FIBRINOGEN IN HEMOSTASIS

FIBRINOGEN IS A 340-kD glycoprotein that circulates in the blood at approximately 3 mg/mL, making it one of the most abundant blood proteins. It is composed of six polypeptide chains, (α, β, γ)2, which are held together by disulfide bonds and organized in a symmetrical dimeric fashion (Fig 1A).1-6 Limited plasmin proteolysis of fibrinogen generates fragments E and D.7Fragment E represents the central domain of the molecule and contains the amino termini of all six chains. The chains extend in coiled coils toward the distal globular regions (fragments D), which contain the carboxyl-terminal regions of the β and γ chains. The carboxyl-terminal region of the α chain has been shown to be noncovalently associated with the E domain.8,9 

Fig. 1.

Schematic representation of human fibrinogen. The α chain is shown in blue, the β chain in green, and the γ chain in red. The black arrowheads indicate thrombin cleavage sites on the α chain. Glycosylation sites are shown as purple hexagons, while the calcium ions within the γ chains are represented as purple spheres (A). Schematic diagram showing the initial process of fibrin polymerization. The central nodules contain the amino-termini of all six chains(α,β,γ)2 and are referred to as the “E” regions, named after a fragment obtained by limited plasmin digestion of fibrin. They are flanked by two symmetric coiled coils that terminate in the distal “D” nodules. After the cleavage of fibrinopeptide A by thrombin, the newly exposed polymerization site “A” binds to the polymerization pocket “a” that is part of the γ chain of fibrin(ogen). The fibrin monomers thus align in a half-staggered, two-stranded arrangement to form long fibrils. Branch points and junctions occur sporadically (only one type is depicted here), contributing to the formation of a three-dimensional mesh (B).

Fig. 1.

Schematic representation of human fibrinogen. The α chain is shown in blue, the β chain in green, and the γ chain in red. The black arrowheads indicate thrombin cleavage sites on the α chain. Glycosylation sites are shown as purple hexagons, while the calcium ions within the γ chains are represented as purple spheres (A). Schematic diagram showing the initial process of fibrin polymerization. The central nodules contain the amino-termini of all six chains(α,β,γ)2 and are referred to as the “E” regions, named after a fragment obtained by limited plasmin digestion of fibrin. They are flanked by two symmetric coiled coils that terminate in the distal “D” nodules. After the cleavage of fibrinopeptide A by thrombin, the newly exposed polymerization site “A” binds to the polymerization pocket “a” that is part of the γ chain of fibrin(ogen). The fibrin monomers thus align in a half-staggered, two-stranded arrangement to form long fibrils. Branch points and junctions occur sporadically (only one type is depicted here), contributing to the formation of a three-dimensional mesh (B).

In the last stage of blood coagulation, thrombin cleaves the amino-termini of the α and β chains of fibrinogen, releasing fibrinopeptides A and B, respectively, and converting fibrinogen to fibrin monomers. The spontaneous polymerization of fibrin monomers initiates fibrin clotting with the formation of protofibrils. The newly exposed α-chain amino-terminus (GPR) of one fibrin molecule, which is referred to as the “A” polymerization site, binds noncovalently to a complementary polymerization pocket, termed the “a” site, in the carboxyl-terminal region of the γ chain of a second fibrin(ogen) molecule. This “A-a” interaction aligns the fibrin monomers into half-staggered, two-stranded protofibrils (Fig 1B). Subsequently, the growing fibrils aggregate in a lateral fashion to form fibers, and this presumably involves interactions between the amino terminus of the β chain, the “B” polymerization site, and a complementary site, “b,” whose location is uncertain.10-14 Under physiological conditions, the release of fibrinopeptide B follows the release of fibrinopeptide A and correlates with lateral aggregation.15-17 This sequential release of fibrinopeptides, such that the “A” site appears before the “B” site, may control the final clot structure.18 

After polymerization, the transglutaminase factor XIIIa19-21 forms covalent bonds between specific lysine and glutamine residues located within the carboxyl-terminal regions of adjacent γ chains22,23 and between α chains24-27 to form γ-γ dimers and α-polymers.28 These intermolecular bonds crosslink the fibrin gel network and, together with the factor XIIIa–mediated crosslinking of α2-antiplasmin to fibrin, solidify the clot, and render it more resistant to fibrinolysis.29-32 

Fibrinolysis, or the lysis of the clot, is initiated by tissue-type plasminogen activator (t-PA)–mediated activation of plasminogen to plasmin. Both t-PA and plasminogen bind to fibrin,33 and the activation of plasminogen by t-PA is stimulated by its substrate, fibrin, which acts as an “effector” for plasmin formation.34,35 The rate of t-PA–catalyzed activation of plasminogen has been shown to depend on the formation of double-stranded protofibrils during fibrin polymerization.36,37 Plasmin then proteolyzes the fibrin mesh, thereby dissolving the clot. Hemostasis is achieved through maintenance of the delicate balance between the procoagulant, anticoagulant, and fibrinolytic reaction pathways. In whole blood, fibrin(ogen) also binds to the receptor GPIIbIIIa (αIIbβ3)38 on the surface of activated platelets and thereby mediates the formation of the platelet plug.

Fig. 2.

Bead model of the globular carboxyl-terminal region of the human fibrinogen γ chain, from Val143 to Val411. Mutations responsible for γ-dysfibrinogenemias are presented in red. Colored beads indicate stretches of α helix or β strand structure.

Fig. 2.

Bead model of the globular carboxyl-terminal region of the human fibrinogen γ chain, from Val143 to Val411. Mutations responsible for γ-dysfibrinogenemias are presented in red. Colored beads indicate stretches of α helix or β strand structure.

Apart from its role in hemostasis, fibrinogen is also involved in inflammation, wound healing, cell migration, and cell proliferation via a number of interactions. These include the binding of fibrinogen to endothelial cells,39-43 to leukocytes,44-47 and to components of the extracellular matrix.48 

γ-CHAIN–RELATED FUNCTIONS

Many of the important intermolecular interactions of fibrin(ogen) are localized partly or entirely within the globular carboxyl-terminal region of the γ chain. The primary polymerization pocket “a,” the γ-chain calcium-binding site, and the γ-γ factor XIIIa crosslinking site all reside in this region.22,23,49-56 The platelet-surface integrin GPIIbIIIa57-60 and the leukocyte integrin MAC-1 (αMβ2, CD11b/CD18)61-63 have been shown to interact with specific sites within this region of the fibrin(ogen) molecule. The γ chain of fibrin has also been implicated in plasminogen binding64and in t-PA binding and stimulation.65-68 

FIBRINOGEN AND DISEASE

An elevated plasma fibrinogen level is associated with coronary atherosclerosis and is recognized as a significant independent risk factor for cardiovascular diseases.69-73 In fact, the predictive value of high fibrinogen levels for ischemic heart disease,69 myocardial infarction, and stroke74appears to be as important as high cholesterol levels. In vitro, high levels of fibrinogen have been shown, among other things, to correlate with lower fibrin gel porosity,75 to affect the rheological properties of blood,76 and to influence the thickness of the fibrin fibers.77 

Dysfibrinogenemia refers to the presence in plasma of functionally abnormal fibrinogen due to a structural defect in the molecule. Inherited dysfibrinogenemia is recognized as a cause of hemorrhagic diathesis and bruising and is now also emerging as a significant risk factor for familial thrombophilia, heart disease, and stroke.78-86 The assessment of the risks that dysfibrinogenemias represent is complicated by the fact that most of the affected individuals are heterozygous for the mutation, with a circulating mixture of normal and abnormalfibrinogen. The molecular basis for the defect and the resulting symptoms vary greatly and those symptoms, when recognized, are usually episodic in nature. Further, to correlate the fibrinogen abnormality with the disease requires the thorough examination of several family members, often over long periods of time. Finally, the correlation with disease depends inherently on the diagnostic tests and their proper interpretation.

The analysis of dysfibrinogens at the molecular level has been challenging because of the size and complexity of the fibrinogen molecule. Over the last 15 years, the molecular defects of numerous inherited dysfibrinogenemias have been elucidated. These can be separated into two groups: those that affect the release of the fibrinopeptides A and B, and those that do not. The first group includes dysfibrinogenemias that are generally caused by the substitutions of amino acids situated at the amino-terminal regions of the α and β chains, specifically at or near the thrombin-cleavage sites, and tend to associate with bleeding complications.87These mutations interfere with the initial conversion of soluble fibrinogen to fibrin monomer, and they account for the majority of abnormal fibrinogens identified to date.88 

The second group of dysfibrinogenemias is heterogeneous. It comprises mutations within the globular carboxyl-terminal regions of the three chains as well as mutations at the “A” and “B” polymerization sites that affect the “A-a” or “B-b” interactions. The clinical manifestations associated with this class of mutations vary from severe bleeding, to asymptomatic, to severe thrombophilia. Generally speaking, the mechanisms by which mutations in these regions of fibrin may cause these clinical manifestations have not been characterized as thoroughly as those belonging to the first group.

FIBRIN(OGEN) MOLECULAR STRUCTURE

Much valuable information on the dimensions and arrangement of domains within the molecule has been gained through electron microscopic and low-resolution crystallographic studies.89-94 These have provided a wealth of structural information detailing the overall size of the molecule, the arrangement of the six chains into a symmetric trinodular structure, and the structures of intermediates formed during polymerization and fibrinolysis. The final fibrin mesh can be visualized by electron microscopy, providing an important tool for analyzing abnormal fibrinogens. Seemingly minor structural abnormalities in fibrinogen can lead to drastic changes in the clot structure, causing alterations in fiber thickness, length, branching, and porosity.95,96 

Although the high-resolution crystal structure of the entire 340-kD fibrinogen molecule remains an elusive goal, several high-resolution crystallographic structures of fibrinogen fragments have been published recently: the 2.1-Å structure of a recombinant 30-kD protein corresponding to the carboxyl-terminal region of the γ chain,55 the same 30-kD protein complexed with the peptide GPRP,56 the 2.9-Å structure of the 86-kD proteolytic fragment D, and the 172-kD factor XIIIa–crosslinked D-dimer complexed with the peptide GPRP.97 The peptide GPRP mimics the amino terminus of the thrombin-cleaved fibrin α chain (the “A” polymerization site). The protein-peptide complex structures showed the exact position of the polymerization pocket “a” within the γ chain and identified the amino acids that interact with the fibrin α chain GPR residues.56,97 

A recombinant protein (rFbgγC30) encoding the 30-kD carboxyl-terminal fragment of the γ chain of human fibrinogen (Val143-Val411) was expressed in a soluble secreted form98 in the yeastPichia pastoris, purified, and crystallized.55 A similar fragment of the γ chain, (the γ-module Ile148-Val411) was also expressed in Escherichia coli as an insoluble protein and refolded in vitro.63 Both of the recombinant proteins were shown to be functional with respect to calcium binding, crosslinking by factor XIIIa, and platelet binding.63,98 These studies represent a “modular” approach to fibrinogen research where small regions within this complex molecule can be expressed and characterized without the complicating factors such as partial degradation and heterogeneity that are associated with fibrinogen isolated from plasma.

The three-dimensional x-ray structures of human fibrinogen fragment D and of the factor XIIIa–crosslinked D dimer complexed with the peptide GPRP amide were determined to 2.9-Å resolution.97 This 86-kD fragment D comprises the carboxyl-terminal regions of the β and γ chains, as well as a short section of the α chain (α111-197, β134-461, γ88-406). In fragment D, the carboxyl-terminal regions of the β and γ chains are globular, with the γ chain comprising the distal end of the outer nodule.97 The overall fold of the β and γ carboxyl terminal regions is similar, as was expected from their strong sequence similarity.99 

The fragment D dimer was seen to have an extensive γ:γ end-to-end “self-association” interface that is formed as the distal nodules of fibrin become aligned during polymerization, before crosslinking by factor XIIIa. The characteristics of this γ:γ interface are of great interest, as it has been hypothesized that some fibrinogen mutations delay polymerization by interfering with the alignment of the distal nodules at this interface.100,101 The final 8 to 20 residues at the carboxyl-terminal end of the γ chains were not visible in the electron density for the γ-chain fragment and fragment D crystal structures. Several studies have indicated that this region can adopt multiple conformations.55,102-106 

The recent crystallographic studies have led to new insights into the structure-function relationships of the complex molecule that is fibrin(ogen). In particular, we can now correlate the clinical manifestations and the biochemical information available for the dysfibrinogens with what is now known about the three-dimensional structure of the molecule.

γ-DYSFIBRINOGEMIAS

Dysfibrinogenemias and their pathologies have been reviewed previously.78,84,87,107-110 The goal of this review is to examine the dysfibrinogemias, 37 to date, with identified molecular defects situated within the carboxyl-terminal region of the γ chain (Fig 2). Table 1 summarizes the available biochemical and clinical information. For all of the γ-dysfibrinogens, the thrombin time was prolonged, fibrin polymerization was impaired, and the release of fibrinopeptides A and B was normal. The mutations were identified either by proteolytic peptide sequencing or by sequencing of PCR-amplified genomic DNA. The structural information presented below is derived from the rFbgγC30 structures,55,56 unless specified otherwise.

Table 1.

Summary of γ-Dysfibrinogens

Mutation  Name  Sex/ Age Ca2+ Binding  γ-γ FXIIIaCrosslinking  Hetero/ homozygous Clinical Symptoms (no. of episodes)  Relative(s) References  
Fibrin  fbg  
G268E  Kurashiki I M/58   N  A  HM  ASYMPT   101  
R275C Baltimore IV  M/56   N   HT  ASYMPT   116, 117  
 Bologna I  F/20      THROMB  2  84 
 Cedar Rapids I      HT  THROMB(2)-150 2-151 122  
 Milano V  F/51  N    HT ASYMPT   119,199  
 Morioka I   N   HT  ASYMPT   115  
 Osaka II M/48   N   HT  ASYMPT   113  
 Tochigi I M/51   N   HT  ASYMPT   88, 114  
 Tokyo II  F/39   N   HT  ASYMPT   100, 118 
 Villajoyosa I  F/27     HT  ASYMPT  120  
R275H  Barcelona III  M/71     HT THROMB(1)  0  120  
 Barcelona IV  M/28    HT  ASYMPT   120  
 Bergamo II     HT  THROMB(M)  5  126  
 Claro I  F/42 N    HT  ASYMPT-152  128, 199  
 Essen I M     HT  ASYMPT   126  
 Haifa I F/30  N  N   HT  THROMB(≥2)  1  95, 130, 131 
 Osaka III  F/38  N  N   HT  ASYMPT  127  
 Perugia I  M/2     HT  ASYMPT  126  
 Saga I  F/16  N    HT  ASYMPT  129  
G292V  Baltimore I  F/29   N   HT THROMB(M)-153  133-137  
N308I  Baltimore III  F/30  N   HT  ASYMPT   140, 141  
N308K Bicêtre II  M/40      THROMB(≥2)  144  
 Kyoto I  M/45  N  N   HT  ASYMPT  142, 143  
 Matsumoto II  F/49     HT BLEED-155 1  145  
M310T  Asahi I  M/33   A  HT  BLEED(M)   146, 147  
D318G  Giessen IV F/18      THROMB-150,-153¶  0  84  
ΔN319,D320 Vlissingen I  F/23  A    HT  THROMB  ≥6 152  
Q329R  Nagoya I  F/43   N   HT  ASYMPT  153-155  
D330V  Milano I   N  N   HT ASYMPT   156, 199  
D330Y  Kyoto III  F/50    HT  ASYMPT   157  
N337K  Bern I  F/24 N    HT  ASYMPT   158, 159, 199  
∇γ350 Paris I   A  A      160-165  
S358C Milano VII  F/21  N    HT  ASYMPT   166, 199  
D364H  Matsumoto I  M/1     HT  ASYMPT  167  
D364 V  Melun I  F/40     HT THROMB(M)  8  168  
R375G  Osaka V  F/44    HT  ASYMPT   172  
K380N  Kaiserslautern I   N   HM  THROMB   177 
Mutation  Name  Sex/ Age Ca2+ Binding  γ-γ FXIIIaCrosslinking  Hetero/ homozygous Clinical Symptoms (no. of episodes)  Relative(s) References  
Fibrin  fbg  
G268E  Kurashiki I M/58   N  A  HM  ASYMPT   101  
R275C Baltimore IV  M/56   N   HT  ASYMPT   116, 117  
 Bologna I  F/20      THROMB  2  84 
 Cedar Rapids I      HT  THROMB(2)-150 2-151 122  
 Milano V  F/51  N    HT ASYMPT   119,199  
 Morioka I   N   HT  ASYMPT   115  
 Osaka II M/48   N   HT  ASYMPT   113  
 Tochigi I M/51   N   HT  ASYMPT   88, 114  
 Tokyo II  F/39   N   HT  ASYMPT   100, 118 
 Villajoyosa I  F/27     HT  ASYMPT  120  
R275H  Barcelona III  M/71     HT THROMB(1)  0  120  
 Barcelona IV  M/28    HT  ASYMPT   120  
 Bergamo II     HT  THROMB(M)  5  126  
 Claro I  F/42 N    HT  ASYMPT-152  128, 199  
 Essen I M     HT  ASYMPT   126  
 Haifa I F/30  N  N   HT  THROMB(≥2)  1  95, 130, 131 
 Osaka III  F/38  N  N   HT  ASYMPT  127  
 Perugia I  M/2     HT  ASYMPT  126  
 Saga I  F/16  N    HT  ASYMPT  129  
G292V  Baltimore I  F/29   N   HT THROMB(M)-153  133-137  
N308I  Baltimore III  F/30  N   HT  ASYMPT   140, 141  
N308K Bicêtre II  M/40      THROMB(≥2)  144  
 Kyoto I  M/45  N  N   HT  ASYMPT  142, 143  
 Matsumoto II  F/49     HT BLEED-155 1  145  
M310T  Asahi I  M/33   A  HT  BLEED(M)   146, 147  
D318G  Giessen IV F/18      THROMB-150,-153¶  0  84  
ΔN319,D320 Vlissingen I  F/23  A    HT  THROMB  ≥6 152  
Q329R  Nagoya I  F/43   N   HT  ASYMPT  153-155  
D330V  Milano I   N  N   HT ASYMPT   156, 199  
D330Y  Kyoto III  F/50    HT  ASYMPT   157  
N337K  Bern I  F/24 N    HT  ASYMPT   158, 159, 199  
∇γ350 Paris I   A  A      160-165  
S358C Milano VII  F/21  N    HT  ASYMPT   166, 199  
D364H  Matsumoto I  M/1     HT  ASYMPT  167  
D364 V  Melun I  F/40     HT THROMB(M)  8  168  
R375G  Osaka V  F/44    HT  ASYMPT   172  
K380N  Kaiserslautern I   N   HM  THROMB   177 

All propositi were found to have impaired fibrin polymerization and normal fibrinopeptide release. The number of episodes is shown in parentheses, if reported. Relatives refers to the number of relatives showing clinical symptoms similar to those of the propositus (but not necessarily the dysfibrinogenemia). Blanks indicate that data were unavailable or not found.

Abbreviations: M, male; F, female; N, normal; A, abnormal; fbg, fibrinogen; HT, heterozygous; HM, homozygous; ASYMPT, asymptomatic; THROMB, thrombosis; BLEED, bleeding; M, multiple.

F0-150

The propositus was also found to be heterozygous for the factor V Leiden defect.

F0-151

All affected relatives were heterozygous for both the dysfibrinogenemia and the factor V Leiden defect.

F0-152

Had two spontaneous abortions.

F0-153

Also suffered from mild bleeding.

F0-155

Easy bruising and postpartum bleeding.

¶Personal communication from Drs F. Haverkate and R.M. Bertina, December 1997.

G268E.

Fibrinogen Kurashiki I was identified in a 58-year-old man who was homozygous for this molecular defect and experienced no major bleeding or thrombosis.101 Crosslinking of the fibrin γ chains appeared to proceed normally. In contrast, the crosslinking of fibrinogen Kurashiki I by factor XIIIa was delayed, compared with normal fibrinogen. The rate of plasminogen activation by t-PA was not enhanced by fibrin Kurashiki I to the same extent as is typically seen with normal fibrin.101 The investigators concluded that the G268E substitution would disturb the D:D association, possibly by introducing repulsive forces at a γ:γ interaction site between two dysfibrinogen molecules. This would in turn lead to alterations in the protofibril alignment and to the observed delay in fibrin polymerization.101 This hypothesis was based in part on the close proximity of G268 to R275, which has been proposed to be an important participant in the D:D interaction.100 

In the crystal structure of rFbgγC30, the backbone nitrogen of G268 is hydrogen bonded to the carbonyl oxygen of R275, and these two residues flank a surface-exposed chain reversal comprising residues 269 through 274, with a sharp β turn at A271 and D272. Mutations at R275 have also been identified in dysfibrinogens (see next section). G268 exhibits no backbone strain, so the effects of mutations at this site are not likely to alter the backbone conformation. Because G268 is exposed to the solvent, a glutamic acid side chain can be modeled in rFbgγC30, without encountering atomic overlaps with neighboring residues. The fact that bovine111 and lamprey112 fibrinogens both encode a threonine at position γ268 further supports the hypothesis that the observed defect in polymerization is likely caused by the introduction of a negative charge here, rather than by strain caused by the bulkiness of the glutamic acid residue.101 

Because G268 is approximately 24 Å from the “a” site and 20 Å from the calcium atom, respectively, it is unlikely that the G268E substitution would affect either the polymerization pocket or the calcium site directly. The normal crosslinking of fibrin Kurashiki I would imply that the D:E interaction provides enough stabilization energy to overcome the proposed D:D repulsion caused by the mutated residue. Thus, the half-staggered arrangement of mutant protofibrils would presumably be close to the normal fibrin structure.

R275C, H.

The amino acid γR275 is the site of mutation for at least 18 dysfibrinogenemias (Table 1), making it the most commonly mutated site within the carboxyl-terminal region of the γ chain. Fibrinogens Osaka II,113 Tochigi I,114 Morioka I,115Baltimore IV,116,117 Tokyo II,100,118 Milano V,119 and Villajoyosa I120 were also characterized as R275C substitutions. In the case of fibrinogen Osaka II,113 the new cysteine residue was shown to be linked to a free cysteine. R275C-fragment D1 that was prepared from fibrinogen Osaka II failed to prolong significantly the fibrinogen clotting time,113 in contrast to normal fragment D1.121 The binding of t-PA and plasminogen to fibrin Villajoyosa I was found to be normal.120 The normal factor XIIIa–catalyzed crosslinking of fibrin Tokyo II was interpreted as indicating a normal D:E interaction.100 Electron microscopic images of crosslinked fibrinogen Tokyo II showed that it failed to form elongated, double-stranded fibrils. Furthermore, in contrast to normal fibrin, fibrin Tokyo II formed tapered terminating fibers with extensive branching of the clot network. The investigators concluded that the mutation R275C causes a functionally abnormal D:D association site100 that results in impaired fibrin assembly.

In contrast to the asymptomatic R275C propositi listed above, fibrinogen Bologna I was identified in a 20-year-old woman who suffered multiple episodes of venous thrombosis. Protein C, protein S, and antithrombin III deficiencies were ruled out as risk factors.84 Recently, fibrinogen Cedar Rapids (R275C) was identified in three second-generation family members with severe pregnancy-associated thrombophilia.122 These patients were also heterozygous for the Factor V Leiden defect.123-125Interestingly, first-generation family members who carried only the Factor V Leiden defect or only the dysfibrinogen had no history of thrombophilia. These findings suggested that thrombophilia is associated via the presence of both defects.122 

The molecular substitution R275H is also commonly encountered. The R275H dysfibrinogens Essen I,126 Perugia I,126Osaka III,127 Barcelona IV,120 and Claro I128 were isolated from asymptomatic patients. Fibrinogen Bergamo II was isolated from a patient who, along with several members of her family, suffered from recurrent thromboembolism,126while Fibrinogen Saga I was found in a patient with hematuria.129 Fibrinogen Haifa I was described in a 30-year-old woman who experienced severe intermittent claudication after short walks, due to bilateral occlusion of the superficial femoral arteries.130 The Fibrinogen Barcelona III propositus suffered a single postsurgical thrombotic event at age 32,120 and the Claro I propositus had two spontaneous abortions.128 These findings are suggestive, but not conclusive, of a possible association between mutations at R275 and a tendency to thrombosis.

Calcium binding, platelet aggregation, and factor XIIIa–mediated crosslinking appeared to be normal in Fibrinogen Haifa.131However, the presence of calcium ions did not protect its fragment D against plasmin cleavage at position K302-F303,131 as would be expected for normal fragment D.132 An electron microscopic study of polymerized fibrin Haifa I showed an abnormal, highly branched matrix, with the fibers appearing generally thinner than those seen in normal polymerized fibrin.95 In contrast to fibrinogen Haifa I, fragment D1 derived from fibrinogen Saga I was fully protected by calcium ions against plasmin degradation, but it failed to inhibit fibrinogen clotting.129 

In the rFbgγC30 structure, residue R275 is surface-exposed, flanking a chain reversal comprising residues 269-274. As mentioned previously, a β turn between residues 270 and 271 brings the backbone of R275 in close proximity to G268, allowing a hydrogen bond between the carbonyl oxygen of R275 and the nitrogen of G268. A salt link between R275 and D272 further stabilizes this chain reversal. Thus, it is reasonable to assume that amino acid substitutions that alter this β turn may cause similar disruptions in the protein structure. This could in turn affect the interactions of this region with other proteins. The participation of R275 in a D:D interface was confirmed by the three-dimensional structure of the factor XIII-crosslinked D dimer.97 

The side chain of R275 is involved in another very important interaction, because it forms two hydrogen bonds with the backbone of G309, thus clearly providing extra stabilization for this region of the protein. The backbone nitrogen atom of G309 is hydrogen bonded to the backbone carbonyl atom of L276 as well as to the side chain oxygen of N308. The N308 side chain in turn shares two hydrogen bonds with the backbone of Y278. The importance of the proper alignment of residues in this region is highlighted further by the localization here of several other molecular defects associated with dysfibrinogenemias; ie, N308I, N308K, and M310T (see below).

G292V.

Dysfibrinogen Baltimore I was diagnosed more than 30 years ago in a 29-year-old woman suffering from femoral vein thrombosis after minor trauma.133-135 The patient had a history of mild hemorrhagic diathesis characterized by frequent bruising, epitaxis, and hemorrhage as well as previous severe recurrent thrombosis and pulmonary embolism. Fibrinogen Baltimore I showed delayed fibrin polymerization that could be partially corrected by addition of calcium.133 The defective formation of factor XIIIa–crosslinked α-polymers was corrected by increased calcium concentration or lowered pH conditions.136 Plasmin degradation of fibrinogen Baltimore I was normal and its fragment D was protected effectively by calcium. The amino acid substitution G292V was identified as the molecular defect.137 

G292 lies in the middle of a highly conserved stretch of amino acids,99 approximately 10 Å from the polymerization pocket and 18 Å from the calcium atom.55 Upon first inspection of the crystal structures,55,56 G292 appears to be isolated from the clusters of residues linked to other dysfibrinogenemias. G292 is located at the surface of the protein, and the backbone dihedral angles of (φ,ψ) = (101o,164o) indicate that substitution of a nonglycine residue at this site would lead to backbone strain and destabilize the structure. Further, the side chain of N337, which points away from the polymerization pocket, is only 7 Å from G292. It is plausible that insertion of a bulky valine side chain at position 292 would alter the region surrounding N337. N337 is part of an unusual, highly strained region of the the γ chain. Its dihedral angles indicate backbone strain, and it lies immediately adjacent to a cis peptide bond between residues K338 and C339. This strained conformation enables the backbone of C339 to form an additional hydrogen bond with the fibrin α chain amino-terminal residues GPR.56 This strained conformation is stabilized in part by an extensive network of hydrogen bonds, including the hydrogen bonds between the N337 side chain and its neighbors. Therefore, any disruption of the environment of the N337 side chain could destabilize its backbone, altering the polymerization pocket and thereby fibrin polymerization.

Residues F303 and F304 are positioned in between G292 and N337. Both the phenyl rings are directed to the surface of the molecule. It has been well established from solvent transfer studies138,139that the phenylalanine side chain is reasonably soluble in both polar and nonpolar solvents; hence, its occurrence both in the hydrophobic cores of proteins and at their solvent-exposed surfaces. The valine side chain, by contrast, occurs almost exclusively in hydrophobic environments, and is typically buried in solvent-inaccessible protein cores. The substitution of a valine for G292 would promote hydrophobic interactions between the valine and phenylalanine side chains, thus altering the molecular surface significantly. Further, the surface exposure of the hydrophobic valine side chain would decrease the entropy, and thus the stability of the protein. Therefore, we propose that the substitution G292V leads to the structural destabilization of a fairly large region of the protein, and that this affects the polymerization pocket directly.

N308I, K.

The dysfibrinogenemia Baltimore III was diagnosed in an asymptomatic 30-year-old woman who had a prolonged thrombin time that was corrected by excess calcium.140 The defect was identified as an N308I mutation.141 The fibrinogen showed normal crosslinking by FXIIIa, normal calcium binding, and delayed fibrin monomer polymerization.140,141 

The N308K mutation was identified in fibrinogens Kyoto I,142,143 Bicêtre II,144 and Matsumoto II.145 The clinical manifestations associated with the N308K substitution varied greatly. Fibrinogen Kyoto I was isolated from an asymptomatic, hypofibrinogenic male who had a family history of both thrombotic and bleeding problems142,143. Fibrinogen Bicêtre II was isolated from a man who suffered a spontaneous deep-vein thrombophlebitis and a pulmonary embolism.144Finally, fibrinogen Matsumoto II was isolated from a woman with Graves' disease who had a tendency to bruise easily and who had experienced moderate to severe bleeding after each of her three deliveries.145 For fibrinogen Bicêtre II, it was shown that the mutation did not affect binding to t-PA or to plasminogen, thus eliminating these possible explanations for the thrombotic incidents.144 

The residue N308 is situated on the surface of rFbgγC30. For that reason, the introduction of a hydrophobic amino acid such as isoleucine at this position would appear to be very destabilizing. In structure-based sequence alignments, N308 is conserved in all of the α-extended, β-, and γ-chain sequences except in the lamprey fibrinogen γ chain, where it is replaced by a leucine.55,97,99 Although a lysine residue should be accommodated easily on the surface of rFbgγC30, a positive charge here could disrupt interactions with other fibrinogen domains. The new lysine residue could also change the pattern of cleavage by plasmin, thereby affecting fibrinolysis. This possibility has not been addressed experimentally.

The side chain of N308 forms hydrogen bonds with the backbone nitrogen and carbonyl oxygen of Y278.56 An additional hydrogen bond connects the side-chain oxygen of N308 with the backbone nitrogen of G309, as discussed in the context of the mutations at G268 and R275. A cluster of residues including N308, G309, G268, R275, and M310 is exposed to the solvent in both the rFbgγC30 and fragment D structures, and is in the vicinity of the D:D interface of the factor XIIIa-crosslinked D dimer structure.97 This interaction site is presumably buried during the initial contact between the D domains, in the early stages of fibrin polymerization. Thus, the introduction of bulky or charged side chains at N308 would be expected to disrupt the initial alignment of fibrin monomers into protofibrils. We would expect the clot structure in these patients to be altered in a manner similar to that seen with fibrinogen Tokyo II.100 

M310T.

Dysfibrinogenemia Asahi I was diagnosed in a 33-year-old man sufferering from posttraumatic bleeding after a traffic accident. He had experienced moderate hemorrhage related to injuries and delayed wound healing since adolescence.146 The mutation M310T creates a consensus sequence, N308-G309-T310, that directs N-linked glycosylation, and, indeed, the attachment of a carbohydrate moiety to N308 was confirmed.146 The factor XIIIa–mediated γ-γ crosslinking of fibrinogen and of fibrin Asahi I were both markedly delayed, even though the γ chain amine acceptor Q398 functioned normally when assayed by monodansylcadaverine incorporation. The delayed fibrin(ogen) crosslinking rate indicated therefore that the abnormal molecules were unable to align properly.146,147 

The extent of fibrinogen glycosylation affects fibrin polymerization and lateral aggregation, as well as the structural and mechanical properties of the clot.148 For example, desialylated fibrin polymerizes faster than normal fibrin149 and clots containing deglycosylated fibrin(ogen) form faster and display thicker, underbranched fibers, resulting in a more porous mesh.150Conversely, clots formed from hyperglycosylated fibrinogen (as would be the case for fibrinogen Asahi I) assemble more slowly.151 

Alignment of the human γ-chain sequence with a series of homologous sequences55,97,99 shows that the methionine at position 310 is strictly conserved among the fibrinogen sequences. Nevertheless, the substitution M310T would appear to be structurally benign. Because the residue is surface-exposed and is not involved in any hydrogen bonds, it is difficult to argue compellingly for the unique contribution of M310 to the structural integrity of this region. Therefore, we propose that the primary consequence of mutations at this site is in the disruption of D:D interactions by the new glycosylation, and that the effects of this mutation are analogous to those resulting from substitutions at the γ chain positions R275, G268, G292, and N308.

D318G.

Fibrinogen Giessen IV (sometimes referred to as Kassel), was identified in an 18-year-old woman who suffered from recurrent venous thrombosis as well as mild bleeding.84 She was later found to be heterozygous for the factor V Leiden defect (personal communication from Drs F. Haverkate and R.M. Bertina, Leiden, The Netherlands, December 1997). The only relative of the patient who was found to have this dysfibrinogenemia did not experience thrombosis; it is not known whether this individual was also heterozygous for the Factor V Leiden defect. The side chain of aspartate 318 is directly involved in binding to the calcium ion in the γ chain.55The removal of this carboxyl group would obviously have a detrimental effect on calcium binding, presumably impairing the regulation of fibrin polymerization and the protection of the γ chain by calcium ions.

ΔΝ319,D320.

Fibrinogen Vlissingen I was identified in a 23-year-old woman who was hospitalized with a massive pulmonary embolism.152 The father and daughter of the patient also showed prolonged fibrinogen clotting times, although they were asymptomatic. The same 2–amino acid deletion was found in fibrinogen Frankfurt IV, in a patient with thrombosis. A subsequent family history showed that these two patients were related.84 Fibrin polymerization was delayed both in the presence and absence of calcium. Calcium was only partially effective in protecting this fibrinogen against plasmin degradation. Calcium binding was measured by equilibrium dialysis, and showed the loss of one high-affinity Ca2+-binding site within fibrinogen Vlissingen I fragment D.152 DNA sequence analysis showed that the patient was heterozygous for a deletion of the six nucleotides encoding N319 and D320. It was concluded that these residues were essential for γ calcium ion binding. Clotting of the mutant fibrinogen in the presence of EDTA was also delayed relative to normal fibrinogen, suggesting that the deletion affected not only the calcium-binding site, but also the polymerization site “a” within the γ chain. In rFbgγC30, the polymerization site appears to function relatively independently from the calcium-binding site. This is illustrated by the fact that the fragment can bind GPRP even after treatment with EDTA.98 Nevertheless, the Vlissingen I data suggest that a disruption of the structure at one site could well affect the folding of the other, leading to a polymerization defect.

Q329R.

Fibrinogen Nagoya I was found in three generations of a clinically asymptomatic Japanese family.153 Fibrin monomer polymerization was defective, while factor XIIIa–catalyzed crosslinking of fibrin was normal. The γ chain of fibrinogen Nagoya I showed abnormal behavior on CM-cellulose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).154 A single amino acid substitution of glutamine by arginine at position 329 was shown by peptide amino acid sequence analysis.155 

A comparison of the 30-kD γ-chain structures, both uncomplexed55 and complexed with the fibrin α chain-mimicking peptide GPRP,56 shows that upon binding of GPRP, Q329 shifts its position to accommodate arginine 3 of the peptide and forms a hydrogen bond with it.56 The recombinant γ chain fragment with the substitution Q329R (rFbgγC30-Q329R) was not protected against plasmin degradation in the presence of the peptide GPRP and EDTA, presumably because the mutation abolished GPRP binding.98 This suggests that the mutant arginine side chain blocks access to the “a” site of fibrin(ogen), quite possibly by occupying the preexisting arginine-binding site within the polymerization pocket.

D330V, Y.

Fibrinogen Milano I (D330V) was identified in a young girl and her father, both of whom were clinically asymptomatic.156 A second dysfibrinogenemia involving this amino acid, fibrinogen Kyoto III (D330Y), has been described in an asymptomatic 50-year-old woman.157 Her two sons showed the same abnormality, and fragment D1 isolated from the second son was not effective in inhibiting fibrinogen clotting. Because aspartic acid and tyrosine have very different pKa's, it was hypothesized that the point mutation perturbed the local folding of the γ chain, thus altering the structure required for fibrin polymerization.157 

The crystal structure of the rFbgγC30-GPRP complex56showed that D330 forms a salt link with the arginine side chain of the peptide, providing an interaction essential for D:E binding. Furthermore, the carboxyl group of D330 is hydrogen-bonded to the side chain of H340. The backbone carbonyl oxygen of H340 forms an additional hydrogen bond with the peptide amino terminus, and the backbone nitrogen is hydrogen-bonded to the peptide glycine carbonyl oxygen. Therefore, the replacement of D330 by an uncharged amino acid residue such as tyrosine or valine would significantly alter the electrostatic environment of the polymerization pocket. The addition of these bulky side chains would necessarily alter the extensive and precise network of hydrogen bonds within the polymerization pocket. These disruptions are consistent with the markedly prolonged clotting times of patients carrying this mutation.

N337K.

Fibrinogen Bern I was discovered in a 24-year-old asymptomatic woman.158 Two-dimensional gel electrophoresis showed an abnormal mobility for the γ chain of fragment D derived from fibrinogen Bern I.159 N337 lies in a region of the γ chain that is highly conserved among mammals. It is part of a very constrained chain reversal within the γ chain,56 as described earlier in the G292V section. The side chain of N337 participates in three hydrogen bonds to S306 and F303 and points away from the polymerization pocket, toward the solvent. Although a lysine residue could be accommodated in this position, several N337 hydrogen bonds that stabilize the strained backbone conformation at the polymerization pocket would be lost.

Paris I (insertion at γ350).

Fibrinogen Paris I is characterized by impaired fibrin polymerization and clot retraction.160 Fibrin Paris I monomers inhibited the polymerization of normal fibrin monomers, did not form γ-γ dimers, and did not incorporate dansylcadaverine in the presence of factor XIIIa.161,162 Approximately 2/3 of the Paris I fibrinogen γ chain exhibited an apparent molecular weight higher (≈2.5 kD) than normal.161 Electron microscopic studies on fibrin Paris I showed abnormal clumps connected by thin irregular fibers, with frequent terminations.163 Fibrinogen Paris I also failed to support adenosine diphosphate (ADP)-induced platelet aggregation.164 The molecular defect was determined by polymerase chain reaction (PCR) analysis to be an A → G point mutation within intron 8 of the γ chain gene. It is hypothesized that alternative splicing, due to this nucleotide change in the Paris I mRNA, leads to the insertion of 15 amino acids after Q350, including two cysteine residues, and to the substitution of G351 for a serine.165 No association with bleeding or thrombosis has been reported for this dysfibrinogenemia.

The insertion of 15 amino acids at residue Q350 cannot be modeled with reliability. Nevertheless, we can speculate that this large insertion within the polymerization domain would cause a major structural reorganization, quite possibly precluding the formation of functional polymerization and crosslinking sites.

S358C.

Fibrinogen Milano VII was identified in an asymptomatic 21-year-old woman.166 No family member had a hemorrhagic or thrombotic tendency, but several had a prolonged thrombin time. The fibrin Milano VII clots had an abnormal “transparent” appearance when compared with clots obtained from normal fibrinogen.166 The S358C mutation creates an unpaired cysteine residue, and immunoblotting analysis determined that the abnormal fibrinogen circulated as a disulfide-linked complex with the abundant blood protein albumin. Interestingly, removal of albumin failed to normalize the fibrin polymerization profile. This suggested that the defect was not due to steric hindrance created by albumin, but rather was attributable directly to the substitution.166 

The three-dimensional structure of rFbgγC30 shows that S358 is indeed on the protein surface, making the new cysteine residue available to react with other free cysteines.55,56 S358 does not interact directly with either the polymerization pocket or the calcium site, and it does not appear to be part of the D:D interaction site. It is possible that the substitution leads to a general structural destabilization of the polymerization domain, but there is no direct evidence for this. The observed transparency of fibrin Milano VII clot suggests to us a possible effect on the lateral aggregation of fibrils.

D364H,V.

Dysfibrinogenemia Matsumoto I was identified recently in a 1-year-old boy who had Down Syndrome and congenital heart disease. The young patient showed no signs of bleeding or thrombotic tendencies, nor did his two relatives who also showed prolonged thrombin times. The D364H mutation was identified in the propositus and his affected relatives.167 

Another dysfibrinogenemia involving amino acid D364, Melun I, was identified in a 40-year-old woman following a routine blood coagulation assay.168 The patient had no sign of hemorrhage or thrombosis at the time of the assay, but at 67 years of age the patient developed superficial venous thrombosis on her right foot. This was followed 2 years later by another episode of superficial venous thrombosis in her right leg. The propositus later had a stroke that did not cause long-term effects.168 Further tests eliminated other possible inherited causes of thrombophilia such as protein C, protein S, or antithrombin III deficiencies, or activated protein C (APC) resistance. The patient's mother, sisters, and brother all suffered from repeated deep vein thrombosis or pulmonary thromboembolic episodes. An exhaustive study of four generations showed that most, but not all, of the family members carrying the dysfibrinogenemia suffered from various forms of thrombosis, with episodes beginning as young as age 11. DNA sequencing of the three chains of fibrinogen Melun I led to the discovery of a D364V mutation.168 Again, two dysfibrinogenemias involving the same amino acid were associated with different phenotypes, one (D364H) devoid of symptoms and the other (D364V) associated with thrombophilia.

Two recent independent investigations showed the crucial role of D364 in the early stages of fibrin polymerization, ie, during the “A-a site” interaction.98,169 In the first study, the substitution D364A was introduced into the 30-kD fragment rFbgγC30 and, unlike the wild-type rFbgγC30, this mutant molecule (rFbgγC30-D364A) did not inhibit fibrinogen clotting. rFbgγC30 was protected against plasmin digestion by addition of the peptide GPRP or by calcium, as is the case for fibrinogen.170,171 The rFbgγC30-D364A mutant molecule was not protected by the peptide, suggesting that the “a” site is substantially altered in this mutant.98 In the second study, fibrin polymerization of recombinant, fully assembled fibrinogen containing the D364A and D364H mutations was significantly impaired, as expected if the “a” site is not functional.169 Fibrin polymerization of recombinant fibrinogen with the D364H mutation was almost undetectable. Clottability of the D364A mutant was essentially normal but that of the D364H mutant was substantially reduced. In fact, a fibrin gel did not form when the D364H-mutant fibrinogen was clotted.169 These data suggest that both protofibril formation and lateral aggregation were disrupted in the D364H mutant, indicating that the carboxyl-terminal region of the γ chain plays a role in both polymerization steps.169 

The crystal structures of rFbgγC30-GPRP56 and fragment D dimer-GPRPamide97 showed that the side chain of D364 forms a critical salt link with the charged amino terminus of the α-chain sequence GPR. The loss of the carboxylate group in the D364H and D364V mutations would eliminate an electrostatic bond that is crucial to the “A-a” interaction.

R375G.

Fibrinogen Osaka V was identified fortuitously in an asymptomatic 44-year-old woman.172 The polymerization of fibrin monomers was prolonged in the absence of calcium but normal polymerization occurred at 5 mmol/L CaCl2. Digestion of fibrinogen Osaka V fragment D by plasmin resulted in a more complete degradation of the molecule than was observed for normal fragment D. Further, equilibrium dialysis and Scatchard analysis showed that the mutant fibrinogen contained only one calcium-binding site, whereas three calcium sites have been found in normal fibrinogen.173-176 

In the GPRP-complexed structures,56,97 the side chain of R375 forms hydrogen bonds that are critical for the stability of the polymerization pocket. The loss of this large side chain would drastically alter the shape and charge of this region. It would also remove an important salt link between R375 and D297. R375 is absolutely conserved among all the known γ chain sequences,55,97,99indicating the importance of this residue. The polymerization pocket appears to be biochemically98 and structurally56,97 distinct from the calcium-binding site. However, we suggest that the loss of the R375 side chain, which lines one side of the polymerization pocket, would exert a destabilizing effect on the entire region. This would explain the observed calcium-binding defect.

K380N.

Fibrinogen Kaiserslautern was identified in a 34-year-old woman who suffered from thrombosis in the cerebral sinus after a cesarean delivery, prior to which she had no history of bleeding or thrombosis.177 Her family included individuals who were homozygous as well as heterozygous for this defect, all of whom were asymptomatic. A K380N substitution was shown and glycosylation at N380, directed by the new consensus sequence (N-K-T), was confirmed.177 

K380 is a surface-exposed residue, occurring at a site that is well removed from the polymerization pocket, the calcium-binding site, and the D:D interaction surfaces. Therefore, the polymerization defect is likely to be an altered packing of fibrils caused by the addition of an extra carbohydrate moiety.

FUNCTIONAL SITES WITHIN THE FIBRINOGEN γ CHAIN

In view of the biochemical, clinical, and structural information available to date on fibrinogen, at least five major functional sites can be distinguished within the globular carboxyl-terminal region of the γ chain. These are: (1) the calcium-binding site; (2) the polymerization site “a”; (3) the D:D interaction surface; (4) the γ:γ crosslinking site; and (5) the platelet-binding site. Other functions have been reported to involve the γ chain of fibrinogen, such as t-PA binding, plasminogen binding, and interactions with cell-surface receptors; these await further characterization at the molecular level. Each of the five sites appears to function fairly independently from the others. However, a mutation at one site may destabilize the overall structure and thus affect function at a second site.

Calcium-binding site.

A single high-affinity calcium binding site has been found within fragment D,49,174-176,178 and localized to the globular carboxyl-terminal region of the γ chain.51,52,55Figure3 presents a schematic of the interactions between the metal ion and the protein. The calcium ion is liganded by the side chains of D318 and D320, as well as by the backbone carbonyl oxygens of F322 and G324.55 Two water molecules provide the fifth and sixth coordinating ligands. Although fibrinogen can clot in the presence of EDTA, it does so less efficiently than in the presence of calcium.179 Thus, the calcium-bound conformation of the γ chain favors fibrin polymerization and protects this region from plasmin degradation.132,173 Two dysfibrinogens, Giessen IV (D318G) and Vlissingen I (ΔN319,D320), alter the calcium-binding site. These mutations disrupt the metal binding, and both of these dysfibrinogenemias correlate with a thrombotic tendency. Fibrinogen Osaka V (R375G) is also associated with altered calcium binding, presumably resulting from a regional disruption of the protein structure, but not with thrombosis.

Fig. 3.

Specific molecular interactions between the fibrinogen γ chain and the calcium ion. The calcium ion is liganded by two aspartate side chains, two carbonyl oxygen atoms, and two water molecules.55 

Fig. 3.

Specific molecular interactions between the fibrinogen γ chain and the calcium ion. The calcium ion is liganded by two aspartate side chains, two carbonyl oxygen atoms, and two water molecules.55 

Fig. 4.

Close-up stereo views of the γ chain polymerization site “a” showing the interactions within the polymerization pocket for both the uncomplexed protein (A) and for the complex with GPRP (B), based on the rFbgγC30 structures. The mutation sites that affect the polymerization pocket are indicated in ball-and-stick representation. In (A), the water molecules that form hydrogen bonds with the displayed side chains are shown as pink spheres. In (B), the peptide GPRP is represented in pink. The calcium ion is shown in green, and is enlarged for emphasis.

Fig. 4.

Close-up stereo views of the γ chain polymerization site “a” showing the interactions within the polymerization pocket for both the uncomplexed protein (A) and for the complex with GPRP (B), based on the rFbgγC30 structures. The mutation sites that affect the polymerization pocket are indicated in ball-and-stick representation. In (A), the water molecules that form hydrogen bonds with the displayed side chains are shown as pink spheres. In (B), the peptide GPRP is represented in pink. The calcium ion is shown in green, and is enlarged for emphasis.

Fig. 5.

Space-filling model showing a close-up view of the γ:γ (or D:D) interface between adjacent molecules in the crosslinked D dimer complexed with the peptide GPRP (A). (Adapted and reprinted with permission from Nature [Spraggon G, Everse SJ, Doolittle RF: Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin. Volume 389, page 455, 1997].97 Copyright 1997 Macmillan Magazines Limited.) Space-filling (B) and ribbon (C) models of the globular carboxyl-terminal γ chain region, based on the rFbgγC30 structures (γ143-392). Mutation sites at the γ:γ interface are colored orange (B and C), the calcium ion is green, and the peptide GPRP is shown in magenta. The side chains of residues F303 and F304 are shown in white. We hypothesize that these residues may form part of an extended interaction surface between the D and E regions of fibrin. Residues G292, S358, and K380 are also shown; G268 and G292 are indicated by asterisks (C).

Fig. 5.

Space-filling model showing a close-up view of the γ:γ (or D:D) interface between adjacent molecules in the crosslinked D dimer complexed with the peptide GPRP (A). (Adapted and reprinted with permission from Nature [Spraggon G, Everse SJ, Doolittle RF: Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin. Volume 389, page 455, 1997].97 Copyright 1997 Macmillan Magazines Limited.) Space-filling (B) and ribbon (C) models of the globular carboxyl-terminal γ chain region, based on the rFbgγC30 structures (γ143-392). Mutation sites at the γ:γ interface are colored orange (B and C), the calcium ion is green, and the peptide GPRP is shown in magenta. The side chains of residues F303 and F304 are shown in white. We hypothesize that these residues may form part of an extended interaction surface between the D and E regions of fibrin. Residues G292, S358, and K380 are also shown; G268 and G292 are indicated by asterisks (C).

Polymerization site “a.”

The localization of the primary polymerization site “a” to the carboxyl-terminal region of the γ chain was established several years ago.49,50,52,54 The “a” site binds the GPR sequence that is exposed upon the release of fibrinopeptide A from the α chain, and several short peptides having sequences related to GPR also bind to the “a” site.12,180-182 The peptides GPRP and GPRP-amide bind to the γ chain of fragment D with an affinity that actually exceeds that of the peptide having the native sequence, GPRV.182 In addition, the peptide GPRP protects the γ chain of fragment D against plasmic degradation, even in the presence of EDTA.63,98,170 Figure 4 shows in detail some of the important molecular interactions at the polymerization site before and after binding to the GPRP sequence.

Several dysfibrinogens involve mutations of γ-chain residues that either bind to the GPR sequence directly, or that constitute the architecture of the polymerization site. These include Fibrinogens Nagoya I (Q329R), Milano I (D330V), Kyoto III (D330Y), Bern I (N337K), Matsumoto I (D364H), Melun I (D364V), and Osaka V (R375G).

D:D interaction surface.

During the alignment of the fibrin monomers into fibrils (Fig 1), surface-exposed regions on the γ chains of two adjacent molecules abut each other and form the D:D interface (Fig 5A). The γ-γ (or D:D) interface is extensive,97 and encompasses a number of amino acids that are substituted in dysfunctional fibrinogens (Fig 5B andC). These dysfibrinogens include Fibrinogens Kurashiki I (G268E), Baltimore IV, Bellingham I, Bologna I, Milano V, Morioka I, Osaka II, Tochigi I, Tokyo II, and Villajoyosa I (R275C), Barcelona III and IV, Bergamo II, Claro I, Essen I, Haifa I, Osaka III, Perugia I, and Saga I (R275H), Japanese I (R275S), Baltimore III (N308I), Bicêtre I, Kyoto I, and Matsumoto II (N308K), and Asahi I (M310T). Disruption of this γ:γ interaction has been hypothesized to disrupt the fibrin alignment.100,101 Scanning electron microscopic images of fibrin Tokyo II (Fig 6) show unequivocally that the structure of the fibrin clot was altered drastically by a mutation at the γ:γ interface, R275C.100 

Fig. 6.

Scanning electron micrograph images of Tokyo II fibrin (γR275C) (A and B) and normal fibrin (C and D). Fibrin formed in HEPES pH 7 buffer (A and C) and formed in the same buffer containing 10 mmol/L CaCl2. (Reproduced from The Journal of Clinical Investigation, 1995, vol. 96, p. 1053, by copyright permission of The American Society for Clinical Investigation.100)

Fig. 6.

Scanning electron micrograph images of Tokyo II fibrin (γR275C) (A and B) and normal fibrin (C and D). Fibrin formed in HEPES pH 7 buffer (A and C) and formed in the same buffer containing 10 mmol/L CaCl2. (Reproduced from The Journal of Clinical Investigation, 1995, vol. 96, p. 1053, by copyright permission of The American Society for Clinical Investigation.100)

We also note that F303 and F304 form a surface patch on the γ chain that could interact favorably with a hydrophobic patch on the surface of another molecule, and that these residues lie between the γ:γ surface and the polymerization pocket (Fig 5B). Therefore, F303 and F304 could potentially be involved in D:E and/or D:D interactions.

γ-γ crosslinking site.

Factor XIIIa catalyzes reciprocal covalent crosslinks between the glutamine donors Q398 and/or Q399 of one molecule and the lysine acceptor K406 of an adjacent fibrin(ogen) molecule.22,183,184 There are no reported dysfibrinogenemias associated with mutations in this region of the γ chain (also referred to as γXLXL). Such mutants would likely be “silent” in standard thrombin time assays, and this may explain why they have not been reported in the literature.

Platelet-binding site.

Fibrinogen interacts with the platelet receptor GPIIbIIIa through the last several amino acids at the carboxyl end of the γ chain.57-59,185,186 Although other regions may interact with this receptor as well, the sequence γ408-411 (AGDV) is absolutely required for fibrinogen binding.60 The interaction with platelets has also been visualized through electron microscopic studies.187 Crystallographic studies have not shown a unique conformation for the carboxyl-terminal 19 residues of the γ chain. In the rFbgγC30 protein preparations, this region was very susceptible to proteolysis.55 The gaps in interpretable electron density in some of the crystal structures of this molecule were likely caused by heterogeneity at the carboxyl terminus, and it is probable that this region also has some inherent flexibility.

γ-CHAIN DYSFIBRINOGENEMIAS ASSOCIATED WITH BLEEDING

In general, mutations within the γ chain of fibrinogen are not associated with serious bleeding disorders. Two patients, Baltimore I (G292V) and Giessen IV (D318G), who experienced mild bleeding symptoms, also suffered from thrombotic tendencies. The only γ-dysfibrinogenemia associated with a serious bleeding diathesis is Asahi I (M310T). In this instance, the bleeding symptoms were probably related to the extra glycosylation resulting from the substitution. As discussed earlier, hypoglycosylation increases the rate and extent of clotting.150 Therefore, one can speculate that hyperglycosylation could decrease the clotting rate and thereby cause a bleeding disorder. The molecular explanation would be that the charged carbohydrates lead to a repulsive force between adjacent molecules, thus hindering the assembly and polymerization of fibrin. Also, the bulkiness of the extra carbohydrate could preclude the proper alignment of the fibrin chains.

γ-DYSFIBRINOGENEMIAS ASSOCIATED WITH THROMBOSIS

The proposed mechanisms by which abnormal fibrinogens may contribute to a thrombotic tendency are numerous.80,84 For example, an increased resistance of the mutant fibrin to plasmin proteolysis may arise from alterations in the clot structure and permeability. These mutations may restrict the accessibility of fibrin to plasmin.96,188,189 Other mutations may reduce fibrin-mediated enhancement of fibrinolysis by altering the binding of plasminogen or t-PA to fibrin.190,191 For the γ-dysfibrinogenemias, the most probable reason for the association of mutations with thrombophilia is an altered clot structure. In heterozygous individuals, fibrin polymerization is perturbed but functional, leading to clots with abnormal fiber thickness and porosity. As fibrin structure has been shown to influence the fibrinolytic rate,192 these clots may not be dissolved effectively by the fibrinolytic system, resulting in an increased tendency to thrombosis. We speculate that many of these mutations, if present in the homozygous state, would cause bleeding disorders as well.

DYSFIBRINOGENEMIA AS A HYPERCOAGULABLE STATE

Thrombosis is a major cause of mortality and morbidity. The risk factors for thrombosis can be transient or permanent, acquired, congenital, or inherited. Among the identified inherited risks factors associated with thrombophilia are protein C, protein S, antithrombin III, and heparin cofactor II deficiencies; factor Va resistance to APC; thrombomodulin defects; factor II 20210 allele; and hypoplasminogenemia.79,80,83,85,86,193,194 However, taken together, these conditions account for only 40% to 60% of all familial thrombophilias.

Dysfibrinogenemias are also recognized as possible risk factors for thrombosis. The majority of diagnosed individuals are asymptomatic, whereas approximately 30% of dysfibrinogenemias are associated with bleeding and ≈10% are associated with thrombotic tendencies. However, if one considers only dysfibrinogenemias with mutations in the carboxyl-terminal region of the γ chain, then the distribution changes considerably. An examination of the clinical symptoms associated with γ-dysfibrinogenemias shows that ≈5% (2 of 37) of the individuals experienced significant bleeding, and ≈30% (11 of 37) showed thrombotic tendencies. Approximately 60% (23 of 37) of patients with γ-dysfibrinogenemias were asymptomatic at the time of diagnosis. Although an association is found between certain dysfibrinogenemias and specific symptoms, a direct causal relationship is difficult to demonstrate. Often, the investigation of other risk factors was omitted, incomplete, or not reported. Furthermore, the family history for these mutations, which can be difficult to gather, was often not documented. Unfortunately, data on long-term follow-up of dysfibrinogemic patients and their relatives are rarely available.

Typically, dysfibrinogenemias are discovered fortuitously during routine coagulation tests. At present, there is no practical, simple, and cost-effective clinical test that could allow the diagnosis of thrombophilic dysfibrinogenemias that do not affect fibrin polymerization. One must consider the possibility that mutant dysfibrinogens exist that affect fibrinolysis but not fibrin polymerization. If these dysfibrinogenemias exist, then thrombosis-associated dysfibrinogenemias are surely underdiagnosed, and these may represent a more important determinant of hereditary hypercoagulable states than is generally recognized.

A relatively small number of the diagnosed dysfibrinogenemias have been characterized at the molecular level. Numerous others that are associated with mild to severe familial thrombosis still await characterization at the molecular level. These include fibrinogens Date I,191 Richfield,195 Tampere I,196and London I.197,198 

It has been suggested that an inherited predisposition to thrombosis is often the result of mutations in two or more genes encoding proteins involved in hemostasis.79 Similarly, a γ-dysfibrinogenemia may well act in concert with other risk factors such as stasis, surgery, pregnancy, trauma, lupus, malignancy, oral contraceptives, hyperhomocysteinemia, or elevated factor VIII or fibrinogen levels. Such combinations could trigger the occurrence of a thrombotic episode in an otherwise healthy individual. Examples illustrating this phenomenon would be the heterozygous dysfibrinogemias Cedar Rapids (R275C)122 and Giessen IV (D318G),84 both of which were found in conjunction with a heterozygous factor V Leiden trait. In the Cedar Rapids family, neither defect alone was associated with symptoms, but the double phenotype was strongly associated with pregnancy-related thrombophilia. The complex interactions between potential risk factors would explain to some degree the variability observed in the clinical symptoms associated with γ-dysfibrinogenemias. In the case of the R275, N308, and D364 mutants, the clinical manifestations associated with a given molecular defect varied greatly from one patient to another. A mutation associated with mild hemorrhage in one patient may appear to be silent in another, and yet may be associated with severe thrombophilia in another individual.

Finally, the analysis of the various fibrinogen γ chain mutants is complicated by many factors. First, most dysfibrinogens were identified in heterozygous individuals; therefore, the circulating fibrinogen is a heterozygous mixture of normal and mutant molecules. Second, apart from commonly used assays such as thrombin time and fibrinopeptide release, the biochemical characterization of these defects has not been standardized. Experiments are performed using different protocols, on plasma in some cases, and on purified fibrinogen in others, under varying conditions. This situation complicates the direct comparison of results from different laboratories, and may explain some of the apparent discrepancies between the observed effects of a given molecular defect.

CONCLUSIONS

As proposed in the fibrinogen subcommittee study,84 it would be valuable, in approaching a case of thrombophilic dysfibrinogenemia, to evaluate all other known contributing risk factors and to rigorously document the family history. This would help determine if the diagnosed dysfibrinogenemia is the sole thrombotic risk factor present. The same analysis should be performed for patients with bleeding symptoms and for asymptomatic individuals, if we are to determine with any degree of certainty the influence of the dysfibrinogenemia on the long-term manifestations of these defects. Finally, every effort should be made to accurately establish the diagnosis of a thrombosis. By combining well-documented family histories with correct diagnoses of symptoms, the possible associations between the various dysfibrinogenemias and thrombosis can then be assessed more accurately.

The recent determination of several crystal structures of fibrinogen fragments has shed light on the arrangement of domains at the distal nodules of fibrinogen, the architecture of the “a” polymerization pocket, and the interactions between amino acid residues that are critical for the various functions of this fascinating and complex molecule. As an early outgrowth of this work, the effects of mutations that cause dysfibrinogenemias can now be explored through further structure-function studies. Hypotheses originating from a consideration of the patients' symptoms and from an examination of the fibrinogen structure can now be explored and refined by experiments using recombinant protein expression systems. We expect that future studies will lead to a broader overall understanding of fibrinogen and its pathologies, and hopefully to improvements in the diagnosis and management of dysfibrinogenemic patients.

ACKNOWLEDGMENT

The authors thank Drs E.W. Davie, D.W. Chung, and F. Haverkate for critical reading of the manuscript. We are also grateful to Dr B. Stoddard for access to computing resources and to Jeff Harris for technical assistance. We thank Dr M. Mosesson for providing us with the EM photographs and for helpful comments, and Dr R.F. Doolittle and coworkers for permission to reproduce Fig 5A.

The coordinates of the various fibrinogen fragment crystal structures are available from the Brookhaven Data Bank, http://www.pdb.bnl.gov, with accession nos. 1FIB, 2FIB, 3FIB, 1FZA, and 1FZB.

Supported in part by National Institute of Health Grants No. HL31048 (to S.T.L.) and HL16919. H.C.F.C. was supported in part by a Research Fellowship from the Heart and Stroke Foundation of Canada.

Address reprint requests to Kathleen P. Pratt, PhD, Department of Biochemistry, Box 357350, University of Washington, Seattle, WA; 98195; e-mail: kpratt@u.washington.edu.

REFERENCES

REFERENCES
1
Shafer
JA
Higgins
DL
Human fibrinogen.
Crit Rev Clin Lab Sci
26
1988
1
2
Henschen
AH
Human fibrinogen—structural variants and functional sites.
Thromb Haemost
70
1993
42
3
Doolittle RF: Fibrinogen and fibrin, in Bloom F, Forbes CD, Thomas DP, Tuddenham EGD (eds): Haemostasis and Thrombosis, vol 1. Edinburgh, UK, Churchill Livingstone, 1994, p 491
4
Lord ST: Fibrinogen, in High KA, Roberts HR (eds): Molecular Basis of Thrombosis and Hemostasis. New York, NY, Dekker, 1995, p 51
5
Blombäck
B
Fibrinogen and fibrin-proteins with complex roles in hemostasis and thrombosis.
Thromb Res
83
1996
1
6
Mosesson
M
Fibrinogen and fibrin polymerization: Appraisal of the binding events that accompany fibrin generation and fibrin clot assembly.
Blood Coagul Fibrinol
8
1997
257
7
Nussenweig
V
Seligmann
M
Pelmont
J
Grabar
P
Les produits de dégradation du fibrinogène humain par la plasmine. 1-Séparation et propriétés physico-chimiques.
Ann Inst Pasteur
100
1961
377
8
Veklich
YI
Gorkun
OV
Medved
LV
Nieuwenhuizen
W
Weisel
JW
Carboxyl-terminal portions of the α chains of fibrinogen and fibrin. Localization by electron microscopy and the effects of isolated αC fragments on polymerization.
J Biol Chem
268
1993
13577
9
Gorkun
OV
Veklich
YI
Medved
LV
Henschen
AH
Weisel
JW
Role of the αC domains of fibrin in clot formation.
Biochemistry
33
1994
6986
10
Hantgan
R
McDonagh
J
Hermans
J
Fibrin assembly.
Ann NY Acad Sci
408
1983
344
11
Olexa
SA
Budzynski
AZ
Evidence for four different polymerization sites involved in human fibrin formation.
Proc Natl Acad Sci USA
77
1980
1374
12
Doolittle
RF
Laudano
AP
Synthetic peptide probes on the location of fibrin polymerization sites.
Protides Biological Fluids
28
1980
311
13
Hasegawa
N
Sasaki
S
Location of the binding site “b” for lateral polymerization of fibrin.
Thromb Res
57
1990
183
14
Medved
LV
Litvinovich
SV
Ugarova
TP
Lukinova
NI
Kalikhevich
VN
Ardemasova
ZA
Localization of a fibrin polymerization site complementary to Gly-His-Arg sequence.
FEBS Lett
320
1993
239
15
Blombäck
B
Blombäck
M
Nilsson
IM
Coagulation studies on “Reptilase”, an extract of the venom from Bothrops jararaca.
Thromb Diath Haemor
1
1957
76
16
Shainoff
JR
Page
IH
Cofibrins and fibrin-intermediates as indicators of thrombin activity in vivo.
Circ Res
8
1960
1013
17
Shainoff
JR
Dardik
BN
Fibrinopeptide B in fibrin assembly and metabolism: Physiologic significance in delayed release of the peptide.
Ann NY Acad Sci
408
1983
254
18
Weisel
JW
Veklich
Y
Gorkun
O
The sequence of cleavage of fibrinopeptides from fibrinogen is important for protofibril formation and enhancement of lateral aggregation in fibrin clots.
J Mol Biol
232
1993
285
19
Lorand
L
Konishi
K
Jacobsen
A
Transpeptidation mechanism in blood clotting.
Nature
194
1962
1148
20
Pisano
JJ
Finlayson
JS
Peyton
MP
Cross-link in fibrin polymerized by factor XIII: ε-(γ-glutamyl)lysine.
Science
160
1968
892
21
McKee
PA
Schwartz
ML
Pizzo
SV
Hill
RL
Cross-linking of fibrin by fibrin-stabilizing factor.
Ann NY Acad Sci
202
1972
127
22
Chen
R
Doolittle
RF
γ-γ cross-linking sites in human and bovine fibrin.
Biochemistry
10
1971
4487
23
Cierniewski
CS
Budzynski
AZ
Localization of the cross-linking site of GPRVVERHK in the γ-chain of human fibrinogen.
Eur J Biochem
218
1993
321
24
McDonagh
RP
McDonagh
J
Blombäck
M
Blombäck
B
Crosslinking of human fibrin: Evidence for intermolecular crosslinking involving α-chains.
FEBS Lett
14
1971
33
25
Cottrell
BA
Strong
DD
Watt
KW
Doolittle
RF
Amino acid sequence studies on the α chain of human fibrinogen. Exact location of cross-linking acceptor sites.
Biochemistry
18
1979
5405
26
Matsuka
YV
Medved
LV
Migliorini
MM
Ingham
KC
Factor XIIIa-catalyzed cross-linking of recombinant αC fragments of human fibrinogen.
Biochemistry
35
1996
5810
27
Sobel
JH
Gawinowicz
MA
Identification of the α chain lysine donor sites involved in factor XIIIa fibrin cross-linking.
J Biol Chem
271
1996
19288
28
McKee
PA
Mattock
P
Hill
RL
Subunit structure of human fibrinogen, soluble fibrin, and cross-linked insoluble fibrin.
Proc Natl Acad Sci USA
66
1970
738
29
Shen
L
Lorand
L
Contribution of fibrin stabilization to clot strength. Supplementation of factor XIII-deficient plasma with the purified zymogen.
J Clin Invest
71
1983
1336
30
Jansen
JW
Haverkate
F
Koopman
J
Niewenhuisen
HK
Kluft
C
Boschman
TA
Influence of factor XIIIa activity on whole blood clot lysis in vitro.
Thromb Haemost
57
1987
171
31
Francis
CW
Marder
VJ
Increased resistance to plasmic degradation of fibrin with highly crosslinked α-polymer chains formed at high factor XIII concentrations.
Blood
71
1988
1361
32
Siebenlist
KR
Mosesson
MW
Progressive cross-linking of fibrin γ chains increases resistance to fibrinolysis.
J Biol Chem
269
1994
28414
33
Wiman
B
Collen
D
Molecular mechanism of physiological fibrinolysis.
Nature
272
1978
549
34
Hoylaerts
M
Rijken
DC
Lijnen
HR
Collen
D
Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin.
J Biol Chem
257
1982
2912
35
Ranby
M
Studies on the kinetics of plasminogen activation by tissue plasminogen activator.
Biochim Biophys Acta
704
1982
461
36
Suenson
E
Bjerrum
P
Holm
A
Lind
B
Meldal
M
Selmer
J
Petersen
LC
The role of fragment X polymers in the fibrin enhancement of tissue plasminogen activator-catalyzed plasmin formation.
J Biol Chem
265
1990
22228
37
Bauer
R
Hansen
SL
Jones
G
Suenson
E
Thorsen
S
Ogendal
L
Fibrin structures during tissue-type plasminogen activator-mediated fibrinolysis studied by laser light scattering: Relation to fibrin enhancement of plasminogen activation.
Eur Biophys J
23
1994
239
38
Phillips
DR
Charo
IF
Parise
LV
Fitzgerald
LA
The platelet membrane glycoprotein IIb-IIIa complex.
Blood
71
1988
831
39
Dejana
E
Colella
S
Languino
LR
Balconi
G
Corbascio
GC
Marchisio
PC
Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro.
J Cell Biol
104
1987
1403
40
Dejana
E
Zanetti
A
Conforti
G
Biochemical and functional characteristics of fibrinogen interaction with endothelial cells.
Haemostasis
18
1988
262
41
Languino
LR
Plescia
J
Duperray
A
Brian
AA
Plow
EF
Geltosky
JE
Altieri
DC
Fibrinogen mediates leukocyte adhesion to vascular endothelium through an ICAM-1-dependent pathway.
Cell
73
1993
1423
42
Sriramarao
P
Languino
LR
Altieri
DC
Fibrinogen mediates leukocyte-endothelium bridging in vivo at low shear forces.
Blood
88
1996
3416
43
Suehiro
K
Gailit
J
Plow
EF
Fibrinogen is a ligand for integrin α5β1 on endothelial cells.
J Biol Chem
272
1997
5360
44
Altieri
DC
Bader
R
Mannucci
PM
Edgington
TS
Oligospecificity of the cellular adhesion receptor Mac-1 encompasses an inducible recognition specificity for fibrinogen.
J Cell Biol
107
1988
1893
45
Wright
SD
Weitz
JI
Huang
AJ
Levin
SM
Silverstein
SC
Loike
JD
Complement receptor type three (CD11b/CD18) of human polymorphonuclear leukocytes recognizes fibrinogen.
Proc Natl Acad Sci USA
85
1988
7734
46
Trezzini
C
Jungi
TW
Kuhnert
P
Peterhans
E
Fibrinogen association with human monocytes: Evidence for constitutive expression of fibrinogen receptors and for involvement of Mac-1 (CD18, CR3) in the binding.
Biochem Biophys Res Commun
156
1988
477
47
Gustafson
EJ
Lukasiewicz
H
Wachtfogel
YT
Norton
KJ
Schmaier
AH
Niewiarowski
S
Colman
RW
High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets.
J Cell Biol
109
1989
377
48
Godyna
S
Diaz Ricart
M
Argraves
WS
Fibulin-1 mediates platelet adhesion via a bridge of fibrinogen.
Blood
88
1996
2569
49
Nieuwenhuizen
W
Voskuilen
M
Vermond
A
Haverkate
F
Hermans
J
A fibrinogen fragment D (D intermediate) with calcium binding but without anticlotting properties.
Biochim Biophys Acta
707
1982
190
50
Southan
C
Thompson
E
Panico
M
Etienne
T
Morris
HR
Lane
DA
Characterization of peptides cleaved by plasmin from the C-terminal polymerization domain of human fibrinogen.
J Biol Chem
260
1985
13095
51
Dang
CV
Ebert
RF
Bell
WR
Localization of a fibrinogen calcium binding site between γ-subunit positions 311 and 336 by terbium fluorescence.
J Biol Chem
260
1985
9713
52
Váradi
A
Scheraga
HA
Localization of segments essential for polymerization and for calcium binding in the γ-chain of human fibrinogen.
Biochemistry
25
1986
519
53
Shimizu
A
Nagel
GM
Doolittle
RF
Photoaffinity labeling of the primary fibrin polymerization site: isolation and characterization of a labeled cyanogen bromide fragment corresponding to γ-chain residues 337-379.
Proc Natl Acad Sci USA
89
1992
2888
54
Yamazumi
K
Doolittle
RF
Photoaffinity labeling of the primary fibrin polymerization site: localization of the label to γ-chain Tyr-363.
Proc Natl Acad Sci USA
89
1992
2893
55
Yee
VC
Pratt
KP
Côté
HCF
Le Trong
I
Chung
DW
Davie
EW
Stenkamp
RE
Teller
DC
Crystal structure of a 30 kDa C-terminal fragment from the γ chain of human fibrinogen.
Structure
5
1997
125
56
Pratt
KP
Côté
HCF
Chung
DW
Stenkamp
RE
Davie
EW
The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro.
Proc Natl Acad Sci USA
94
1997
7176
57
Kloczewiak
M
Timmons
S
Hawiger
J
Recognition site for the platelet receptor is present on the 15-residue carboxy-terminal fragment of the γ chain of human fibrinogen and is not involved in the fibrin polymerization reaction.
Thromb Res
29
1983
249
58
Kloczewiak
M
Timmons
S
Lukas
TJ
Hawiger
J
Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the γ chain.
Biochemistry
23
1984
1767
59
Hettasch
JM
Bolyard
MG
Lord
ST
The residues AGDV of recombinant γ chains of human fibrinogen must be carboxy-terminal to support human platelet aggregation.
Thromb Haemost
68
1992
701
60
Rooney
MM
Parise
LV
Lord
ST
Dissecting clot retraction and platelet aggregation. Clot retraction does not require an intact fibrinogen γ chain C terminus.
J Biol Chem
271
1996
8553
61
Trezzini
C
Jungi
TW
Maly
FE
Vittoz
M
Peterhans
E
Low-affinity interaction of fibrinogen carboxy-gamma terminus with human monocytes induces an oxidative burst and modulates effector functions.
Biochem Biophys Res Commun
165
1989
7
62
Altieri
DC
Plescia
J
Plow
EF
The structural motif glycine 190-valine 202 of the fibrinogen γ chain interacts with CD11b/CD18 integrin (αMβ2, Mac-1) and promotes leukocyte adhesion.
J Biol Chem
268
1993
1847
63
Medved
L
Litvinovich
S
Ugarova
T
Matsuka
Y
Ingham
K
Domain structure and functional activity of the recombinant human fibrinogen γ-module (γ148-411).
Biochemistry
36
1997
4685
64
Váradi
A
Patthy
L
Location of plasminogen-binding sites in human fibrin(ogen).
Biochemistry
22
1983
2440
65
Schielen
WJ
Adams
HP
van Leuven
K
Voskuilen
M
Tesser
GI
Nieuwenhuizen
W
The sequence γ-(312-324) is a fibrin-specific epitope.
Blood
77
1991
2169
66
Yonekawa
O
Voskuilen
M
Nieuwenhuizen
W
Localization in the fibrinogen γ-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen.
Biochem J
283
1992
187
67
Kaczmarek
E
Lee
MH
McDonagh
J
Initial interaction between fibrin and tissue plasminogen activator (t-PA). The Gly-Pro-Arg-Pro binding site on fibrin(ogen) is important for t-PA activity.
J Biol Chem
268
1993
2474
68
Nieuwenhuizen
W
Sites in fibrin involved in the acceleration of plasminogen activation by t-PA. Possible role of fibrin polymerisation.
Thromb Res
75
1994
343
69
Meade
TW
Mellows
S
Brozovic
M
Miller
GJ
Chakrabarti
RR
North
WR
Haines
AP
Stirling
Y
Imeson
JD
Thompson
SG
Haemostatic function and ischaemic heart disease: Principal results of the Northwick Park Heart Study [see comments].
Lancet
2
1986
533
70
Kannel
WB
Wolf
PA
Castelli
WP
D'Agostino
RB
Fibrinogen and risk of cardiovascular disease. The Framingham Study.
JAMA
258
1987
1183
71
Kannel
WB
D'Agostino
RB
Belanger
AJ
Update on fibrinogen as a cardiovascular risk factor.
Ann Epidemiol
2
1992
457
72
Ernst
E
Fibrinogen: An important risk factor for atherothrombotic diseases.
Ann Med
26
1994
15
73
de la Serna
G
Fibrinogen: A major new risk factor for cardiovascular disease. A review of the literature.
J Fam Pract
39
1994
468
74
Ernst
E
Fibrinogen: Its emerging role as a cardiovascular risk factor.
Angiology
45
1994
87
75
Fatah
K
Hamsten
A
Blombäck
B
Blombäck
M
Fibrin gel network characteristics and coronary heart disease: Relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis.
Thromb Haemost
68
1992
130
76
Dintenfass
L
Erythrocyte aggregation and cardiovascular risk factors.
Clin Hemorheol
8
1988
237
77
Blombäck
B
Carlsson
K
Fatah
K
Hessel
B
Procyk
R
Fibrin in human plasma: Gel architectures governed by rate and nature of fibrinogen activation.
Thromb Res
75
1994
521
78
Galanakis
DK
Fibrinogen anomalies and disease. A clinical update.
Hematol Oncol Clin North Am
6
1992
1171
79
Miletich
JP
Prescott
SM
White
R
Majerus
PW
Bovill
EG
Inherited predisposition to thrombosis.
Cell
72
1993
477
80
Eby
CS
A review of the hypercoagulable state.
Hematol Oncol Clin North Am
7
1993
1121
81
[see comments in Ann Intern Med 119:819, 1993]
Nachman
RL
Silverstein
R
Hypercoagulable states.
Ann Intern Med
119
1993
819
82
Sham
RL
Francis
CW
Evaluation of mild bleeding disorders and easy bruising.
Blood Rev
8
1994
98
83
Schafer
AI
Hypercoagulable states: Molecular genetics to clinical practice.
Lancet
344
1994
1739
84
Haverkate
F
Samama
M
Familial dysfibrinogenemia and thrombophilia. Report on a study of the SSC Subcommittee on Fibrinogen.
Thromb Haemost
73
1995
151
85
Rosendaal
FR
Risk factors for venous thrombosis: Prevalence, risk, and interaction.
Semin Hematol
34
1997
171
86
Koeleman
BPC
Reitsma
PH
Bertina
RM
Familial thrombophilia: A complex genetic disorder.
Semin Hematol
34
1997
256
87
Koopman J, Haverkate F: Hereditary variants of human fibrinogens, in Bloom F, Forbes CD, Thomas DP, Tuddenham EGD (eds): Haemostasis and Thrombosis, vol 1. Edinburgh, UK, Churchill Livingstone, 1994, p 515
88
Ebert RF (ed): Index of Variant Human Fibrinogens. Boca Raton, FL, CRC, 1994
89
Hall
C
Slayter
H
The fibrinogen molecule: Its size, shape, and mode of polymerization.
J Biophys Biochem Cyt
5
1959
11
90
Stryer
L
Cohen
C
Langridge
R
Axial period of fibrinogen and fibrin.
Nature
197
1963
793
91
Cohen
C
Tooney
NM
Crystallisation of a modified fibrinogen.
Nature
251
1974
659
92
Weisel
JW
Warren
SG
Cohen
C
Crystals of modified fibrinogen: Size, shape and packing of molecules.
J Mol Biol
126
1978
159
93
Fowler
WE
Erickson
HP
Trinodular structure of fibrinogen. Confirmation by both shadowing and negative stain electron microscopy.
J Mol Biol
134
1979
241
94
Weisel
JW
Stauffacher
CV
Bullitt
E
Cohen
C
A model for fibrinogen: Domains and sequence.
Science
230
1985
1388
95
Siebenlist
KR
Mosesson
MW
Di Orio
JP
Tavori
S
Tatarsky
I
Rimon
A
The polymerization of fibrin prepared from fibrinogen Haifa (γ275Arg → His).
Thromb Haemost
62
1989
875
96
Koopman
J
Haverkate
F
Grimbergen
J
Lord
ST
Mosesson
MW
DiOrio
JP
Siebenlist
KS
Legrand
C
Soria
J
Soria
C
Caen
JP
Molecular basis for fibrinogen Dusart (Aα 554 Arg → Cys) and its association with abnormal fibrin polymerization and thrombophilia.
J Clin Invest
91
1993
1637
97
Spraggon
G
Everse
SJ
Doolittle
RF
Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin.
Nature
389
1997
455
98
Côté
HCF
Pratt
KP
Davie
EW
Chung
DW
The polymerization pocket “a” within the carboxyl-terminal region of the γ chain of human fibrinogen is adjacent to but independent from the calcium-binding site.
J Biol Chem
272
1997
23792
99
Doolittle
RF
A detailed consideration of a principal domain of vertebrate fibrinogen and its relatives.
Protein Sci
1
1992
1563
100
Mosesson
MW
Siebenlist
KR
DiOrio
JP
Matsuda
M
Hainfeld
JF
Wall
JS
The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (γ275 Arg →Cys).
J Clin Invest
96
1995
1053
101
Niwa
K
Takebe
M
Sugo
T
Kawata
Y
Mimuro
J
Asakura
S
Sakata
Y
Mizushima
J
Maeda
A
Endo
H
Matsuda
M
A γ Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I.
Blood
87
1996
4686
102
Mayo
KH
Burke
C
Lindon
JN
Kloczewiak
M
1H NMR sequential assignments and secondary structure analysis of human fibrinogen γ-chain C-terminal residues 385-411.
Biochemistry
29
1990
3277
103
Blumenstein
M
Matsueda
GR
Timmons
S
Hawiger
J
A β-turn is present in the 392-411 segment of the human fibrinogen γ-chain. Effects of structural changes in this segment on affinity to antibody 4A5.
Biochemistry
31
1992
10692
104
Donahue
JP
Patel
H
Anderson
WF
Hawiger
J
Three-dimensional structure of the platelet integrin recognition segment of the fibrinogen γ chain obtained by carrier protein-driven crystallization.
Proc Natl Acad Sci USA
91
1994
12178
105
Fan
F
Mayo
KH
Effect of pH on the conformation and backbone dynamics of a 27-residue peptide in trifluorethanol. An NMR and CD study.
J Biol Chem
270
1995
24693
106
Mayo
KH
Fan
F
Beavers
MP
Eckardt
A
Keane
P
Hoekstra
WJ
Andrade-Gordon
P
Integrin receptor GPIIb/IIIa bound state conformation of the fibrinogen γ-chain C-terminal peptide 400-411: NMR and transfer NOE studies.
Biochemistry
35
1996
4434
107
Dang
CV
Bell
WR
Shuman
M
The normal and morbid biology of fibrinogen.
Am J Med
87
1989
567
108
Matsuda M, Yoshida N, Terukina S, Yamazumi K, Maekawa H: Molecular abnormalities of fibrinogen—The present status of structure elucidation, in Matsuda M, Iwanaga S, Takada A, Henschen A (eds): Fibrinogen 4. Current Basic and Clinical aspects. Amsterdam, The Netherlands, Elsevier, 1990, p 139
109
Galanakis
DK
Inherited dysfibrinogenemia: Emerging abnormal structure associations with pathologic and nonpathologic dysfunctions.
Semin Thromb Hemost
19
1993
386
110
Matsuda
M
The structure-function relationship of hereditary dysfibrinogens.
Int J Hematol
64
1996
167
111
Brown
WM
Dziegielewska
KM
Foreman
RC
Saunders
NR
Nucleotide and deduced amino acid sequence of a γ subunit of bovine fibrinogen.
Nucleic Acids Res
17
1989
6397
112
Strong
DD
Moore
M
Cottrell
BA
Bohonus
VL
Pontes
M
Evans
B
Riley
M
Doolittle
RF
Lamprey fibrinogen γ chain: Cloning, cDNA sequencing, and general characterization.
Biochemistry
24
1985
92
113
Terukina
S
Matsuda
M
Hirata
H
Takeda
Y
Miyata
T
Takao
T
Shimonishi
Y
Substitution of γ Arg-275 by Cys in an abnormal fibrinogen, “fibrinogen Osaka II.” Evidence for a unique solitary cystine structure at the mutation site.
J Biol Chem
263
1988
13579
114
Yoshida
N
Ota
K
Moroi
M
Matsuda
M
An apparently higher molecular weight γ-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of γ arginine-275 by cysteine.
Blood
71
1988
480
115
Terukina
S
Matsuda
M
Yoshida
N
Yamazumi
K
Takeda
Y
Takano
T
Two abnormal fibrinogens designated as Osaka II and Morioka with a hitherto unidentified amino acid substitution; γArg-275 by Cys (abstr).
Thromb Haemost
58
1987
515
116
Ebert
RF
Bell
WR
Fibrinogen Baltimore IV: Congenital dysfibrinogenemia with delayed fibrin monomer polymerization.
Thromb Res
38
1985
121
117
Schmelzer
CH
Ebert
RF
Bell
WR
Fibrinogen Baltimore IV: Congenital dysfibrinogenemia with a γ275 (Arg → Cys) substitution.
Thromb Res
56
1989
307
118
Matsuda
M
Baba
M
Morimoto
K
Nakamikawa
C
“Fibrinogen Tokyo II”. An abnormal fibrinogen with an impaired polymerization site on the aligned DD domain of fibrin molecules.
J Clin Invest
72
1983
1034
119
Steinmann
C
Bögli
C
Jungo
M
Lämmle
B
Heinemann
G
Wermuth
B
Redaelli
R
Baudo
F
Furlan
M
Fibrinogen Milano V: A congenital dysfibrinogenaemia with a γ275 Arg → Cys substitution.
Blood Coagul Fibrinol
5
1994
463
120
Borrell
M
Garı́
M
Coll
I
Vallvé
C
Tirado
I
Soria
JM
Sala
N
Muñoz
C
Oliver
A
Garcı́a
A
Fontcuberta
J
Abnormal polymerization and normal binding of plasminogen and t-PA in three new dysfibrinogenaemias: Barcelona III and IV (γArg 275 → His) and Villajoyosa (γArg 275 → Cys).
Blood Coagul Fibrinol
6
1995
198
121
Haverkate
F
Timan
G
Nieuwenhuizen
W
Anticlotting properties of fragments D from human fibrinogen and fibrin.
Eur J Clin Invest
9
1979
253
122
Mosesson MW, Siebenlist KR, Olson JD: Thrombophilia associated with dysfibrinogenemia [fibrinogen Cedar Rapids (γR275C)] and a heterozygous factor V Leiden defect. Thromb Haemost OC-1560:382, 1997 (abstr, suppl)
123
van den Meer FJ, Koster T, Vandenbroucke JP, Briët E, Rosendaal FR: The Leiden thrombophilia study (LETS), Thromb Haemost 78:631, 1997
124
Simioni
P
Prandoni
P
Lensing
AW
Scudeller
A
Sardella
C
Prins
MH
Villalta
S
Dazzi
F
Girolami
A
The risk of recurrent venous thromboembolism in patients with an Arg506 → Gln mutation in the gene for factor V (factor V Leiden).
N Engl J Med
336
1997
399
125
Dahlback
B
Resistance to activated protein C as risk factor for thrombosis: Molecular mechanisms, laboratory investigation, and clinical management.
Semin Hematol
34
1997
217
126
Reber
P
Furlan
M
Henschen
A
Kaudewitz
H
Barbui
T
Hilgard
P
Nenci
GG
Berrettini
M
Beck
EA
Three abnormal fibrinogen variants with the same amino acid substitution (γ275 Arg → His): Fibrinogens Bergamo II, Essen and Perugia.
Thromb Haemost
56
1986
401
127
Yoshida
N
Imaoka
S
Hirata
H
Matsuda
M
Asakura
S
Heterozygous abnormal fibrinogen Osaka III with the replacement of γ arginine-275 by histidine has an apparently higher molecular weight γ-chain variant.
Thromb Haemost
68
1992
534
128
Steinmann
C
Jungo
M
Beck
EA
Lämmle
B
Furlan
M
Fibrinogen Claro—Another dysfunctional fibrinogen variant with γ275 arginine → histidine substitution [published erratum appears in Thromb Res 82:107, 1996].
Thromb Res
81
1996
145
129
Yamazumi
K
Terukina
S
Onohara
S
Matsuda
M
Normal plasmic cleavage of the γ-chain variant of “fibrinogen Saga” with an Arg-275 to His substitution.
Thromb Haemost
60
1988
476
130
Brook
JG
Tabori
S
Tatarsky
I
Hashmonai
M
Schramek
A
Fibrinogen “Haifa”—A new fibrinogen variant. A case report.
Haemostasis
13
1983
277
131
Soria
J
Soria
C
Samama
M
Tabori
S
Kehl
M
Henschen
A
Nieuwenhuizen
W
Rimon
A
Tatarski
I
Fibrinogen Haifa: Fibrinogen variant with absence of protective effect of calcium on plasmin degradation of gamma chains.
Thromb Haemost
57
1987
310
132
Haverkate
F
Timan
G
Protective effect of calcium in the plasmin degradation of fibrinogen and fibrin fragments D.
Thromb Res
10
1977
803
133
Beck
EA
Charache
P
Jackson
DP
A new inherited coagulation disorder caused by an abnormal fibrinogen (‘Fibrinogen Baltimore’).
Nature
208
1965
143
134
Mosesson MW, Beck EA: Chromatographic, ultracentrifugal, and related studies of fibrinogen “Baltimore.” J Clin Invest 48:1656, 1969
135
Beck EA, Shainoff JR, Vogel A, Jackson DP: Functional evaluation of an inherited abnormal fibrinogen: Fibrinogen “Baltimore.” J Clin Invest 50:1874, 1971
136
Brown
CH
Crowe
MF
Defective α-polymerization in the conversion of fibrinogen Baltimore to fibrin.
J Clin Invest
55
1975
1190
137
Bantia
S
Mane
SM
Bell
WR
Dang
CV
Fibrinogen Baltimore I: Polymerization defect associated with a γ292Gly → Val (GGC→GTC) mutation.
Blood
76
1990
2279
138
Wolfenden
R
Andersson
L
Cullis
PM
Southgate
CC
Affinities of amino acid side chains for solvent water.
Biochemistry
20
1981
849
139
Wesson
L
Eisenberg
D
Atomic solvation parameters applied to molecular dynamics of proteins in solution.
Protein Sci
1
1992
227
140
Ebert
RF
Bell
WR
Fibrinogen Baltimore III: Congenital dysfibrinogenemia with a shortened γ-subunit.
Thromb Res
51
1988
251
141
Bantia
S
Bell
WR
Dang
CV
Polymerization defect of fibrinogen Baltimore III due to a γ Asn308 → Ile mutation.
Blood
75
1990
1659
142
Yoshida
N
Okuma
M
Moroi
M
Matsuda
M
A lower molecular weight γ-chain variant in a congenital abnormal fibrinogen (Kyoto).
Blood
68
1986
703
143
Yoshida
N
Terukina
S
Okuma
M
Moroi
M
Aoki
N
Matsuda
M
Characterization of an apparently lower molecular weight γ-chain variant in fibrinogen Kyoto I. The replacement of γ-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site.
J Biol Chem
263
1988
13848
144
Grailhe
P
Boyer-Neumann
C
Haverkate
F
Grimbergen
J
Larrieu
MJ
Anglés-Cano
E
The mutation in fibrinogen Bicêtre II (γ Asn308 → Lys) does not affect the binding of t-PA and plasminogen to fibrin.
Blood Coagul Fibrinol
4
1993
679
145
Okumura
N
Furihata
K
Terasawa
F
Ishikawa
S
Ueno
I
Katsuyama
T
Fibrinogen Matsumoto II: γ308 Asn → Lys (AAT → AAG) mutation associated with bleeding tendency.
Br J Haematol
94
1996
526
146
Yamazumi
K
Shimura
K
Terukina
S
Takahashi
N
Matsuda
M
A γ methionine-310 to threonine substitution and consequent N-glycosylation at γ asparagine-308 identified in a congenital dysfibrinogenemia associated with posttraumatic bleeding, fibrinogen Asahi.
J Clin Invest
83
1989
1590
147
Yamazumi
K
Shimura
K
Maekawa
H
Muramatsu
S
Terukina
S
Matsuda
M
Delayed intermolecular γ-chain cross-linking by factor XIIIa in fibrinogen Asahi characterized by a γ-Met-310 to Thr substitution with an N-glycosylated γ-Asn-308.
Blood Coagul Fibrinol
1
1990
557
148
Dang
CV
Shin
CK
Bell
WR
Nagaswami
C
Weisel
JW
Fibrinogen sialic acid residues are low affinity calcium-binding sites that influence fibrin assembly.
J Biol Chem
264
1989
15104
149
Martinez
J
Palascak
J
Peters
C
Functional and metabolic properties of human asialofibrinogen.
J Lab Clin Med
89
1977
367
150
Langer
BG
Weisel
JW
Dinauer
PA
Nagaswami
C
Bell
WR
Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers.
J Biol Chem
263
1988
15056
151
Maekawa
H
Yamazumi
K
Muramatsu
S
Kaneko
M
Hirata
H
Takahashi
N
Arocha-Piñango
CL
Rodriguez
S
Nagy
H
Perez-Requejo
JL
Matsuda
M
Fibrinogen Lima: A homozygous dysfibrinogen with an Aα-arginine-141 to serine substitution associated with extra N-glycosylation at Aα-asparagine-139. Impaired fibrin gel formation but normal fibrin-facilitated plasminogen activation catalyzed by tissue-type plasminogen activator.
J Clin Invest
90
1992
67
152
Koopman
J
Haverkate
F
Briët
E
Lord
ST
A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the γ-chain gene, causing defective calcium binding and impaired fibrin polymerization.
J Biol Chem
266
1991
13456
153
Takamatsu
J
Iwanaga
S
Studies on abnormal fibrinogen.
Nippon Ketsueki Gakkai Zasshi Acta Haematologica Japan
43
1980
1203
154
Mizuochi
T
Taniguchi
T
Asami
Y
Takamatsu
J
Okude
M
Iwanaga
S
Kobata
A
Comparative studies on the structures of the carbohydrate moieties of human fibrinogen and abnormal fibrinogen Nagoya.
J Biochem Tokyo
92
1982
283
155
Miyata
T
Furukawa
K
Iwanaga
S
Takamatsu
J
Saito
H
Fibrinogen Nagoya, a replacement of glutamine-329 by arginine in the γ-chain that impairs the polymerization of fibrin monomer.
J Biochem Tokyo
105
1989
10
156
Reber
P
Furlan
M
Rupp
C
Kehl
M
Henschen
A
Mannucci
PM
Beck
EA
Characterization of fibrinogen Milano I: Amino acid exchange γ330 Asp → Val impairs fibrin polymerization.
Blood
67
1986
1751
157
Terukina
S
Yamazumi
K
Okamoto
K
Yamashita
H
Ito
Y
Matsuda
M
Fibrinogen Kyoto III: A congenital dysfibrinogen with a γ aspartic acid-330 to tyrosine substitution manifesting impaired fibrin monomer polymerization.
Blood
74
1989
2681
158
Rupp
C
Kuyas
C
Haeberli
A
Furlan
M
von Fliedner
V
Beck
EA
Fibrinogen Bern I and fibrinogen Bern II: 2 hereditary fibrinogen variants with diverse biochemical properties.
Schweiz Med Wochenschr
111
1981
1543
159
Steinmann
C
Reber
P
Jungo
M
Lämmle
B
Heinemann
G
Wermuth
B
Furlan
M
Fibrinogen Bern I: Substitution γ 337 Asn → Lys is responsible for defective fibrin monomer polymerization.
Blood
82
1993
2104
160
(suppl)
Ménaché
D
Constitutional and familial abnormal fibrinogen.
Thromb Diath Haemorrh
13
1964
173
161
Budzynski
AZ
Marder
VJ
Ménaché
D
Guillin
M-C
Defect in the gamma polypeptide chain of a congenital abnormal fibrinogen.
Nature
252
1974
66
162
Mosesson
MW
Amrani
DL
Ménaché
D
Studies on the structural abnormality of fibrinogen Paris I.
J Clin Invest
57
1976
782
163
Mosesson
MW
Feldmann
G
Ménaché
D
Electron microscopy of fibrin Paris I.
Blood
56
1980
80
164
Denninger
M-H
Jandrot-Perrus
M
Elion
J
Bertrand
O
Homandberg
GA
Mosesson
MW
Guillin
M-C
ADP-induced platelet aggregation depends on the conformation or availability of the terminal gamma chain sequence of fibrinogen. Study of the reactivity of fibrinogen Paris 1.
Blood
70
1987
558
165
Rosenberg
JB
Newman
PJ
Mosesson
MW
Guillin
M-C
Amrani
DL
Paris I dysfibrinogenemia: A point mutation in intron 8 results in insertion of a 15 amino acid sequence in the fibrinogen γ-chain.
Thromb Haemost
69
1993
217
166
Steinmann
C
Bögli
C
Jungo
M
Lämmle
B
Heinemann
G
Wermuth
B
Redaelli
R
Baudo
F
Furlan
M
A new substitution, γ358 Ser → Cys, in fibrinogen Milano VII causes defective fibrin polymerization.
Blood
84
1994
1874
167
Okumura
N
Furihata
K
Terasawa
F
Nakagoshi
R
Ueno
I
Katsuyama
T
Fibrinogen Matsumoto I: A γ364 Asp → His (GAT → CAT) substitution associated with defective fibrin polymerization.
Thromb Haemost
75
1996
887
168
Bentolila
S
Samama
MM
Conard
J
Horellou
MH
Ffrench
P
Association of dysfibrinogenemia and thrombosis. Apropos of a family (Fibrinogen Melun) and review of the literature.
Ann Med Interne Paris
146
1995
575
169
Okumura
N
Gorkun
OV
Lord
ST
Severely impaired polymerization of recombinant fibrinogen γ364 Asp → His, the substitution discovered in a heterozygous individual.
J Biol Chem
272
1997
29596
170
Yamazumi
K
Doolittle
RF
The synthetic peptide Gly-Pro-Arg-Pro-amide limits the plasmic digestion of fibrinogen in the same fashion as calcium ion.
Protein Sci
1
1992
1719
171
Dang
CV
Bell
WR
Ebert
RF
Starksen
NF
Protective effect of divalent cations in the plasmin degradation of fibrinogen.
Arch Biochem Biophys
238
1985
452
172
Yoshida
N
Hirata
H
Morigami
Y
Imaoka
S
Matsuda
M
Yamazumi
K
Asakura
S
Characterization of an abnormal fibrinogen Osaka V with the replacement of γ-arginine 375 by glycine. The lack of high affinity calcium binding to D-domains and the lack of protective effect of calcium on fibrinolysis.
J Biol Chem
267
1992
2753
173
Purves
LR
Lindsey
GG
Brown
G
Franks
J
Stabilization of the plasmin digestion products of fibrinogen and fibrin by calcium ions.
Thromb Res
12
1978
473
174
Nieuwenhuizen
W
Vermond
A
Nooijen
WJ
Haverkate
F
Calcium-binding properties of human fibrin(ogen) and degradation products.
FEBS Lett
98
1979
257
175
Marguerie
G
Ardaillou
N
Potential role of the Aα chain in the binding of calcium to human fibrinogen.
Biochim Biophys Acta
701
1982
410
176
Nieuwenhuizen
W
Haverkate
F
Calcium-binding regions in fibrinogen.
Ann NY Acad Sci
408
1983
92
177
Ridgway
HJ
Brennan
SO
Loreth
RM
George
PM
Fibrinogen Kaiserslautern (γ380 Lys to Asn): A new glycosylated fibrinogen variant with delayed polymerization.
Br J Haematol
99
1997
562
178
Lindsey
GG
Brown
G
Purves
LR
Calcium binding to human fibrinogen—Localization of two calcium specific sites.
Thromb Res
13
1978
345
179
Boyer
MH
Shainoff
JR
Ratnoff
OD
Acceleration of fibrin polymerization by calcium ions.
Blood
39
1972
382
180
Laudano
AP
Doolittle
RF
Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements, number of binding sites, and species differences.
Biochemistry
19
1980
1013
181
Laudano
AP
Doolittle
RF
Influence of calcium ion on the binding of fibrin amino terminal peptides to fibrinogen.
Science
212
1981
457
182
Laudano
AP
Cottrell
BA
Doolittle
RF
Synthetic peptides modeled on fibrin polymerization sites.
Ann NY Acad Sci
408
1983
315
183
Lorand
L
Losowsky
MS
Miloszewski
KJ
Human factor XIII: Fibrin-stabilizing factor.
Prog Hemost Thromb
5
1980
245
184
Purves L, Purves M: The structure of the human fibrin gamma-chain crosslink, in Mosesson MW, Amrani DL, Siebenlist KR, DiOrio JP (eds): Fibrinogen 3. Biochemistry, Biological Functions, Gene Regulation and Expression. Amsterdam, The Netherlands, Elsevier Science, 1988, p 95
185
Kloczewiak
M
Timmons
S
Hawiger
J
Localization of a site interacting with human platelet receptor on carboxy-terminal segment of human fibrinogen γ chain.
Biochem Biophys Res Commun
107
1982
181
186
Farrell
DH
Thiagarajan
P
Chung
DW
Davie
EW
Role of fibrinogen α and γ chain sites in platelet aggregation.
Proc Natl Acad Sci USA
89
1992
10729
187
Weisel
JW
Nagaswami
C
Vilaire
G
Bennett
JS
Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy.
J Biol Chem
267
1992
16637
188
Carrell
N
Gabriel
DA
Blatt
PM
Carr
ME
McDonagh
J
Hereditary dysfibrinogenemia in a patient with thrombotic disease.
Blood
62
1983
439
189
Collet
J-P
Soria
J
Mirshahi
M
Hirsch
M
Dagonnet
FB
Caen
J
Soria
C
Dusart syndrome: A new concept of the relationship between fibrin clot architecture and fibrin clot degradability: Hypofibrinolysis related to an abnormal clot structure.
Blood
82
1993
2462
190
Lijnen
HR
Soria
J
Soria
C
Collen
D
Caen
JP
Dysfibrinogenemia (fibrinogen Dusard) associated with impaired fibrin-enhanced plasminogen activation.
Thromb Haemost
51
1984
108
191
Ieko
M
Sawada
K
Sakurama
S
Yamagishi
I
Isogawa
S
Nakagawa
S
Satoh
M
Yasukouchi
T
Matsuda
M
Fibrinogen Date: Congenital hypodysfibrinogenemia associated with decreased binding of tissue-plasminogen activator.
Am J Hematol
37
1991
228
192
Gabriel
DA
Muga
K
Boothroyd
EM
The effect of fibrin structure on fibrinolysis.
J Biol Chem
267
1992
24259
193
Nachman
RL
Silverstein
R
Hypercoagulable states [see comments].
Ann Intern Med
119
1993
819
194
Lane
DA
Mannucci
PM
Bauer
KA
Bertina
RM
Bochkov
NP
Boulyjenkov
V
Chandy
M
Dahlback
B
Ginter
EK
Miletich
JP
Rosendaal
FR
Seligsohn
U
Inherited thrombophilia: Part 1.
Thromb Haemost
76
1996
651
195
Schorer
AE
Singh
J
Basara
ML
Dysfibrinogenemia: A case with thrombosis (fibrinogen Richfield) and an overview of the clinical and laboratory spectrum.
Am J Hematol
50
1995
200
196
Hessel
B
Silveira
AM
Carlsson
K
Oksa
H
Rasi
V
Vahtera
E
Procyk
R
Blombäck
B
Fibrinogenemia Tampere—A dysfibrinogenemia with defective gelation and thromboembolic disease.
Thromb Res
78
1995
323
197
Lane
DA
VanRoss
M
Kakkar
VV
Bottomley
KJ
Dhir
K
Holt
LP
MacIver
JE
An abnormal fibrinogen with delayed fibrinopeptide A release.
Br J Haematol
46
1980
89
198
Ruf
W
Bender
A
Lane
DA
Preissner
KT
Selmayr
E
Müller-Berghaus
G
Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: Influence of inhibitors of fibrin polymerization.
Biochim Biophys Acta
965
1988
169
199
Furlan
M
Stucki
B
Steinmann
C
Jungo
M
Lämmle
B
Normal binding of calcium to five fibrinogen variants with mutations in the carboxy terminal part of the γ-chain.
Thromb Haemost
76
1996
377