A novel mutation (deletion of Aalpha-Asn 80) in an abnormal fibrinogen: fibrinogen Caracas VI. Consequences of disruption of the coiled coil for the polymerization of fibrin: peculiar clot structure and diminished stiffness of the clot.

An abnormal fibrinogen was identified in a 10-year-old male with a mild bleeding tendency; several years later, the patient developed a thrombotic event. Fibrin polymerization of plasma from the propositus and his mother, as measured by turbidity, was impaired. Plasmin digestion of fibrinogen and thrombin bound to the clot were both normal. The structure of clots from both plasma and purified fibrinogen was characterized by permeability, scanning electron microscopy and rheological measurements. Permeability of patients' clots was abnormal, although some measurements were not reliable because the clots were not mechanically stable. Consistent with these results, the stiffness of patients' clots was decreased approximately two-fold. Electron microscopy revealed that the patients' clots were very heterogeneous in structure. DNA sequencing of the propositus and his mother revealed a new unique point mutation that gives rise to a fibrinogen molecule with a missing amino acid residue at Aalpha-Asn 80. This new mutation, which would disrupt the alpha-helical coiled-coil structure, emphasizes the importance of this part of the molecule for fibrin polymerization and clot structure. This abnormal fibrinogen has been named fibrinogen Caracas VI.     

A novel fibrinogen variant (fibrinogen Seoul II; AalphaGln328Pro) characterized by impaired fibrin alpha-chain cross-linking

We report a novel fibrinogen variant (fibrinogen Seoul II), which has a heterozygous point mutation from CAA to CCA leading to AalphaGln328Pro. The mutation site is among several glutamine residues that serve as alpha-chain cross-linking acceptor sites. Fibrinogen Seoul II was found in a 51-year-old male patient and his family in Seoul, Korea. The patient was diagnosed with myocardial infarction at age 43. Eight years later he was admitted to the emergency room due to recurrence of the disease, where he expired under treatment with tissue plasminogen activator (t-PA). Fibrin polymerization curves, made using purified fibrinogen from the patient's relatives, showed a decreased final turbidity, suggesting Seoul II fibrin clots are composed of thinner fibers. This supposition was verified using scanning electron microscopy. Alpha-polymer formation by the mutant fibrinogen upon thrombin treatment in the presence of factor XIII and calcium was distinctly impaired. This result confirms that the residue Aalpha328 plays a pivotal role in alpha-chain cross-linking.     

Functional characterization of fibrinogen Bicêtre II: a gamma 308 Asn-->Lys mutation located near the fibrin D:D interaction sites.

The effects of the gamma-308 Asn-->Lys substitution of fibrinogen Bicêtre II on clot formation, structure and properties were determined to elucidate the role of this part of the molecule in fibrin polymerization. This process was followed by measurement of turbidity, and the structure and biophysical characteristics of the clots were studied by permeation, scanning electron microscopy, and rheological techniques. Turbidity studies revealed an increased lag period and greater final turbidity for fibrin BII clots, indicating impaired oligomer formation. By permeation it was found that BII clots had greater network porosity, four times more than that of the control. The clot architecture visualized by scanning electron microscopy was similar to that of control clots with pore size and fiber diameter slightly increased. BII clots had a stiffness decreased by more than half, and an increased loss tangent, a measure of the inelastic deformation of the clot. All these results suggest a disruption of the proper alignment of fibrin monomers during oligomer formation. Consistent with these results, fibrin cross-linking by adding the physiological concentration of factor XIII to the purified protein showed that gamma and alpha chain cross-linking was impaired in BII clots. This amino acid substitution defines distinctive effects on the surface of the D:D interaction sites that are reflected in the clot structure and functional properties.     

Fibrinogen Philadelphia, a hypodysfibrinogenemia characterized by abnormal polymerization and fibrinogen hypercatabolism due to gamma S378P mutation

Fibrinogen Philadelphia, a hypodysfibrinogenemia described in a family with a history of bleeding, is characterized by prolonged thrombin time, abnormal fibrin polymerization, and increased catabolism of the abnormal fibrinogen. Turbidity studies of polymerization of purified fibrinogen under different ionic conditions reveal a reduced lag period and lower final turbidity, indicating more rapid initial polymerization and impaired lateral aggregation. Consistent with this, scanning and transmission electron microscopy show fibers with substantially lower average fiber diameters. DNA sequence analysis of the fibrinogen genes A, B, and G revealed a T>C transition in exon 9 resulting in a serine-to-proline substitution near the gamma chain C-terminus (S378P). The S378P mutation is associated with fibrinogen Philadelphia in this kindred and was not found in 10 controls. This region of the gamma chain is involved in fibrin polymerization, supporting this as the polymerization defect causing the mutation. Thus, this abnormal fibrinogen is characterized by 2 unique features: (1) abnormal polymerization probably due to a major defect in lateral aggregation and (2) hypercatabolism of the mutant protein. The location, nature, and unusual characteristics of this mutation may add to our understanding of fibrinogen protein interactions necessary for normal catabolism and fibrin formation.     

Structure of fibrin network of two abnormal fibrinogens with mutations in the αC domain on the human dermal microvascular endothelial cells 1

There is evidence that clot structure can be modulated by endothelial cells, wherein the fibrinogen αC domain plays a major role in the fibrin-cell interaction. The spatial distribution of fibrin fibers from fibrinogen Caracas V and Caracas I, with heterozygous mutation in the αC domain (Aα Ser432Cys and Aα Ser466stop, respectively) on human dermal microvasculature endothelial cells (HMEC-1), was studied by laser scanning confocal microscopy. In order to assess fibrin-cell interaction and the role of the αC domain, preliminary experiments were done with inert microspheres and RGD peptide included in the clotting reaction, and forming clots with fibrinogen fragment X (fibrinogen without αC domain). Groups of stressed fibers were observed near the cell surface and were related to fibrin-cell interactions, which were abolished by the RGD peptide, and by the absence of the αC domain. The fibrin network of fibrinogen Caracas V and Caracas I was very different from that of normal fibrinogen. In general, patient's clots were characterized by very thin, tightly packed fibrin fibers, with a substantially reduced network porosity. Near the cell's surface, both abnormal fibrinogens formed a very fine meshwork, with stressed fibers 'anchored' to the cell surface, a pattern that was lost far from the cell surface. The structure of normal and patient clots performed in the absence of cells resembled that observed far from the cell surface, concluding that Caracas V and Caracas I fibrin was modulated by the presence of endothelial cells.     

Common variation in the C-terminal region of the fibrinogen beta-chain: effects on fibrin structure, fibrinolysis and clot rigidity

Fibrinogen BbetaArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bbeta-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BbetaLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BbetaArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.     

A novel fibrinogen variant--Liberec: dysfibrinogenaemia associated with gamma Tyr262Cys substitution

Objective:  A 22-yr-old woman had abnormal preoperative coagulation test results and congenital dysfibrinogenaemia was suspected.
Patients and methods:  The patient from Liberec (Czech Republic) had a low fibrinogen plasma level as determined by Clauss method, normal fibrinogen level as determined by immunoturbidimetrical method, and prolonged thrombin time. To identify the genetic mutation responsible for this dysfibrinogen, genomic DNA extracted from the blood was analysed. Fibrin polymerisation measurement, kinetics of fibrinopeptide release, fibrinogen clottability measurement and scanning electron microscopy were performed.
Results:  DNA sequencing showed the heterozygous fibrinogen γ Y262C mutation. Kinetics of fibrinopeptide release was normal, however fibrin polymerisation was impaired. Fibrinogen clottability measurement showed that only about 45% molecules of fibrinogen are involved in the clot formation. Scanning electron microscopy revealed thicker fibres, which were significantly different from the normal control.
Conclusion:  A case of dysfibrinogenaemia, found by routine coagulation testing, was genetically identified as a novel fibrinogen variant (γ Y262C) that has been named Liberec.     

Fibrinogen gamma-chain splice variant gamma' alters fibrin formation and structure

Fibrinogen gammaA/gamma' results from alternative splicing of mRNA. This variant, which constitutes approximately 8% to 15% of plasma fibrinogen, contains FXIII and thrombin binding sites. Our objective was to investigate whether gammaA/gamma' differs in fibrin formation and structure from the more common variant gammaA/gammaA. Both variants were separated and purified by anion-exchange chromatography. Fibrin formation and clot structure of the variants and unfractionated fibrinogen were investigated by turbidity and scanning electron microscopy (SEM). Thrombin cleavage of fibrinopeptides was analyzed by high-performance liquid chromatography (HPLC). Turbidity analysis showed significantly altered polymerization rates and overall fiber thickness in gammaA/gamma' clots compared with gammaA/gammaA and unfractionated fibrinogen. This finding was consistent with a range of thrombin concentrations. HPLC demonstrated reduced rates of fibrinopeptide B (FpB) release from gammaA/gamma' fibrinogen compared with gammaA/gammaA. Delayed FpB release was associated with delayed lateral aggregation of protofibrils and significant differences were found on SEM, with gammaA/gamma' clots consisting of smaller diameter fibers and increased numbers of branch points compared with both gammaA/gammaA and unfractionated fibrinogen. These results demonstrate that the gammaA/gamma' splice variant of fibrinogen directly alters fibrin formation and structure, which may help to explain the increased thrombotic risk associated with this variant.     

Homophenotypic Aalpha R16H fibrinogen (Kingsport): uniquely altered polymerization associated with slower fibrinopeptide A than fibrinopeptide B release.

We detail for the first time the uniquely altered fibrin polymerization of homophenotypic Aalpha R16H dysfibrinogen. By polymerase chain reaction amplification and DNA sequencing, our new proposita's genotype consisted of a G>A transition encoding for Aalpha R16H, and an 11 kb Aalpha gene deletion. High-performance liquid chromatography disclosed fibrinopeptide A release approximately six times slower than its fibrinopeptide B. Turbidimetric analyses revealed unimpaired fibrin repolymerization, and abnormal thrombin-induced polymerization (1-7 mumol/l fibrinogen, > 96% coagulable), consisting of a prolonged lag time, slow rate, and abnormal clot turbidity maxima, all varying with thrombin concentration. For example, at 0.2-3 U/ml, the resulting turbidity maxima ranged from lower to higher than normal control values. By scanning electron microscopy, clots formed by 0.3 and 3 thrombin U/ml displayed mean fibril diameters 42 and 254% of the respective control values (n = 400). Virtually no such differences from control values were demonstrable, however, when clots formed in the presence of high ionic strength (micro = 0.30) or of monoclonal antibeta(15-42)IgG. The latter also prolonged the thrombin clotting time approximately three-fold. Additionally, thrombin-induced clots displayed decreased elastic moduli, with G' values of clots induced by 0.3, 0.7 and 3 thrombin U/ml corresponding to 11, 34, and 45% of control values. The results are consistent with increased des-BB fibrin monomer generation preceding and during polymerization. This limited the inherent gelation delay, decreased the clot stiffness, and enabled a progressively coarser, rather than finer, network induced by increasing thrombin concentrations. We hypothesize that during normal polymerization these constitutive des-BB fibrin monomer properties attenuate their des-AA fibrin counterparts.     

Immunofluorescent identification of fibronectin and fibrinogen/fibrin in experimental myocardial infarction.

Distribution of fibrinogen/fibrin and fibronectin in regions of experimental myocardial infarction were studied by the immunofluorescence technique. Distinct from normal myocardium 3 and 12 to 24 h after coronary artery ligation infiltration of necrotized cardiomyocytes by fibrinogen/fibrin and plasma fibronectin was detected. Fibrinogen/fibrin and plasma fibronectin constitute "primary matrix" of granulation tissue. On the third day after experimental infarction, synthesis of cellular fibronectin begins. Its content in the extracellular matrix (ECM of granulation tissue significantly increases on days 7 to 15. The amount of fibronectin in the ECM of developing scar tissue dramatically decreases 30 days after infarction, Fibrinogen/fibrin was continually identified in granulation tissue in zones of myocardial infarction. However, its amount in the ECM of developing scar tissue gradually decreased.     

End-linked homodimers in fibrinogen Osaka VI with a B beta-chain extension lead to fragile clot structure

The authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bbeta gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient. (Blood. 2000;96:3779-3785)     

Familial thrombophilia associated with fibrinogen paris V: Dusart syndrome

We report on a family with a history of venous thromboembolism associated with fibrinogen Paris V (fibrinogen Aalpha-Arg554-->Cys). Ten members experienced thrombotic events, including 4 with fatal pulmonary emboli. Pulmonary embolism was the presenting feature in 4. Those with the mutation and a history of thrombosis had somewhat higher fibrinogen concentrations than those with the mutation and no thrombosis (294 +/- 70 mg/dL vs 217 +/- 37 mg/dL, respectively). The Paris V mutation consistently caused a prolongation of the reptilase time, and fibrin clots containing the abnormal fibrinogen were more translucent than normal clots. Given the early onset of symptoms and the initial presentation with pulmonary embolism in some family members, it was justifiable to offer prophylactic anticoagulation with warfarin to carriers of the mutation. Fibrinogen Paris V has now been reported in 4 apparently unrelated families, indicating that it is a relatively common cause of dysfibrinogenemia-associated thrombosis.     

Age dependency of the sialic acid content of fibrinogen. Consequences for erythrocyte aggregation

Sialic acid (SA) content of plasma proteins and fibrinogen was investigated in 60 persons of different age and health status. SA/mg fibrin rose with age and morbidity in plasma, rinsed clots of fibrin from plasma and after prothrombin adsorption as well as in fibrin clots prepared from purified fibrinogen. The higher plasma SA is caused by increasing concentrations of glycoproteins like fibrinogen, but also by a higher content of SA/mg protein, as has been shown for fibrinogen. SA is considered to be an unspecific marker of acute phase reactions. Changing SA content of glycoproteins may have functional consequences. An increased red cell aggregation with fibrinogen of healthy elderly correlated with its SA content, but SA is most probably only indicating an altered protein heterogeneity in the aged and not a causative factor that influences erythrocyte aggregation.     

Immunochemical characterization of fibrinogen, fibrin I, and fibrin II in human thrombi and atherosclerotic lesions

Arterial thrombi and atherosclerotic lesions were analyzed immunochemically and examined histologically. The extent of in vivo proteolytic cleavage of the amino-terminal end of fibrinogen by thrombin and plasmin was determined and quantitated by specific radioimmunoassays. The samples were treated with cyanogen bromide (CNBr), and the total amount of fibrinogen and fibrin-derived protein was determined as NDSK, the NH2-terminal disulfide knot of fibrinogen. Thrombin-releasable fibrinopeptides A and B were used to quantitate fibrinogen and fibrin I. Previous plasmin cleavage of the B beta chain was inferred from the amount of B beta 1-42 and B beta 15-42 in undigested NDSK. The results obtained in both acute and organized thrombi indicate that approximately 60% of the total protein (as determined by amino acid analysis) was fibrinogen-derived and that 70% to 80% of the fibrinogen-derived material was fibrin II. These findings support the hypothesis that fibrin II as distinct from fibrin I is the predominant component in a thrombus. In samples from normal and atherosclerotic aortas, fibrinogen-derived protein comprised less than 10% of the total protein. Samples from grossly normal aortas contained only fibrinogen and fibrin I. Fibrinogen concentration decreased and fibrin II concentration increased with increasing severity of the lesions, suggesting that increased fibrin II formation is associated with progression of atheromas.     

Alteration of fibrin network by activated protein C

The antithrombotic plasma enzyme, activated protein C (APC), may play a role in thrombolysis. In vitro, acceleration of clot lysis by APC depends on its ability to inhibit the activation of prothrombin. The effect of APC on the assembly and dispersion of fibrin network was studied using turbidimetry, plasmin digestion of fibrin, and electron microscopy of plasma clots. The addition of APC before clotting but not after clotting accelerated clot lysis. The rate of increase in the turbidity of clotting plasma was reduced by APC. The turbidity of plasma clots containing APC was directly related to the clot lysis time. Fibrin from plasma clots that were formed in the presence of APC yielded less fibrin degradation products than fibrin from clots without added APC. Furthermore, APC reduced the diameter and relative number of fibrin fibers in plasma clots during gel assembly. We propose that APC may enhance the efficacy of thrombolysis by reducing the relative mass of fibrin within maturing thrombi.     

The alphaC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fibrinolysis

The functions of the alphaC domains of fibrinogen in clotting and fibrinolysis, which have long been enigmatic, were determined using recombinant fibrinogen truncated at Aalpha chain residue 251. Scanning electron microscopy and confocal microscopy revealed that the fibers of alpha251 clots were thinner and denser, with more branch points than fibers of control clots. Consistent with these results, the permeability of alpha251 clots was nearly half that of control clots. Together, these results suggest that in normal clot formation, the alphaC domains enhance lateral aggregation to produce thicker fibers. The viscoelastic properties of alpha251 fibrin clots differed markedly from control clots; alpha251 clots were much less stiff and showed more plastic deformation, indicating that interactions between the alphaC domains in normal clots play a major role in determining the clot's mechanical properties. Comparing factor XIIIa cross-linked alpha251 and control clots showed that gamma chain cross-linking had a significant effect on clot stiffness. Plasmin-catalyzed lysis of alpha251 clots, monitored with both macroscopic and microscopic methods, was faster than lysis of control clots. In conclusion, these studies provide the first definitive evidence that the alphaC domains play an important role in determining the structure and biophysical properties of clots and their susceptibility to fibrinolysis.     

Fibrinogen Matsumoto II: γ308Asn → Lys (AAT → AAG) mutation associated with bleeding tendency

Fibrinogen Matsumoto II is a hereditary dysfibrinogenaemia identified in a woman with Basedow's disease and a bleeding tendency. Coagulation tests of the patient's plasma revealed a prolonged thrombin time and a decreased fibrinogen level determined by functional method. Release of fibrinopeptide A and B was normal, whereas fibrin monomer polymerization was delayed. Fibrinogen γ-chain gene of the propositus was heterozygous for a missense mutation that resulted in Asn → Lys substitution at codon 308. Though the same amino acid substitution was also attributed to fibrinogen Kyoto I and Bicetre II, fibrinogen Matsumoto II showed different clinical manifestations from them.     

Incorporation of fibrin molecules containing fibrinopeptide A alters clot ultrastructure and decreases permeability

Previous studies have shown that a heterozygous mutation in the fibrinogen Aalpha chain gene, which results in an Aalpha R16C substitution, causes fibrinolytic resistance in the fibrin clot. This mutation prevents thrombin cleavage of fibrinopeptide A from mutant Aalpha R16C chains, but not from wild-type Aalpha chains. However, the mechanism underlying the fibrinolytic resistance is unclear. Therefore, this study investigated the biophysical properties of the mutant fibrin that contribute to fibrinolytic resistance. Fibrin clots made from the mutant fibrinogen incorporated molecules containing fibrinopeptide A into the polymerised clot, which resulted in a 'spiky' clot ultrastructure with barbed fibrin strands. The clots were less stiff than normal fibrin and were cross-linked slower by activated FXIII, but had an increased average fiber diameter, were more dense, had smaller pores and were less permeable. Protein sequencing showed that unclottable fibrinogen remaining in the supernatant consisted entirely of homodimeric Aalpha R16C fibrinogen, whereas both cleaved wild-type alpha chains and uncleaved Aalpha R16C chains were in the fibrin clot. Therefore, fibrinolytic resistance of the mutant clots is probably a result of altered clot ultrastructure caused by the incorporation of fibrin molecules containing fibrinopeptide A, resulting in larger diameter fibers and decreased permeability to fibrinolytic enzymes.     

Fibrinogen Saint-Germain I: a case of the heterozygous Aalpha GLY 12 --> VAL fibrinogen variant.

A fibrinogen variant was suspected based on the results of routine coagulation tests in a 2-year-old asymptomatic child. Coagulation studies showed marked prolongation of both the thrombin and reptilase times, and discrepancy was noted between the level of plasma fibrinogen as measured by a kinetic versus immunological determination. Family studies revealed that the father beared the same abnormality. Studies of purified fibrinogen revealed an impaired release of both fibrinopeptides by thrombin. Fibrin monomer polymerization and fibrin stabilization were normal. DNA sequencing revealed a heterozygous G --> T point mutation in exon 2 of the gene coding for the Aalpha chain, which substituted a Gly for Val at position 12. Although the mutation is the same as in fibrinogen Rouen, fibrinogen Saint-Germain I shows a different fibrinopeptide release pattern and a mild factor V deficiency.     

Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin

Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not β-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.