Adhesion of Blood Clots Can Be Enhanced When Copolymerized with a Macromer That Is Crosslinked by Coagulation Factor XIIIa.

The adhesion of blood clots to blood vessels, such as through the adhesion of fibrin, is essential in hemostasis. While numerous strategies for initiating clot formation and preventing clot lysis are being developed to create improved hemostatic agents, strategies for enhancing clot adhesion have not been widely explored. Here, we show that adhesion of blood clots can be increased by adding a previously characterized synthetic polymer that is crosslinked by coagulation factor XIIIa during clotting. Addition of the polymer to normal plasma increased the adhesive strength of clots by 2-fold. It also recovered the adhesive strength of nonadhesive fibrinogen-deficient whole blood clots from <0.06 kPa to 1.9 ± 0.14 kPa, which is similar to the adhesive strength of a fibrinogen-rich clot (1.8 ± 0.64 kPa). The polymer also enabled plasma clots to remain adhered under fibrinolytic conditions. By demonstrating that the adhesive strength of clots can be increased with a synthetic material, this provides a potential strategy for creating advanced hemostatic materials, such as treatments for fibrinogen deficiency in trauma-induced coagulopathy.

[Functional study of abnormal fibrinogen caused by Arg275His mutation in fibrinogen γ chain].

OBJECTIVE: To investigate the function of abnormal fibrinogen in two inherited dysfibrinogenemia pedigrees. METHODS: Routine coagulation tests were conducted in the probands and related family members. The antigen and activity levels of fibrinogen were detected by immunoturbidimetry assay and clauss assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes and antithrombin gene（AT3）were analyzed by PCR amplification and direct sequencing. Routine thrombelastography (TEG) test and functional fibrinogen TEG test were both used to make a comprehensive evaluation of coagulation status and functional fibrinogen level in patients. The molecular weights of the three peptides from fibrinogen were measured by Western blot. The function of abnormal fibrinogen was assessed by fibrinogen dynamic polymerization and fibrinolysis velocity. RESULTS: The coagulation routine tests were normal in two probands except for prolonged thrombin time (TT) and reptilase time (RT), as well as reduced activity levels of 0.5 g/L and 0.6 g/L fibrinogen, respectively. The antigen levels of fibrinogen were 2.32 g/L and 2.66 g/L in two probands, which were in the normal reference range. The genotype analysis showed that Arg275His in fibrinogen γ chain (γ Arg275His) existed in both probands and patients in these two pedigrees. Meanwhile, proband B's grandfather and aunt also carried heterozygote g.5876T>C (Ser116Pro) mutation in AT3. The results of routine TEG test demonstrated that the α values of proband B and his father were close to and lower than the lower limit of reference range, respectively, while the MA values were normal in both of them. However, functional fibrinogen TEG test revealed obviously reduced MA value. All the probands and patients demonstrated prolonged lag-off time and reduced peak value in fibrinogen dynamic polymerization tests. Meanwhile, most of fibrin formed from the patients' plasma could not be dissolved completely by plasminogen (PLG) and urokinase-typeplasminogenactivator (u-PA) at a certain time. CONCLUSION: We first reported cases of inherited dysgibrinogenemia associated with inherited AT deficiency. γArg275His mutation caused the abnormal fibrinogen in terms of fibrin mono polymerization and possibly in fibrinolysis. Combined use of routine TEG test and functional fibrinogen TEG test with comprehensive analyses of the parameters in both tests could better evaluate the level of functional fibrinogen and predict the risk of hemorrhage and thrombosis in patients with inherited dysfibrinogenemia.

The pleiotropic role of the fibrinogen gamma' chain in hemostasis.

A fraction of fibrinogen contains a differently spliced gamma chain called gamma', which presents itself mainly as heterodimer with the common gammaA chain as gammaA/gamma' fibrinogen. The gamma' chain differs from the gammaA chain in its C-terminus and has important functional implications for fibrinogen. The presence of the gamma' chain modulates thrombin and FXIII activity, influences clot architecture, and eliminates a platelet-binding site. Associations of gammaA/gamma' fibrinogen levels with arterial and venous thrombosis have been reported, indicating that the functional effects of gammaA/gamma' fibrinogen may contribute to the pathology of thrombosis. This review summarizes the key biologic aspects of this interesting variant of fibrinogen and discusses inconsistencies in current reports.

[Functional analysis of heterozygous plasma dysfibrinogens derived from two families of gammaArg275Cys and three families of gammaArg275His, and haplotype analysis for these families].

We have identified five heterozygous dysfibrinogenemias, two families with variant fibrinogen gammaArg275Cys (CGC > TGC; Matsumoto III and Sendai) and three families with gammaArg275His (CGC > CAC; Otsu II, Iida, and Shizuoka), from PCR-amplified DNA fragments and direct sequence analysis. gammaArg275 is the most important residue in fibrinogen for the so-called "D-D interface" in protofibril elongation. We compared the functions of plasma fibrinogen purified from affected family members with gammaArg275Cys and gammaArg275His. Both fibrinogens showed markedly impaired thrombin-catalyzed fibrin polymerization in comparison with normal controls. The degree of impairment of gammaArg275Cys fibrinogen was greater than that of gammaArg275His. These results were consistent with the fibrinogen concentration ratio (thrombin time method/immunological method). That is, the ratio of gammaArg275Cys was significantly lower than that of gammaArg275His. Moreover, scanning electron microscopy indicated significantly thicker fibers in fibrin clots made from gammaArg275Cys than in those of normal controls or gammaArg275His, and abnormal bundles with tapered ends. Factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chain (in the absence of thrombin) showed a similar delay for gammaArg275Cys and gammaArg275His. We report markedly impaired fibrin polymerization of gammaArg275Cys compared to gammaArg275His, and speculate that the difference is due to the disulfide-linked Cys in gammaArg275Cys, as we have already demonstrated for plasma and recombinant mutant fibrinogens. These results also indicate that an amino acid substitution of gammaArg275 disrupts D:D interactions in fibrin fiber formation. Furthermore haplotype analysis for three families with gammaArg275His suggested that founder of Iida family might be different from that of Otsu II or Shizuoka family.

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.

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.

Biophysical characterization of fibrinogen Caracas I with an Aalpha-chain truncation at Aalpha-466 Ser: identification of the mutation and biophysical characterization of properties of clots from plasma and purified fibrinogen.

Fibrinogen Caracas I is a dysfibrinogenemia with a mild bleeding tendency; a novel nonsense mutation, in the gene coding the Aalpha-chain, identified in this study as G4731T, giving rise to a new stop codon at Aalpha-Glu 467. Fibrinogen from two family members, the mother and sister of the propositus, both heterozygous for the mutation were studied, analyzing clots made from both plasma and purified fibrinogen. Clot structure and properties were characterized by turbidity, permeation, scanning electron microscopy and rheological studies. Permeation through Caracas I plasma clots was decreased, consistent with the decreased final turbidity. As shown by scanning electron microscopy, plasma clots from the patients were composed of very thin fibers, with increased fibrin density and reduced pore size. Viscoelastic measurements revealed that fibrinogen Caracas I plasma clots were much stiffer and less subject to compaction. These results demonstrate a key role of the carboxyl-terminal alpha chains of fibrin in lateral aggregation during polymerization and reinforce the utility of studying plasma clots. It is important to point out that the biophysical studies with fibrinogen purified by two different methods yielded contradictory results, which can be accounted for by selective purification of certain molecular species as seen by two-dimensional electrophoresis.

Thrombophilic dysfibrinogen Tokyo V with the amino acid substitution of gammaAla327Thr: formation of fragile but fibrinolysis-resistant fibrin clots and its relevance to arterial thromboembolism.

Thrombophilic dysfibrinogen Tokyo V was identified in a 43-year-old man with recurrent thromboembolism. Based on analyses of the patient fibrinogen genes, the amino acid sequence of the aberrant fibrinogen peptide, and deglycosylation experiments, fibrinogen Tokyo V was shown to have an amino acid substitution of gamma Ala327Thr and possibly extra glycosylation at gamma Asn325 because the mutation confers the N-linked glycosylation consensus sequence Asn-X-Thr. The mutation resulted in impaired function and hypofibrinogenemia (hypodysfibrinogen). Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium, resulting in very low clottability. Additionally, a large amount of soluble cross-linked fibrin was formed upon thrombin treatment in the presence of factor XIII and calcium. However, Tokyo V-derived fibrin was resistant to degradation by tissue plasminogen activator (tPA)-catalyzed plasmin digestion. The structure of Tokyo V fibrin appeared severely perturbed, since there are large pores inside the tangled fibrin networks and fiber ends at the boundaries. Taken together, these data suggest that Tokyo V fibrin clots are fragile, so that fibrinolysis-resistant insoluble fibrin and soluble fibrin polymers may be released to the circulation, partly accounting for the recurrent embolic episodes in the patient.

Fibrinogens Kosai and Ogasa: Bbeta15Gly-->Cys (GGT-->TGT) substitution associated with impairment of fibrinopeptide B release and lateral aggregation.

We found two heterozygous dysfibrinogenemias, designated fibrinogen Kosai and fibrinogen Ogasa. Kosai was associated with arteriosclerosis obliterans but Ogasa showed no bleeding or thrombotic tendencies. The plasma fibrinogen concentrations from the two propositi (Ogasa and Kosai) were much lower when determined by the thrombin-time method (0.94 and 1.06 g L(-1), respectively) than when determined by the immunological method (2.87 and 2.72 g L(-1), respectively). We performed DNA sequencing and functional analyses to clarify the relationship between the structural and functional abnormalities. Genetic analysis of PCR-amplified DNA from the propositi identified the heterozygous substitution Bbeta15Gly-->Cys (GGT-->TGT). Western blotting analysis of purified fibrinogen revealed the existence of albumin-fibrinogen complexes. Functional analyses indicated that compared with the normal control, the propositi's fibrinogen released only half the normal amount of fibrinopeptide B and showed markedly impaired polymerization. In addition, the observation of thinner fibers in fibrin clots (by scanning electron microscopy) indicated markedly defective lateral aggregation in the variant fibrinogens. The impaired functions may be due to the substitution of Cys for Bbetao15Gly plus the existence of some additional disulfide-bonded forms.

Structure and function of human fibrinogen inferred from dysfibrinogens.

Fibrinogen is a 340-kDa plasma protein that is composed of two identical molecular halves, each consisting of three non-identical subunit polypeptides designated as A alpha, B beta- and gamma-chains held together by multiple disulfide bonds. Fibrinogen has a trinodular structure, i.e., one central E domain comprizing the amino-terminal regions of paired individual three polypeptides, and two identical outer D domains. These three nodules are linked by two coiled-coil regions [1,2]. After activation with thrombin, a tripeptide segment consisting of Gly-Pro-Arg is exposed at the amino-terminus of each alpha-chain residing at the center of the E domain and combines with its complementary binding site, called the 'a' site, residing in the carboxyl-terminal region of the gamma-chain in the outer D domain of another molecule. By crystallographic analysis [3], the alpha-amino group of alpha Gly-1 is shown to be juxtaposed between the carboxyl group of gamma Asp-364 and the carboxyamide of Gln-329 in the 'a' site. Half molecule-staggered, double-stranded fibrin protofibrils are thus formed [4,5]. Upon abutment of two adjacent D domains on the same strand, D-D self association takes place involving Arg-275, Tyr-280 and Ser-300 of the gamma-chain on the surface of the abutting two D domains [3]. Thereafter, carboxyl-terminal regions of the fibrin a-chains are thought to be untethered and interact with those of other protofibrils leading to the formation of thick fibrin bundles and interwoven networks after appropriate branching [6-9]. Although many enigmas still remain regarding the mechanisms of these molecular interactions, fibrin assembly proceeds in a highly ordered fashion. In my talk, I would like to discuss these molecular interactions of fibrinogen and fibrin based on the up-date data provided by analyses of normal as well as hereditary dysfibrinogens, particularly in the latter by introducing representative molecules at each step of fibrin clot formation.

Fibrinogen Hillsborough: a novel gammaGly309Asp dysfibrinogen with impaired clotting.

We present a novel gamma-chain dysfibrinogen that was discovered in a 32-year-old asymptomatic man admitted to the hospital after a car accident. He presented with a low fibrinogen concentration, 0.5 mg/mL, and a prolonged thrombin clotting time, 58 seconds. Analysis of purified fibrinogen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a gamma-chain variant with an apparently higher molecular weight. Isoelectric focusing (IEF) demonstrated an anodal shift in the banding pattern of the chains and electrospray ionization mass spectrometry (ESIMS) showed a 27-Da increase in the average mass of the unresolved variant and normal gamma chains. DNA sequence analysis showed a heterozygous mutation of GGC (Gly)-->GAC (Asp) at codon 309 of the gamma chain gene. This Gly--> Asp substitution was consistent with the charge change shown by IEF as well as the mass change identified by ESIMS. Functional analysis revealed that thrombin-catalyzed polymerization occurred with a longer lag time, lower rate of lateral aggregation, and similar final turbidity compared to normal and that factor XIII cross-linking was normal. The polymerization results suggest that residue gamma309 is necessary for proper alignment of fibrinogen molecules, specifically in protofibril formation and D:D interactions. gammaGly309 is highly conserved and x-ray structures support the conclusion that the lack of a side chain at this position helps facilitate the close contact between abutting gammaD domains of condensing fibrin monomers during polymerization.

Coagulative, fibrinolytic and metabolic pattern in patients with central retinal vein occlusion.

Central retinal vein occlusion (CRVO) is an important cause of visual loss. Many risk factors have been associated with CRVO onset at various ages. Among them diabetes mellitus, hypertension, immunologic disorders, increase in blood viscosity and coagulation, decrease of fibrinolysis have been reported in many subjects. The aim of our study was to detect the metabolic, coagulative and fibrinolytic pattern in 54 patients (26 men, 28 women, mean age 50.4 +/- 12.3) affected by CRVO. We excluded from the study patients with other ocular disorders. A fibrinolytic impairment is the most common feature in our population. It occurs either in dysmetabolic or in nonmetabolic subjects. Such data suggest a prominent role of the fibrinolytic system in the pathogenesis of CRVO.

Structure and function of fibrinogen inferred from hereditary dysfibrinogens.

The structure-function relationships of human fibrinogen and their clinical implications are discussed on the basis of data provided by biochemical and electron microscopic analyses of normal and abnormal fibrinogen and by recent crystallographic studies on certain functional domains and segments of normal fibrinogen. Particularly, ultrastructure studies of individual fibrinogen molecules and fibrin networks of structurally elucidated dysfibrinogens enable us to understand the structure-function relationships of these dysfibrinogens more clearly than ever. Electron micrographs of some representative dysfibrinogen molecules are provided.

The molecular basis of renal amyloidosis in Irish-American and Polish-Canadian kindreds.

Hereditary amyloidosis of an unusual form has been reported in two separate kindreds; one was Polish-Canadian and the other was Irish-American (Am J Med 1975; 59:121 and Trans Assoc Am Physicians 1981; 94:211). In both kindreds, affected members developed hypertension and nephrotic syndrome due to amyloidosis in their forties or fifties, but the genetic background responsible for the condition has been left undetermined. To identify the genetic defect in these kindreds, a portion of exon 5 of the fibrinogen alpha-chain gene in members of these kindreds was examined for a mutation by single-strand conformation polymorphism analysis and direct DNA sequencing. DNA analyses revealed an A-->T transversion at the second base of codon 526 of the fibrinogen alpha-chain gene in both of these kindreds. Analysis of DNA polymorphisms in the fibrinogen alpha-chain gene locus (TCTT repeat in intron 3, Rsal site in exon 5, and Taql site in the 3' flanking region of the gene) showed the haplotype B5-Rsal(+)-Taql(-) for the Val 526 mutant gene in both kindreds studied here, as well as in two kindreds previously described (J Clin Invest 1994; 93:731). The fibrinogen alpha-chain gene mutation (Val 526) is the genetic defect responsible for hereditary renal amyloidosis in these two kindreds, and the mutant genes in the Val 526 kindreds may have been derived from a single founder.

The structure-function relationship of hereditary dysfibrinogens.

Fibrinogen is a 340-kDa multi-subunit glycoprotein present in plasma and tissues of all classes of vertebrates. Fibrinogen exerts a variety of physiologically important functions, and most of them, if not all, are assigned to certain structures of fibrin including double-stranded fibrin protofibrils and highly crosslinked fibrin networks. Fibrin formation is indeed a series of highly ordered molecular interactions. The mechanisms underlying these molecular interactions have been extensively studied in the last 40 years, but there still remain many enigmas. In this mini-review, the structure-function relationships of hereditary dysfibrinogens are discussed.

Técnicas diagnósticas de laboratorio en trombofilias congénitas / Diagnostic laboratory techniques for hereditary trombophillias

Las alteraciones hereditarias de los inhibidores naturales de la coagulación y de los componentes del sistema fibrinolítico, están asociadas con enfermedad tromboembólica. Como no siempre es posible determinar la causa de la trombofilia, se analizan aquí distintas metodologías que pueden ser de utilidad para arrojar más luz sobre este problema; y que están disponibles en la bibliografía, para medir la actividad funcional y la concentración de antitrombina III, proteína C, proteína S, cofactor II de la heparina, glicoproteína rica en histidina, lipoproteína A, fibrinógeno, plasminógeno y homocisteína. En todos los casos es conveniente comenzar primero con la determinación de la actividad funcional de la proteína en estudio y, en caso de encontrar una alteración, se completará el estudio con una cuantificación inmunológica u otra metodología que permita obtener mayor información sobre la alteración

Técnicas diagnósticas de laboratorio en trombofilias congénitas / Diagnostic laboratory techniques for hereditary trombophillias

Las alteraciones hereditarias de los inhibidores naturales de la coagulación y de los componentes del sistema fibrinolítico, están asociadas con enfermedad tromboembólica. Como no siempre es posible determinar la causa de la trombofilia, se analizan aquí distintas metodologías que pueden ser de utilidad para arrojar más luz sobre este problema; y que están disponibles en la bibliografía, para medir la actividad funcional y la concentración de antitrombina III, proteína C, proteína S, cofactor II de la heparina, glicoproteína rica en histidina, lipoproteína A, fibrinógeno, plasminógeno y homocisteína. En todos los casos es conveniente comenzar primero con la determinación de la actividad funcional de la proteína en estudio y, en caso de encontrar una alteración, se completará el estudio con una cuantificación inmunológica u otra metodología que permita obtener mayor información sobre la alteración (AU)