Release Of Fibrinopeptide Research Articles

Establishing the inhibitory effects of bradykinin on thrombin

Bradykinin, RPPGFSPFG, has been reported to be an inhibitor of thrombin’s roles in blood clotting, platelet activation, and cellular permeability. The exact target, magnitude, and type of inhibition occurring are not well characterized. Based on the individual kinetic parameters calculated here, bradykinin is classified as a weak competitive inhibitor against hydrolysis of S-2238 and of a PAR4-like peptide. The K m values increased twofold in the presence of bradykinin, whereas the k cat values remained constant. The K i values ranged from 170 to 326 μM. Other biochemical studies indicated that bradykinin inhibits release of fibrinopeptide A from fibrinogen. Furthermore, bradykinin hindered the time required for fibrin clot formation. The weak inhibitions observed in vitro suggest that the direct effects of bradykinin on the thrombin active site become significant only at high concentrations, levels that may be difficult to achieve physiologically. Clearly, bradykinin can target thrombin but whether this direct interaction can be achieved in vivo and is sufficient to elicit a response without contributions from other cofactors requires further investigation.

Allosteric Effects Potentiating the Release of the Second Fibrinopeptide A from Fibrinogen by Thrombin

Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of alpha-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified alpha-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na(+). The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na(+), whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form.

Mechanism of factor VIIa–dependent coagulation in hemophilia blood: Presented in part at the 42nd Annual Meeting of the American Society of Hematology, December 1-5, 2000, San Francisco, CA.

AbstractThe ability of factor VIIa to initiate thrombin generation and clot formation in blood from healthy donors, blood from patients with hemophilia A, and in anti–factor IX antibody–induced (“acquired”) hemophilia B blood was investigated. In normal blood, both factor VIIa–tissue factor (TF) complex and factor VIIa alone initiated thrombin generation. The efficiency of factor VIIa was about 0.0001 that of the factor VIIa–TF complex. In congenital hemophilia A blood and “acquired” hemophilia B blood in vitro, addition of 10 to 50 nM factor VIIa (pharmacologic concentrations) corrected the clotting time at all TF concentrations tested (0-100 pM) but had little effect on thrombin generation. Fibrinopeptide release and insoluble clot formation were only marginally influenced by addition of factor VIIa. TF alone had a more pronounced effect on thrombin generation; an increase in TF from 0 to 100 pM increased the maximum thrombin level in “acquired” hemophilia B blood from 120 to 480 nM. Platelet activation was considerably enhanced by addition of factor VIIa to both hemophilia A blood and “acquired” hemophilia B blood. Thus, pharmacologic concentrations of factor VIIa cannot restore normal thrombin generation in hemophilia A and hemophilia B blood in vitro. The efficacy of factor VIIa (10-50 nM) in hemophilia blood is dependent on TF.

An antibody that binds to primary specific pocket-associated structure in the active site of bovine thrombin.

We attempted to produce a monoclonal antibody (MAb) against the active site of native thrombin. Bovine thrombin was treated with diisopropyl fluorophosphate, and prepared diisopropylphosphoryl-thrombin was used for the immunization to BALB/c mice. Spleen cells of immunized mice were hybridized with mouse myeloma cells P3U1, and a hybridoma clone CC2, which produced a MAb against bovine thrombin was established. The MAb produced by hybridoma clone CC2 (MAb(CC2)), consisting of IgG(1) and kappa light chain, was purified using protein A affinity chromatography. Purified MAb(CC2) prolonged the fibrin forming time of bovine thrombin and inhibited the release of fibrinopeptide A from rabbit fibrinogen. In addition, it was found that argatroban partially, but competitively, interfere the binding between MAb(CC2) and bovine thrombin. It was then considered that MAb(CC2) would bind to the molecular structure associating primary specific pocket in the active site of bovine thrombin.

The dysfibrinogenaemias.

The dysfibrinogenaemias.

An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin.

Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: Aalpha R16C and gamma G165R. As seen previously with other heterozygous Aalpha R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to Aalpha 16 C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the alpha-helix content in the variant D3 fragment. It is concluded that the Aalpha-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the gamma-chain induced a conformational change in the D3 fragment.

Fibrinogen Naples I (Bβ A68T) Nonsubstrate Thrombin-Binding Capacities

Fibrinogen Naples I (Bβ A68T) is characterized by defective thrombin binding and fibrinopeptide cleavage at the fibrinogen substrate site in the E domain. We evaluated the fibrinogen of three homozygotic members of this kindred (II.1, II.2, II.3) who have displayed thrombophilic phenotypes and two heterozygotic subjects (I.1, I.2) who were asymptomatic. Electron microscopy of Naples I fibrin networks showed relatively wide fiber bundles, probably due to slowed fibrin assembly secondary to delayed fibrinopeptide release. We evaluated 125I-thrombin binding to the fibrin from subjects I.1, I.2, II.1, and II.2 by Scatchard analysis with emphasis on the high-affinity site in the D domain of fibrin(ogen) molecules containing a γ chain variant termed γ′. Homozygotic subjects II.1 and II.2 showed virtually absent low-affinity binding, consistent with the Bβ A68T mutation, whereas heterozygotes I.1 and I.2 showed only moderately reduced low-affinity binding. The homozygotes also showed impaired high-affinity thrombin binding, whereas that of the heterozygotes was nearly the same as normal. Genomic sequencing of the γ′ coding sequence (I.2, II.2), ELISA measurements of two γ′ chain epitopes (L2B, γ′409–412, and IF10, γ′417–427) (I.2, II.1, II.2, II.3), and mass spectrometry of Naples I fibrinogen (II.2) showed no differences from normal, thus indicating that there were no abnormal structural modifications of the γ′ chain residues in Naples I fibrinogen. However, thrombin reportedly utilizes both of its available exosites for binding to high- and low-affinity sites on normal fibrin, suggesting that binding is cooperative. Thus, reduced high-affinity thrombin binding to homozygotic Naples I fibrin may be related to the absence of low-affinity binding sites.

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.

Fibrinogen Longmont

B beta Arg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo-Cys residues were found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Thrombin and batroxobin induced fibrin polymerization were impaired, despite normal release of fibrinopeptides A and B. Moreover, the polymerization defect was not corrected by removing the dimeric species or adding calcium. Fibrinogen Longmont had normal polymerization site a, as evidenced by normal GPRP-peptide binding. Thus, the sites A and a can interact to form protofibrils, as evidenced by dynamic light scattering measurements. These protofibrils, however, do not associate laterally in a normal manner, leading to an abnormal clot formation.

Platelet Glycoprotein Ibα Binds to Thrombin Anion-binding Exosite II Inducing Allosteric Changes in the Activity of Thrombin

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate d-Phe-Pro-Arg-pNA. The K(d) for the glycocalicin-thrombin interaction was 1 microm at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 microm at 100 mm NaCl (Gamma(salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K(d) values for their interactions with GPIb alpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K(d) values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate d-Phe-Pro-Arg-pNA.

Fibrinogen Matsumoto V: a Variant with Aα19 Arg → Gly (AGG → GGG)

Fibrinogen Matsumoto V (M-V) is a dysfibrinogen identified in a 52-year-old woman with systemic lupus erythematous. The triplet AGG encoding the amino acid residue Aalpha19 was replaced by GGG, resulting in the substitution of Arg-->Gly. Residue Aalpha19 has been shown to be one of the most important amino acids in the so-called 'A' site or alpha-chain knob. The thrombin-catalyzed release of fibrinopeptide A from M-V fibrinogen was only slightly delayed yet release of fibrinopeptide B was significantly delayed. Both thrombin-catalyzed fibrin polymerization and fibrin monomer polymerization were markedly impaired compared to normal fibrinogen. In addition, reptilase-catalyzed fibrin polymerization of M-V was much more impaired than thrombin-catalyzed fibrin polymerization. These results indicate 'B' and/or 'b' site of M-V fibrinogen play a more important role in thrombin-catalyzed fibrin polymerization than that of normal control fibrinogen.

Fibrinogen Alès: a homozygous case of dysfibrinogenemia (γ-Asp330 →Val) characterized by a defective fibrin polymerization site “a”

Congenital homozygous dysfibrinogenemia was diagnosed in a man with a history of 2 thrombotic strokes before age 30. His hemostatic profile was characterized by a dramatically prolonged plasma thrombin clotting time, and no clotting was observed with reptilase. Complete clotting of the abnormal fibrinogen occurred after a prolonged incubation of plasma with thrombin. The release of fibrinopeptides A and B by thrombin and of fibrinopeptide A by reptilase were both normal. Thrombin-induced fibrin polymerization was impaired, and no polymerization occurred with reptilase. The polymerization defect was characterized by a defective site "a," resulting in an absence of interaction between sites A and a, indicated by the lack of fragment D(1) (or fibrinogen) binding to normal fibrin monomers depleted in fibrinopeptide A only (Des-AA fm). By SDS-PAGE, the defect was detected on the gamma-chain and in its fragment D(1). The molecular defect determined by analysis of genomic DNA showed a single base change (A-->T) in exon VIII of the gamma-chain. The resulting change in the amino acid structure is gamma 330 aspartic acid (GAT) --> valine (GTT). It is concluded that the residue gamma-Asp(330) is essential for the normal functioning of the polymerization site a on the fibrinogen gamma-chain.

Fibrinogen Alès: a homozygous case of dysfibrinogenemia (γ-Asp330 →Val) characterized by a defective fibrin polymerization site “a”

AbstractCongenital homozygous dysfibrinogenemia was diagnosed in a man with a history of 2 thrombotic strokes before age 30. His hemostatic profile was characterized by a dramatically prolonged plasma thrombin clotting time, and no clotting was observed with reptilase. Complete clotting of the abnormal fibrinogen occurred after a prolonged incubation of plasma with thrombin. The release of fibrinopeptides A and B by thrombin and of fibrinopeptide A by reptilase were both normal. Thrombin-induced fibrin polymerization was impaired, and no polymerization occurred with reptilase. The polymerization defect was characterized by a defective site “a,” resulting in an absence of interaction between sites A and a, indicated by the lack of fragment D1 (or fibrinogen) binding to normal fibrin monomers depleted in fibrinopeptide A only (Des-AA fm). By SDS-PAGE, the defect was detected on the γ-chain and in its fragment D1. The molecular defect determined by analysis of genomic DNA showed a single base change (A→T) in exon VIII of the γ-chain. The resulting change in the amino acid structure is γ 330 aspartic acid (GAT) → valine (GTT). It is concluded that the residue γ-Asp330 is essential for the normal functioning of the polymerization site a on the fibrinogen γ-chain.

Recombinant Fibrinogen Studies Reveal That Thrombin Specificity Dictates Order of Fibrinopeptide Release

During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A'beta fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA' (G14V). Analyses of fibrinopeptide release from A'beta fibrinogen showed that FpA release and FpA' release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA' were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA' release from A'beta fibrinogen was unaffected. Thus, with A'beta fibrinogen, fibrinopeptide release from the beta chain is similar to fibrinopeptide release from the alpha chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A'beta fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A'beta fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the beta chain does not affect polymerization of fibrinogen.

Decreased Lateral Aggregation of a Variant Recombinant Fibrinogen Provides Insight into the Polymerization Mechanism

We analyzed the polymerization of BbetaA68T fibrinogen, the recombinant counterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, BbetaA68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V(max), than normal recombinant fibrinogen, but a similar final turbidity. At thrombin concentrations that equalized the rates of fibrinopeptide A release, BbetaA68T fibrinogen polymerized with a lag time and V(max) similar to normal, but reached a significantly lower final turbidity. Similar results were produced when BbetaA68T was polymerized with Ancrod, which cleaves fibrinopeptide A at the same rate from either fibrinogen, and when BbetaA68T desA monomers were polymerized. The polymerization of desAB fibrin monomers, which circumvents fibrinopeptide release, was the same for both fibrinogens. We confirmed that turbidity was indicative of fiber thickness by scanning electron microscopy of fibrin clots. Here, we present the first experimental evidence of fibrin polymerization with a normal period of protofibril formation and rate of lateral aggregation, but with a significantly decreased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in BbetaA68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during normal polymerization a subtle conformational change in the E domain occurs, between the release of FpA and FpB, and that this change modulates the mechanism of lateral aggregation. Without this change, the lateral aggregation of BbetaA68T fibrinogen is impaired such that variant clots have thinner fibers than normal clots.

Models of blood coagulation.

We have used three models to study the process of tissue factor-initiated blood coagulation. These are: synthetic 'plasma' mixtures prepared with the proteins and membranes involved in the reaction and its regulation; mathematical models based on the reaction kinetics, binding constants and stoichiometries of individual procoagulant and inhibitor reactions, and contact pathway-inhibited coagulation of minimally altered whole blood in vitro. In all of these models, the procoagulant process may be divided into two phases: an initiation phase and a propagation phase. The initiation phase is characterized by the appearance of thrombin and other coagulation enzymes, and the activation of pro-cofactors V and VIII. The propagation phase is characterized by explosive and extensive prothrombin activation. During normal blood coagulation, the bulk of thrombin generation occurs after clot formation, while most release of fibrinopeptide A is observed just at the conclusion of the initiation phase. In the case of haemophilia A and B, the initiation phase is slightly extended, while thrombin generation during the propagation phase is significantly suppressed. The clot time, as well as fibrinopeptide release, is delayed in these patients. Data obtained in our laboratory, employing the above models, indicate that they are efficient tools for blood coagulation studies.

Transglutaminase-catalyzed crosslinking of the Aalpha and gamma constituent chains in fibrinogen.

Studies on transglutaminases usually focus on the polymerization of protein substrates by intermolecular N(epsilon)(gamma-glutamyl)lysine bridges, without considering the possibility that the monomeric protein units, themselves, could also become crosslinked internally. Both types of crosslinks are produced in the reaction of fibrinogen with red cell transglutaminase. We isolated the transglutaminase-modified, mostly monomeric form (92-96%) of fibrinogen with a N(epsilon)(gamma-glutamyl)lysine content of approximately 1.6 moles/mole of fibrinogen. The preparation was fully clottable by thrombin, but the rates of release of fibrinopeptides and clotting times were delayed compared with control. Hybrid Aalpha.gamma type of crosslinking, the hallmark of the reaction of the transglutaminase with fibrinogen, occurred by bridging the Aalpha(408-421) chain segment of the protein to that of gamma(392-406). Rotary shadowed electron microscope images showed many monomers to be bent, and the crosslinks seemed to bind the otherwise flexible alphaC domain closer to the backbone of fibrinogen.

A Novel Fibrinogen-clotting Enzyme, TL-BJ, from the Venom of the Snake Bothrops jararaca: Purification and Characterization

Three chromatographically distinct forms of a novel fibrinogen-clotting serine endopeptidase, TL-BJ1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-Arg-pNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.

Dysfibrinogenemia and thrombosis.

Congenital abnormal fibrinogen molecules (dysfibrinogenemias) are due to structural defects in the molecule. The molecular structure of the fibrinogen molecule is to a great extent known and this has allowed identification of the abnormalities at a molecular level. While most patients with dysfibrinogenemia are clinically asymptomatic, some present with a bleeding diathesis, others with thrombophilia, and occasionally with both, bleeding and thromboembolism. In principle, the dysfibrinogenemias are due to either impaired release of the fibrinopeptides, defective fibrin polymerization, or abnormal cross-linking by factor XIIIa. Dysfibrinogenemias associated with thrombophilia have been reported in those related to abnormal fibrinopeptide release or defective polymerization. In addition, abnormal interactions with platelets, defective fibrinolysis, defective assembly of the fibrinolytic system, and abnormal calcium binding have been described. The presently identified dysfibrinogenemias associated with thrombosis and their molecular defects are described in this review. It must also be recognized that some patients with abnormal fibrinogen molecules have additional hemostasis defects, such as abnormalities of antithrombin, protein C, protein S, factor V Leiden, and others. On rare occasions, dysfibrinogenemias can be associated with hypofibrinogenemia.