of such concurrent conditions is to be found in fibrinogen Giessen IV (gD318G194). The exact mechanism by which two coexisting conditions contribute to the thrombophilic state is not clear, but such conditions may ultimately be found in certain families with thrombophilia, especially those with variable clinical expression of thromboembolic disease among families or individuals (e.g., fibrinogens Kumamoto, Melun I, Kaiserslautern). This subject is covered in detail in Chap. 127.
Defects in the Calcium Binding Site of the g Chain A high-affinity calcium binding site in the g chain is located between residues 311 and 336, and involves residues at g318D, g320D, g322F, g324G, and g328E.165,205 The calcium site is important for the structural integrity of the D domain and provides a protective effect against plasmin cleavage of the g chain.206 Fibrinogen Vlissingen (gdel319N,320D165) was identified in a woman who had been hospitalized because of pulmonary embolism. Fibrin polymerization was delayed both in the presence or absence of calcium. The deletion of g chain residues 319 and 320 resulted in defective calcium binding and probable allosterically mediated dysfunction of the Da polymerization site and possibly the D:D site as well. Abnormal ADP-induced platelet aggregation has also been reported for this abnormal fibrinogen,164 implying impaired function at the gXL site. The functional relationship between the calcium-binding defect and thrombophilia, as in many situations, is not clear. Similarly, the fibrinogen Giessen IV (gD318G)194 defect results in defective calcium binding and polymerization and was reported in an 18-year-old woman with recurrent venous thrombosis as well as mild bleeding, who also was heterozygous for the factor V Leiden defect.
Truncation Mutations Involving the Aa Chain Fibrinogen Marburg169 is a homozygous hypodysfibrinogenemia lacking amino acid residues Aa461-610 due to a stop codon at position 461 of the Aa chain. Its unpaired cysteine residue at position 442 forms a disulfide bridge with albumin and other substances.192 The Marburg patient suffered from severe uterine bleeding after caesarian section, pelvic vein thrombosis, and recurrent thromboembolic disease. Another homozygous truncation mutant, fibrinogen Milano III,170,171 is also associated with recurrent venous thrombosis. Unlike fibrinogen Marburg, this mutant fibrinogen circulates at normal levels. The abnormality is associated with defective lateral fibril association and comes about by a single base insertion after Aa451, resulting in a premature stop at 453S. Because of premature Aa chain termination, there is an unpaired Cys residue at position 442, and like Marburg, this is associated with covalant linkage of albumin to the Aa chain. The thrombophilia may be causally related to formation of fine fibrin clots, which reportedly are resistant to fibrinolysis.177,207,208
The Dusart Syndrome Fibrinogen Dusart (AaR554C-albumin172,173,174,175,176 and 177) manifests marked thrombophilia and has been studied extensively. The defect is the same as that in fibrinogen Chapel Hill III,128,209 which also presented with thrombophilia. Dusart fibrinogen displays reduced plasminogen binding,173,176 impaired fibrin-dependent tPA activation,173 and abnormal fibrin polymerization and clot structure172,174,175,177 that is normalized by removing the affected region of the molecule.175 In addition to these abnormalities, fibrinogen Dusart molecules show an enhanced self-association tendency that is directly related to the Aa chain defect, and the fibrinogen cross-linking rate is accelerated.176 It has been suggested that the thrombophilia is attributable to hypofibrinolysis caused by the abnormal clot structure itself,177 but the other factors mentioned above may be just as important in causing the syndrome.
The fibrinogen Caracas V abnormality (AaS532C194) is located in about the same region of the Aa chain as the Dusart and Chapel Hill III anomalies. This dysfibrinogenemia has been associated with both venous and arterial thrombotic diseases in several members of the kindred.210 Electron microscopy of Caracas V fibrin revealed no differences from normal,211 suggesting that expression of the defect is different from that of Dusart.
Thrombin Binding Defects Low-affinity nonsubstrate thrombin-binding sites are in the fibrin E domain and represent, at least in part, a residual aspect of fibrinogen substrate recognition.65 Both fibrinogen New York I (desBb9-72)99 and fibrinogen Naples I (BbA68T),100,101 presented with striking thromboembolic disease, and both fibrins showed impaired thrombin binding, seemingly at the low-affinity thrombin binding site. Fibrinogen New York I exhibited recurrent venous thrombosis and fatal pulmonary embolism. Homozygous members of the fibrinogen Naples I kindred suffered juvenile arterial stroke, thrombotic abdominal aortic occlusions, and postoperative deep venous thrombosis. Fibrinogens Kumamoto and Aarhus I (both AaR19G) are each associated with thrombosis, and reduced thrombin binding to fibrin has been reported for Kumamoto.130 However, fibrinogen Mannheim I, with the same Aa chain defect, has a bleeding tendency and no thrombosis.128
Melun I The defect in fibrinogen Melun I (D364V145) is situated in a position that interferes with Da site function. This family has a pervasive history of venous thromboembolic disease in the heterozygous state. One may presume that defective Da:EA site interactions contribute to the thrombophilia that has been observed. However, an anomaly (D364H) at the same site (fibrinogen Matsumoto I)144 is not associated with thrombophilia. As in other situations of this kind, more information will be needed to solve this paradoxical situation.
Oslo I The Oslo I dysfibrinogenemia involves a Bb chain defect and is associated with a shortened thrombin time and increased fibrinogen activity as a cofactor for platelet aggregation.128 This augmented behavior of fibrinogen may be presumed to be causally related to the thrombophilic state, but until the structural defect is known, it will not be clear how the anomaly brings about these effects.
Kaiserslautern Fibrinogen Kaiserslautern (gK380N-glycosylated212,213) was described in a 34-year-old woman who developed a cerebral sinus thrombosis after caesarean section and is included in the thrombophilia section for that reason, even though other members of her family with the functional defect were asymptomatic. The site of the abnormality is quite removed from either the Da polymerization pocket or the D:D association site. The polymerization defect is normalized with calcium or by removing sialic acid residues, and thus appears to be due to electrostatic repulsion between condensing fibrils. The mechanism for the thrombophilia is unclear.
Therapy Patients with thrombophilic dysfibrinogenemias are heterogeneous both with respect to their molecular abnormalities as well as their thromboembolic problems. Bleeding, especially when associated with hypofibrinogenemia (e.g., fibrinogen Marburg), may be managed by infusing fibrinogen concentrates as well as by blood replacement. Patients with potentially life-threatening thrombophilic manifestations, such as occurs with fibrinogen Cedar Rapids,195 have been successfully managed with plasma exchange prior to major surgery. However, there is insufficient evidence to suggest that this type of treatment is more effective than more general measures for managing potential thromboembolism, such as treatment with anticoagulants. Long-term management strategies for thrombophilic dysfibrinogenemia are the same as those in patients with recurrent thromboembolism and include life-long anticoagulation with vitamin K antagonists.
*A one letter abbreviation for amino acids is used in this chapter. A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.

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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology




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