Thrombin-catalyzed Polymerization Research Articles

On the Mechanism of Self-Assembly of Fibrinogen in Thrombin-free Aqueous Solution.

Fibrinogen dissolved in 0.12 M aqueous NaCl solution at a pH of 6.6 exhibits self-assembly in response to a lowering of the NaCl concentration to values equal to or lower than 60 mM. As has been established in a preceding work (Langmuir 2019, 35, and 12113), a characteristic signature of the self-assembly triggered by a drop in ionic strength is the formation of large globular particles. Growth of these particles most likely obeys a coalescence-like process also termed a step growth process. In order to extend this knowledge, the present work first optimized the protocol, leading to highly reproducible self-assembly experiments. Based on this optimization, the work succeeded in identifying an initial stage, not yet accessible, during which rigid short fibrils grow in close analogy to the thrombin-catalyzed polymerization of fibrin. In addition, first suggestions could be made on the transformation of these fibrils into larger aggregates, which upon drying turn into thick fiber-like ropes.

Altered structure and function of fibrinogen after cleavage by Factor VII Activating Protease (FSAP)

Factor VII Activating Protease (FSAP) is a plasma protease affecting both coagulation and fibrinolysis. Although a role in hemostasis is still unclear, the identification of additional physiologic substrates will help to elucidate its role in this context. FSAP has been reported to cleave fibrinogen, but the functional consequences of this are not known. We have therefore undertaken this study to determine the implications of this cleavage for fibrin-clot formation and its lysis. Treatment of human fibrinogen with FSAP released an N-terminal peptide from the Bβ chain (Bβ1-53) and subsequently the fibrinopeptide B; within the Aα chain a partial truncation of the αC-region by multiple cleavages was seen. The truncated fibrinogen showed a delayed thrombin-catalyzed polymerization and formed fibrin clots of reduced turbidity, indicative of thinner fibrin fibers. Confocal laser scanning and scanning electron microscopy of these clots revealed a less coarse fibrin network with thinner fibers and a smaller pore size. A lower pore size was also seen in permeability studies. Unexpectedly, FSAP-treated fibrinogen or plasma exhibited a significantly faster tPA-driven lysis, which correlated exclusively with cleavage of fibrinogen and not with activation of plasminogen activators. Similar observations were also made in plasma after activation of endogenous zymogen FSAP, but not in plasma of carrier of the rare Marburg I single nucleotide polymorphism. In conclusion, altering fibrin clot properties by fibrinogenolysis is a novel function of FSAP in the vasculature, which facilitates clot lysis and may in vivo contribute to reduced fibrin deposition during thrombosis.

Fibrin Association at Hybrid Biointerfaces Made of Clot-Binding Peptides and Polythiophene.

The properties as biointerfaces of electroactive conducting polymer-peptide biocomposites formed by poly(3,4-ethylenedioxythiophene) (PEDOT) and CREKA or CR(NMe)EKA peptide sequences (where Glu has been replaced by N-methyl-Glu in the latter) have been compared. CREKA is a linear pentapeptide that recognizes clotted plasma proteins and selectively homes to tumors, while CR(NMe)EKA is an engineer to improve such properties by altering peptide-fibrin interactions. Differences between PEDOT-CREKA and PEDOT-CR(NMe)EKA reflect dissemblance in the organization of the peptides into the polymeric matrix. Both peptides affect fibrinogen thrombin-catalyzed polymerization causing the immediate formation of fibrin, whereas in the absence of thrombin this phenomenon is only observed for CR(NMe)EKA. Consistently, the fibrin-adsorption capacity is higher for PEDOT-CR(NMe)EKA than for PEDOT-CREKA, even though in both cases adsorbed fibrin exhibits round-like morphologies rather than the characteristic fibrous structure. PEDOT-peptide films coated with fibrin are selective in terms of cell adhesion, promoting the attachment of metastatic cells with respect to normal cells.

Protective effects of (−)-epicatechin against nitrative modifications of fibrinogen

Fibrinogen appears to be particularly sensitive to toxic action of peroxynitrite; a potent oxidizing and nitrating species. An increased nitration of fibrinogen has been reported in cardiovascular diseases. The defense mechanisms against PN are crucial for complex hemostasis process. Flavonoids have antioxidative properties and could protect biomolecules against action of peroxynitrite. The aim of our studies was to establish, if (−)-epicatechin may in vitro protect fibrinogen molecule against peroxynitrite-induced nitration of tyrosines and change its thrombin-catalyzed polymerization. The exposure of purified fibrinogen (6μM) to peroxynitrite (1–100μM) resulted in both structural modifications and clotting ability of this glycoprotein. Peroxynitrite at the concentration of 1μM increased maximum velocity of Fg polymerization, whereas exposure to 100μM PN resulted in a significant decrease of Vmax. (−)-Epicatechin (1–100μM) caused a dose-dependent inhibition of 3-nitrotyrosine formation in fibrinogen treated with peroxynitrite (100μM) in both Western blot assays and C-ELISA assays. At the highest concentration of (−)-epicatechin (100μM) the level of 3-NT in fibrinogen reached the control values. At lower doses (−)-epicatechin reduced tyrosine nitration by approx. 23% and 40% at the concentration of 1μM and 10μM, respectively. (−)-Epicatechin also abolished the pro-thrombotic effect of peroxynitrite on fibrinogen clotting. The presented in vitro results demonstrated for the first time that (−)-epicatechin might have protective effects against the impairment of structure and properties of Fg, caused by action of the strong biologic oxidant/nitration and inflammatory mediators.

From Fibrinogen to Fibrin: Dynamic Light Scattering to Probe the Mechanism of Protofibril Assembly Into Fibers.

AbstractAbstract 1156The conversion from fibrinogen to fibrin, and fibrin clot structure and function, are important problems in cardiovascular research. The mechanism of fibrin clot formation remains undefined after more than 50 years of study. We have developed a new method to monitor polymerization and examine the products formed prior to gelation. We monitored polymerization by dynamic light scattering (DLS), a sensitive method to measure formation of small complexes. We “stopped” thrombin-catalyzed polymerization at different phases by crosslinking the polymers with formaldehyde and analyzed the products by DLS, agarose gel electophoresis (AGE) and transmission electron microscopy (TEM). DLS of the formaldehyde fixed products showed the products were stable and were analogous to the polymerization reactions with about a 2 minute delay in the average radius. As shown in the table, AGE showed a decrease in the numbers of small molecules and the appearance of larger molecules with polymerization. Preliminary TEM results showed a similar progression with AGE and clearly displayed the polymerization process from fibrin monomers, oligomers, protofibrils, then fibers. About 6% protofibrils were observed at 2 minutes and continuously increased with polymerization. Small amounts of fibers appeared from 4 minutes, and increased both in sizes and numbers with polymerization. At 10 minutes, about 8% fibers were observed and the length of large fibers reached around 3 μm. TEM also showed that after 7 minutes, the polymerization products were relatively heterogeneous with a high dispersity of sizes. With these experiments we followed the polymerization process step by step and determined the changes between steps by comparing adjacent-time products. Additionally, our laboratory specializes in synthesizing recombinant variant fibrinogens that have proven useful in studying functional attributes of fibrinogen. By DLS, we examined polymerization of variants in calcium binding sites: the single substitution gE132A (in the b2 calcium site) and the double substitutions gD318A,D320A (in the high affinity calcium site) and gD298A,D301A (in the g2 calcium site). For gD298A,D301A the DLS profile was similar to normal fibrinogen with a slower formation of protofibrils, as expected for the slightly prolonged lag time. For gE132A DLS showed a more rapid rise and higher plateau than normal fibrinogen. The initial polymerization is the same as normal fibrinogen shown in DLS, indicating the rate of protofibril formation for gE132A is the same as normal. After protofibril formation, the rapid rise in DLS for gE132A preceded the rapid rise for normal fibrin, indicating that assembly of the variant protofibrils into fibers was faster than normal. For gD318A,D230A fibrinogen we saw a slight change in DLS, such that the average radius increased 2-fold in 8.5 hours. This change in radius was not different after 13 hours, indicating a stable complex was formed. Further experiments are needed to characterize this complex. Considered together, these initial studies show that DLS can provide additional insight into the influence of structure on polymerization. The comparison of the normal fibrinogen to variant fibrinogens will help us to better understand the role of functional sites in polymerization and identify the residues of fibrinogen that influence protofibril formation and lateral aggregation.Table 1AGE result of polymerization products crosslinked by formaldehyede at different timepoints.ConstituentPercent composition10 min0 min2 min4 min7 minmonomer97.4%84.4%83.7%80.3%56.8%dimer2.6%14.3%12.5%11.7%22.1%trimer1.3%3.8%3.9%13.0%tetramer2.5%3.6%pentamer1.6%3.5%hexamer1.0% Disclosures:No relevant conflicts of interest to declare.

Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction

Increased levels of protein carbonyls have been measured in plasma of patients following a myocardial infarction (Mocatta et al. J. Am. Coll. Cardiol. 49:1993–2000; 2007). In this study, we have examined representative plasma samples from this group of patients. We show that carbonyls are formed preferentially on fibrinogen and that there is a strong correlation between fibrinogen and total plasma protein carbonyls. Functional properties of fibrinogen isolated from post myocardial plasmas were investigated by measuring thrombin-catalyzed polymerization. Fibrinogen from plasma with upper quartile protein carbonyls (mean 0.16 nmol/mg protein) polymerized ∼1.4 times more rapidly and gave 1.4-fold higher maximum turbidity (12 per group, P < 0.001) than fibrinogen from lower quartile carbonyl plasma (mean 0.007 nmol/mg), which behaved similarly to control plasma. Significant differences were also apparent when related to the carbonyl content of the fibrinogen itself. These changes in the high carbonyl plasma reflect a faster rate of lateral aggregation of small oligomers to form fibrin polymers that comprise thicker, more loosely woven fibers. In vivo this could be translated into a tendency to clot faster and form more fragile clots. High protein carbonyls in fibrinogen were not associated with an increased content of nitrotyrosine or chlorotyrosine. Nitrotyrosine levels were significantly lower in fibrinogen than total plasma protein, with no difference between patients and controls. Chlorotyrosine levels in fibrinogen (but not total protein) were significantly higher for the post myocardial patients. Our data suggest that high fibrinogen protein carbonyls in heart disease could be a prothrombotic risk factor.

B:b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'.

Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val. We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants. These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

The role of ascorbate and histidine in fibrinogen protection against changes following exposure to a sterilizing dose of γ-irradiation

Sodium ascorbate and histidine were employed to protect fibrinogen against modifications followed by a gamma-irradiation process that could potentially inactivate the blood-borne viruses in plasma-derived products. Fibrinogen was irradiated (50 kGy total dose, on dry ice) using a 60Co source. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. Carbonyl groups were measured by the 2,4-dinitrophenylhydrazine-coupled method, and the fibrinogen clotting activity was assessed by different functional assays. In irradiated fibrinogen, the carbonyl group concentration was elevated three-fold versus control; and moderate fragmentation of largely Aalpha and Bbeta chains was revealed. The rate of thrombin-catalyzed fibrinogen polymerization was inhibited (average 50%) with normal fibrinopeptide release and with a minor decrease of total clottable fibrinogen and alpha-polymer formation. Ascorbate reduced the incorporation of carbonyls to the fibrinogen molecule (by > 50% at 50 mmol/l; P < 0.001). Contrary to ascorbate, which alone delayed the fibrinogen polymerization rate, histidine abolished irradiation-induced inhibition of fibrinogen polymerization (by 80% at 50 mmol/l; P < 0.001). In conclusion, even though ascorbate effectively protects fibrinogen from oxidation due to its adverse effects on fibrinogen function, it may not serve as a suitable radioprotective. On the contrary, the first definite evidence is provided that radiation-sterilized fibrinogen in the presence of histidine greatly retains its clotting capability.

Probing the gamma2 calcium-binding site: studies with gammaD298,301A fibrinogen reveal changes in the gamma294-301 loop that alter the integrity of the "a" polymerization site.

To determine the significance of the gamma2 calcium-binding site in fibrin polymerization, we synthesized the fibrinogen variant, gammaD298,301A. We expected these two alanine substitutions to prevent calcium binding in the gamma2 site. We examined the influence of calcium on the polymerization of gammaD298,301A fibrinogen, evaluated its plasmin susceptibility, and solved 2.7 and 2.4 A crystal structures of the variant with the peptide ligands Gly-Pro-Arg-Pro-amide (GPRP) and Gly-His-Arg-Pro-amide (GHRP), respectively. We found that thrombin-catalyzed polymerization of gammaD298,301A fibrinogen was modestly impaired, whereas batroxobin-catalyzed polymerization was significantly impaired relative to normal fibrinogen. Notably, the influence of calcium on polymerization was the same for the variant and for normal fibrinogen. Fibrinogen gammaD298,301A was more susceptible to plasmin proteolysis in the presence of GPRP. This finding suggests structural changes in the near-by "a" polymerization site. Comparisons of the structures revealed minor conformational changes in the gamma294-301 loop that are likely responsible for the weakened "a" site. When considered altogether, the data suggest that the gamma2 calcium-binding site does not significantly modulate polymerization. We cannot, however, rule out the possibility that the weakened "a" polymerization site masks an important role for the gamma2 calcium-binding site in normal polymerization. Somewhat unexpectedly, the structure data showed that GPRP bound to the "b" site and induced the same local conformational changes as GHRP to this site. This structure shows that "A:b" interactions can occur and suggests that these may participate in normal polymerization.

Fibrinogen Seoul (FGG Ala341Asp): A Novel Mutation Associated With Hypodysfibrinogenemia

Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in fibrinogen function. The molecular basis of hypodysfibrinogenemia was investigated in a 66-year-old woman with peripheral artery obstructive disease and in her family members. Plasma level of functional fibrinogen determined using the Clauss method was lower (75 mg/dL; normal, 140-460 mg/dL) than that measured with immunologic nephelometric assay (137 mg/dL; normal, 180-400 mg/dL). Similar results were also observed in two family members through two generations. DNA was extracted from whole blood, and the coding regions and intron/exon boundaries of gamma chain gene (FGG) were amplified. A novel (Fibrinogen Seoul) heterozygous FGG mutation (GCT->GAT, Ala341Asp) was identified in all three affected family members. Thrombin-catalyzed polymerization was found to be defective on the analysis of purified fibinogen from the propositus. Molecular modeling also showed a conformational change of fibrinogen structure.

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

AbstractCrystallographic structures indicate that γ-chain residue Asn308 participates in D:D interactions and indeed substitutions of γAsn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: γAsn308 changed to lysine (γN308K), isoleucine (γN308I), and alanine (γN308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa–catalyzed cross-linking by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of γN308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with γN308K. Factor XIIIa–catalyzed γ-γ dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) γ-γ dimer formation of only γN308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both.

Fibrinogen Hillsborough: a novel γGly309Asp 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.

Analysis of engineered fibrinogen variants suggests that an additional site mediates platelet aggregation and that "B-b" interactions have a role in protofibril formation.

The C-terminal domain of the fibrinogen gamma-chain includes multiple functional sites that have been defined in high-resolution structures and biochemical assays. Calcium binds to this domain through the side chains of gammaD318 and gammaD320 and the backbone carbonyls of gammaF322 and gammaG324. We have examined variant fibrinogens with alanine at position gamma318 and/or gamma320 and found that calcium binding, fibrin polymerization, and fibrinogen-mediated platelet aggregation, but not FXIIIa-catalyzed cross-linking, were abnormal. When measured by turbidity, thrombin-catalyzed polymerization was severely reduced, and batroxobin-catalyzed polymerization was completely obliterated. Moreover, thrombin-catalyzed polymerization was abolished by the peptide GHRP, which binds to the polymerization site in the beta-chain but does not inhibit polymerization of normal fibrinogen. ADP-induced platelet aggregation was also severely impaired. In contrast, as measured by SDS-PAGE, FXIIIa introduced cross-links between gamma-chains for all three variants, as expected if the gamma-chain C-terminal sites were normal. In addition, binding of the monoclonal antibody 4A5, which recognizes the C-terminal residues, was not different from normal. These data suggest two specific conclusions: (1) a site in the gamma-module other than the C-terminus is critical for platelet aggregation and (2) "B-b" interactions have a role in protofibril formation.

Insight from studies with recombinant fibrinogens.

Using a two-step cloning strategy, we have synthesized more than 20 variant human fibrinogens for biochemical studies. In preliminary experiments we showed that normal fibrinogen produced in CHO cells serves as an accurate model for plasma fibrinogen. We focus here on those variants whose characterization has provided insight into the mechanism of thrombin-catalyzed polymerization. Analysis of N-terminal variants showed that thrombin specificity dictates the ordered release of fibrinopeptides. Nevertheless, analysis of C-terminal variants indicated that fibrinopeptide B (FpB) release is dependent on polymerization. Changes in the a polymerization site and the high-affinity calcium-binding site were associated with a complete loss of polymerization. These experiments showed that alterations in the calcium-binding site influenced function of the a site; in contrast, alterations in the a site did not alter calcium binding. Analysis of variants in the N-terminus of the B beta chain provided the first direct evidence that this region impacts predominantly on lateral aggregation, as has long been presumed. These experiments also suggested that lateral aggregation facilitated by this region proceeds without the release of FpB. From these studies we learned that individual sites within fibrinogen do not function in isolation. We conclude that thrombin-catalyzed polymerization is mediated by a continuum of concerted interactions.

Polymerization of Rod-Like Macromolecular Monomers Studied by Stopped-Flow, Multiangle Light Scattering: Set-Up, Data Processing, and Application to Fibrin Formation

Many biological supramolecular structures are formed by polymerization of macromolecular monomers. Light scattering techniques can provide structural information from such systems, if suitable procedures are used to collect the data and then to extract the relevant parameters. We present an experimental set-up in which a commercial multiangle laser light scattering photometer is linked to a stopped-flow mixer, allowing, in principle, the time-resolved extrapolation of the weight-average molecular weight M w and of the z-average square radius of gyration 〈R g 2〉 z of the polymers from Zimm-like plots. However, if elongated structures are formed as the polymerization proceeds, curved plots rapidly arise, from which M w and 〈R g 2〉 z cannot be recovered by linear fitting. To verify the correctness of a polynomial fitting procedure, polydisperse collections of rod-like or worm-like particles of different lengths, generated at various stages during bifunctional polycondensations of rod-like macromolecular monomers, were considered. Then, the angular dependence of their time-averaged scattered intensity was calculated in the Rayleigh–Gans–Debye approximation, with random and systematic noise also added to the data. For relatively narrow size distributions, a third-degree polynomial fitting gave satisfactory results across a broad range of conversion degrees, yielding M w and 〈R g 2〉 z values within 2% and no greater than 10–20%, respectively, of the calculated values. When more broad size distributions were analyzed, the procedure still performed well for semiflexible polymers, but started to seriously underestimate both M w and 〈R g 2〉 z when rigid rod-like particles were analyzed, even at relatively low conversion degrees. The data were also analyzed in the framework of the Casassa approximation, from which the mass per unit length of the polymers can be derived. These procedures were applied to a set of data taken on the early stages of the thrombin-catalyzed polymerization of fibrinogen, a rod-like macromolecule ∼50 nm long. The polymers, grown in the absence of Ca 2+ by rate-limiting amounts of thrombin, appeared to be characterized by a much broader size distribution than the one expected for a classical Flory bifunctional polycondensation, and they seem to behave as relatively flexible worm-like double-stranded chains. Evidence for the formation of fibrinogen–fibrin monomer complexes is also inferred from the time dependence of the mass/length ratio. However, our data are also compatible with the presence of limited amounts of single-stranded structures in the very early stages, either as a secondary, less populated pathway, or as transient intermediates to the classical double-stranded fibrils.

Fibrinogen bellingham: a gamma-chain R275C substitution and a beta-promoter polymorphism in a thrombotic member of an asymptomatic family.

Congenital dysfibrinogenemia is a rare cause of unexplained thrombosis. However, most individuals with dysfibrinogenemia are asymptomatic, suggesting that co-morbid factors contribute to thrombo-embolic events. The potential roles of additional genetic or acquired prothrombotic risk factors are poorly understood because detailed family studies are lacking. Herein, we describe a family whose propositus was a young Caucasian man with recurrent venous thrombo-emboli and dysfibrinogenemia due to heterozygosity for an Arg-->Cys substitution at residue 275 in the gamma-chain. The only additional thrombophilic abnormality found in the proband was heterozygosity for a G/A transition at position -455 in the fibrinogen beta-chain promoter; a genotype associated with high acute phase levels of fibrinogen. The proband's father, who died of a cerebral artery thrombosis, carried the gammaR275C substitution but not the beta-promoter -455 variant. Among 14 living relatives, eight were heterozygous for one or the other mutation and only one, a 21-year-old niece, was dually affected. None had suffered bleeding or thrombosis. In vitro studies of the proband's purified fibrinogen revealed markedly abnormal thrombin-catalyzed polymerization and delayed fibrin clot lysis by tPA-activated plasmin. We hypothesize that the gammaR275C substitution predisposes to thrombosis by generating clots that are relatively resistant to fibrinolysis. The clinical risk is low, however, in the absence of an additional thrombophilic mutation. The beta-promoter variant could, theoretically, contribute to this risk by augmenting expression of the dysfibrinogen under conditions of stress. Like the common hereditary thrombophilias, heterozygous familial dysfibrinogenemia induces thrombosis in the setting of multiple prothrombotic influences.

Analysis of A alpha 251 fibrinogen: the alpha C domain has a role in polymerization, albeit more subtle than anticipated from the analogous proteolytic fragment X.

Numerous experiments have demonstrated that the C-terminal domain of the fibrinogen Aalpha-chain, the alphaC domain, has a role in polymerization. To further examine the role of this domain, we synthesized a recombinant fibrinogen, Aalpha251 fibrinogen, that lacks the alphaC domain. We examined thrombin-catalyzed fibrinopeptide release and found that the rate of FpB release from Aalpha251 fibrinogen was 2.5-fold slower than FpB release from normal fibrinogen, while the rate of FpA release was the same for both proteins. We examined thrombin-catalyzed polymerization and found that the rates of protofibril formation and lateral aggregation were similar for both proteins, although discernible differences in lateral aggregation were clear. The rate of protofibril formation for Aalpha251 fibrinogen was never less than 85% of normal fibrinogen, while the rate of lateral aggregation for Aalpha251 fibrinogen varied from 64 to 74% of normal. We examined polymerization of fibrin monomers and found that polymerization of Aalpha251 fibrin was similar to normal fibrin at 0.4 M NaCl, but clearly different from normal at 0.05 M NaCl. These results indicate that the alphaC domain has a role in lateral aggregation, but this role is more subtle than anticipated from previous experiments, particularly those with fibrinogen fragment X. We interpret this unanticipated finding as indicative of an important contribution from the N-terminus of the beta-chain, such that protein heterogeneity that includes small amounts of fibrin lacking that N-terminus of the beta-chain leads to markedly altered lateral aggregation.

The Conversion of Fibrinogen to Fibrin: Recombinant Fibrinogen Typifies Plasma Fibrinogen

AbstractPlasma fibrinogen is a mixture of multiple molecular forms arising mainly through alternative mRNA processing and subsequent posttranslational modification. Recombinant fibrinogen is synthesized without alternative mRNA processing in a cultured cell system that may generate novel posttranslational modifications. Thus, to show that recombinant fibrinogen can serve as a functional model for plasma fibrinogen, we have examined the conversion of fibrinogen to fibrin, comparing the recombinant with the plasma protein. We examined the kinetics of (1) thrombin-catalyzed fibrinopeptide release, (2) thrombin-catalyzed polymerization of fibrinogen, (3) the polymerization of fibrin monomers, and (4) FXIIIa-catalyzed cross-link formation. We saw small differences in polymerization, suggesting that the ordered assembly of protofibrils and fibers was not identical. In all other analyses, we found that plasma fibrinogen and recombinant fibrinogen were remarkably similar. Using electron microscopy, we examined the structures of individual fibrinogen molecules and fibrin clots. Individual fibrinogen molecules were predominantly three nodule structures for both recombinant and plasma proteins. Both samples also displayed four nodule structures, but fewer four nodule structures were found with recombinant fibrinogen. Fibrin clot structures were essentially indistinguishable. We concluded that recombinant fibrinogen can serve as a accurate model for plasma fibrinogen.