Fibrin Monomer Polymerization Research Articles

Fibrinogen BOE II: dysfibrinogenemia with bleeding and defective thrombin binding

BackgroundVariants of fibrinogen sequences that bind to thrombin’s catalytic sites are mostly associated with bleeding phenotypes, while variants with fibrinogen nonsubstrate-thrombin–binding sites are commonly believed to cause thrombosis. AαGlu39 and BβAla68 play important roles in fibrin(ogen)-thrombin–nonsubstrate binding. The BβAla68Thr variant has been described in several unrelated families with apparent thrombotic phenotypes. ObjectivesHomozygous AαGlu39Lys variant (fibrinogen BOE II) was identified in a boy with dysfibrinogenemia who had multiple cerebral hemorrhages. A series of analyses were performed to assess the variant’s functions and elucidate underlying bleeding mechanisms. MethodsAbnormal fibrinogen was purified from plasma and subjected to Western blot, fibrinogen and fibrin monomer polymerization, clottability, fibrinopeptides release, activated factor (F)XIII (FXIIIa) cross-linking, fibrinolysis, and scanning electron microscopy analyses. ResultsFibrinogen BOE II weakened the binding capacity of thrombin to fibrinogen and delayed the formation of fibrin clots. The release of fibrinopeptides, polymerization of fibrinogen catalyzed by thrombin, and cross-linking of FXIIIa of fibrinogen BOE II were impaired. In contrast, batroxobin-catalyzed fibrinogen polymerization and desA/desAB fibrin monomer polymerization did not differ from those in normal controls. Fibrin clots formed by fibrinogen BOE II were composed of thicker fibrin fibers and showed a faster fibrinolysis rate. ConclusionDefective fibrin(ogen)-thrombin–nonsubstrate binding is not necessarily associated with thrombotic disorders. When the hypercoagulable state created by increased circulating free thrombin is insufficient to compensate for defective hemostasis caused by slowly formed but rapidly lysed clots, the primary concern of thrombin-binding deficiency dysfibrinogenemia appears to be hemorrhage rather than thrombosis.

Assessment of the effect of exogenous fibrin monomer on post-traumatic bleeding in hypofibrinogenemia caused by administration of snake venom Agkistrodon rhodostoma

Aim. To assess the effect of fibrin monomer on the rate of blood loss after controlled liver injury in hypofibrinogenemia induced by systemic administration of Malayan pit viper venom (Agkistrodon rhodostoma).
 Methods. A placebo-controlled study of the hemostatic effect of fibrin monomer administered intravenously at 0.25 mg/kg, and coagulation parameters in the controlled liver injury with profound hypofibrinogenemia caused by administration of Malayan pit viper venom was conducted in 34 male Chinchilla rabbits. The distribution of the studied parameters was investigated by the ShapiroWilk test. Statistical differences between groups were tested by Students t-test, MannWhitney U test, or Wilcoxon test, as appropriate. Differences in mortality rate were examined using Fisher's exact test.
 Results. A model of experimental toxogenic disseminated intravascular coagulation was reproduced, manifested by high mortality of animals (50.0%), severe blood loss (increased blood loss by 1.78 times), hemolysis, a decreased platelet count (by 19.6% of median) and platelet dysfunction, fibrinogen consumption (protein content less than 0.9 g/l), hypocoagulation as well as intensive D-dimer production (increased concentration by 25.0 times of median). A high level of the fibrin derivative demonstrated activation of fibrin formation and fibrinolysis in the bloodstream of the animals. Systemic prophylactic administration of exogenous fibrin monomer after receiving snake venom did not lead to a decrease in post-traumatic bleeding, whereas earlier, during reproduction of disseminated intravascular coagulation caused by streptokinase infusion, such a hemostatic effect of fibrin monomer was shown.
 Conclusion. The absence of fibrin monomer effect (at a dose of 0.25 mg/kg) on the severity of blood loss in toxogenic disseminated intravascular coagulation may be associated with more profound disseminated intravascular coagulation and a sharp 25-fold increase in D-dimer levels that can act as a fibrin monomer polymerization inhibitor.

Molecular and clinical profile of congenital fibrinogen disorders in Iran, identification of two novel mutations.

Congenital fibrinogen disorders (CFDs) comprise the quantitative and qualitative fibrinogen molecule abnormalities that are caused by fibrinogen gene mutations. The objective of this cohort research was to study the molecular and clinical profiles of patients with CFDs. Genomic DNA Sanger sequencing of 14 Iranian patients was performed to determine CFDs-causing mutations. The disorders were diagnosed by routine and specific (fibrinogen antigen and functional assay) coagulation tests, and clinical data were obtained from medical records. Molecular dynamics (MD) simulations were performed to investigate the effect of missense mutation on the protein structure. Thirteen out of 14 patients had afibrinogenemia while the remaining patient had dysfibrinogenemia. Umbilical cord bleeding was the most common clinical presentation (n: 9, ~70%) which led to the diagnosis of afibrinogenemia, while menorrhagia led to the diagnosis of dysfibrinogenemia. Six homozygous mutations were identified in afibrinogenemia: three previously described variants in FGA (p.Trp52Ter, p.Ser312AlafsTer109 and p.Gly316GlufsTer105), one in FBG (p.Gly430Asp), and two novel mutations in FGB (p.Gly430Arg) and FGG (p.His366ThrfsTer40), while the FGA (p.Arg38Thr) heterozygous mutation was identified in dysfibrinogenemia. MD simulation indicated that the FGA p. Arg38Thr mutation probably interferes with polymerization of fibrin monomers. In Iran, with its high rate of consanguinity, autosomal recessive afibrinogenemia with severe clinical presentations is relatively common due to heterogeneous molecular defects.

Combined Effect of By-Pass Agents and a Sequence Identical Analogue of Emicizumab on Fibrin Clot Turbidity and Structure in Factor VIII Deficient Plasma

Development of inhibitors to Factor VIII is a serious and not uncommon (occurs in about 30% of treated patients) complication of the treatment with factor concentrate in hemophilia A. Treatment of patients with inhibitors is complicated, expensive and not always successful. By-pass agents (rFVIIa and aPCC) have been considered as a treatment of choice for those patients. Very recently bispecific factor IXa- and factor X-directed antibody - Emicizumab has been approved for the treatment of hemophilia A patients with inhibitors. However, there are data from the clinical studies which raise concerns about potential prothrombotic effect of Emicizumab in combination with by-pass agents particularly aPCC.To elucidate a potential mechanism of hypercoagulability we have investigated fibrin clot quality in hemophilic plasma after addition of a sequence identical analogue (SIA) of Emicizumab in combination with by-pass agents.Parameters of fibrin clot turbidity (lag time, Max Abs, Slope, Slope time) were measured in recalcified FVIII-deficient plasma samples, after addition of low thrombin concentration (0.04 NIH/mL) and different concentrations of SIA (200 and 600 nM), rFVIIa (1.75 and 5.25 µg/mL) and aPCC (50 and 500 mU/mL). Pooled normal plasma (PNP) was used as control.After fixation, fibrin clots were analyzed by scanning electron microscopy (SEM) (Carl Zeiss, Oberkochen, Germany) and the thickness of individual fibers was measured using SIS iTEM software (FEI Company, Eindhoven, Netherlands). As previously determined, 50 randomly selected fibers in each sample were measured to obtain stable mean.All parameters of fibrin turbidity and structure were analyzed in triplicate and the results are presented as mean values.Fibrin clot in FVIII deficient plasma was difficult to analyze due to the lack of polymerization of fibrin monomers and absence of fiber formation. This structure is loose and prone to fibrinolysis. Interestingly the similar structure was observed after addition of both concentrations of rFVIIa while after addition of SIA fibrin structure improves. The best effect using single agent was noticed after addition of aPCC in concentration of 500 mU/mL. In combination with SIA (both concentrations of 200 nM and 600 nM) aPCC in concentrations of 500 mU/mL generated clots with highest density formed from very thin fibers and small intrinsic pores. These clots are even tighter than those formed from PNP samples.Parameters of fibrin turbidity obtained with higher concentrations of agents as well as representative SEM scans are presented on the Figure 1.Addition of SIA to by-pass agents additionally improved fibrin quality in FVIII deficient plasma. Combination of therapeutic concentrations of aPCC and SIA may produce clots even less porous than those in normal plasma. [Display omitted] DisclosuresChaireti:Shire: Research Funding. Antovic:NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Werfen: Honoraria; Stago: Honoraria; Siemens: Honoraria; Roche: Honoraria; Sysmex: Honoraria.

Zinc promotes clot stability by accelerating clot formation and modifying fibrin structure.

Zinc released from activated platelets binds fibrin(ogen) and attenuates fibrinolysis. Although zinc also affects clot formation, the mechanism and consequences are poorly understood. To address these gaps, the effect of zinc on clot formation and structure was examined in the absence or presence of factor (F) XIII. Zinc accelerated a) plasma clotting by 1.4-fold, b) fibrinogen clotting by 3.5- and 2.3-fold in the absence or presence of FXIII, respectively, c) fragment X clotting by 1.3-fold, and d) polymerisation of fibrin monomers generated with thrombin or batroxobin by 2.5- and 1.8-fold, respectively. Whereas absorbance increased up to 3.3-fold when fibrinogen was clotted in the presence of zinc, absorbance of fragment X clots was unaffected by zinc, consistent with reports that zinc binds to the αC-domain of fibrin(ogen). Scanning electron microscopic analysis revealed a two-fold increase in fibre diameter in the presence of zinc and in permeability studies, zinc increased clot porosity by 30-fold with or without FXIII. Whereas FXIII increased clot stiffness from 128 ± 19 Pa to 415 ± 27 Pa in rheological analyses, zinc reduced clot stiffness by 10- and 8.5-fold in the absence and presence of FXIII, respectively. Clots formed in the presence of zinc were more stable and resisted rupture with or without FXIII. Therefore, zinc accelerates clotting and reduces fibrin clot stiffness in a FXIII-independent manner, suggesting that zinc may work in concert with FXIII to modulate clot strength and stability.

Potential antithrombotic activity detected in amaranth proteins and its hydrolysates

Amaranth protein isolate and fractions were obtained and subjected to proteolysis in order to evaluate its potential antithrombotic activity. The proteins were first hydrolyzed with alcalase (pH 10, 37 °C) and then with trypsin (pH 8, 37 °C). The samples were characterized physicochemically and antithrombotic activity was evaluated using clotting tests (PT, TT and APTT) and the microplates assay. The fractions compared to the hydrolysates exhibited different electrophoretic profiles (tricine-SDSPAGE) and gel filtration chromatograms, evidencing the presence of different molecular species. The hydrolysis improved in every sample the bioactivity detected, excepting for the glutelin fraction, which exhibited the highest antithrombotic activity, significantly superior (p < 0.05) compared to the other fractions and the isolate. This behavior was observed in the two assays that analyzed the common path of the coagulation cascade at similar concentrations: TT (81.0 ± 8.5 s with a control of 19.5 ± 0.7 s) and microplate test (IC50 80 μg/mL), indicating a possible mechanism of action that involves the thrombin activity or the polymerization of fibrin monomers. The glutelin fraction showed a potential capacity to inhibit coagulation, appearing as a promising ingredient to formulate functional foods.

Hemostatic mechanisms of a soluble fraction of plant-derived sodium carboxymethyl cellulose

Sodium carboxymethyl cellulose (Sol-CM) is a water-soluble material derived from plants. The molecular mass of Sol-CM is estimated approximately 170 kDa. In our previous study, we found that Sol-CM were able to hemostatic effects in animals. The Sol-CM was found to enhance polymerization of fibrin monomers in a concentration-dependent fashion, without stimulating the activation of proenzymes, such as prothrombin, that are involved in the blood coagulation process. Fibrin fibers that formed in the presence of Sol-CM appeared to be thicker than those formed in its absence and Sol-CM was incorporated into them in the scanning electron microscopic study. Sol-CM promoted fibrinogen gamma-chain cross-linking mediated by activated blood coagulation factor XIII and also plasminogen activation by tissue-type plasminogen activator bound to fibrin. These results suggested that Sol-CM accelerated fibrin aggregation by hastening of D-D self-association of fibrin monomers at the stage of two-stranded fibrin protofibril formation as well as lateral association between two-stranded fibrin protofibrils. Sol-CM also restored polymerization of abnormal fibrinogen whose fibrin polymerization was inhibited by abnormal D-D self-association. These findings showed that the hemostatic effects of Sol-CM were due to acceleration of fibrin polymerization.

Procoagulant Activity in Hemostasis and Thrombosis

Virchow's triad is traditionally invoked to explain pathophysiologic mechanisms leading to thrombosis, alleging concerted roles for abnormalities in blood composition, vessel wall components, and blood flow in the development of arterial and venous thrombosis. Given the tissue-specific bleeding observed in hemophilia patients, it may be instructive to consider the principles of Virchow's triad when investigating mechanisms operant in hemostatic disorders as well. Blood composition (the function of circulating blood cells and plasma proteins) is the most well studied component of the triad. For example, increased levels of plasma procoagulant proteins such as prothrombin and fibrinogen are established risk factors for thrombosis, whereas deficiencies in plasma factors VIII and IX result in bleeding (hemophilia A and B, respectively). Vessel wall (cellular) components contribute adhesion molecules that recruit circulating leukocytes and platelets to sites of vascular damage, tissue factor, which provides a procoagulant signal of vascular breach, and a surface upon which coagulation complexes are assembled. Blood flow is often characterized by 2 key variables: shear rate and shear stress. Shear rate affects several aspects of coagulation, including transport rates of platelets and plasma proteins to and from the injury site, platelet activation, and the kinetics of fibrin monomer formation and polymerization. Shear stress modulates adhesion rates of platelets and expression of adhesion molecules and procoagulant activity on endothelial cells lining the blood vessels. That no one abnormality in any component of Virchow's triad fully predicts coagulopathy a priori suggests coagulopathies are complex, multifactorial, and interactive. In this review, we focus on contributions of blood composition, vascular cells, and blood flow to hemostasis and thrombosis, and suggest that cross-talk among the 3 components of Virchow's triad is necessary for hemostasis and determines propensity for thrombosis or bleeding. Investigative models that permit interplay among these components are necessary to understand the operant pathophysiology, and effectively treat and prevent thrombotic and bleeding disorders.

Sulfated Sericin is a Novel Anticoagulant Influencing the Blood Coagulation Cascade

Sulfated sericin's influence on factors in the blood coagulation cascade was investigated to elucidate its anticoagulant mechanism. Prolongation of the prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in the presence of sulfated sericin. Fluorogenic peptide substrates on thrombin (factor IIa) and factor Xa were used to study the influence of sulfate sericin on their respective activities. Sulfated sericin inhibited neither thrombin nor factor Xa in the presence of antithrombin III (AT III). Gel electrophoresis was used to examine fibrinogen–fibrin conversion by thrombin in the presence of sulfated sericin. FPA and FPB release from fibrinogen by thrombin proceeded in the presence of sulfated sericin. The behavior of polymerization of fibrin monomer (FM) was affected by the presence of sulfated sericin. No initial lag time in the polymerization process was observed by addition of sulfated sericin to FM. This result means that sulfated sericin will interfere in the build-up of normal double-strand fibrils of FM during formation of fibrin fiber. Scanning electron microscopy (SEM) observations supported this inference. The anticoagulant mechanism of sulfated sericin is inferred to interfere with the initial polymerization process of FM.

Dysfibrinogen Kagoshima with the amino acid substitution γThr-314 to Ile: Analyses of molecular abnormalities and thrombophilic nature of this abnormal molecule

Introduction Emerging lines of evidence have suggested that certain dysfibrinogens present a significant risk of thrombosis. Patient/methods The thrombophilic nature of a new-type of dysfibrinogen Kagoshima identified in a 36-year-old female with deep vein thrombosis during the postpartum period was studied. Results/discussion Based on the analyses of the patient fibrinogen and the fibrinogen genes, fibrinogen Kagoshima was shown to have the amino acid substitution of γThr-314 to Ile that resulted in impaired function and hypofibrinogenemia. Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium ions, causing very low clottability and delayed cross-linking of patient fibrin catalyzed by activated factor XIII. Because of the low clottability, a large amount of soluble fibrin was formed upon thrombin treatment, resulting in an increase of thrombin in the soluble fraction. Additionally, tPA-mediated plasmin generation on fibrin was impaired and calcium-ion-dependent integrity of the γ-chain D domain of Kagoshima fibrinogen was perturbed. The presence of many tapered-fiber ends inside the tangled fibrin networks, observed by scanning electron microscopy, suggested early termination of fibrin polymerization and the structural alteration. Conclusion These data suggest that fibrinogen Kagoshima is dysfunctional, giving rise to formation of fibrinolysis-resistant soluble fibrin polymers and entrance of soluble fibrin associating with thrombin to the circulation, partly accounting for the thrombophilic nature of the affected fibrinogen and fibrin molecules.

Acquired Factor IX (fIX) Deficiency Associated with a Multiple Myeloma.

Multiple myeloma is associated with bleeding disorders in about 15 % of patients. The pathophysiology is multifactorial and may be related to the paraprotein level. The bleeding tendency has been reported to be caused by thrombocytopenia, paraprotein-induced qualitative platelet dysfunction, inhibition of polymerization of fibrin monomers, monoclonal thrombin inhibitor, factor X deficiency, acquired von Willebrand syndrome and local tissue fragility secondary to amyloidosis. Here, we report the first case of acquired fIX deficiency associated with a multiple myeloma (MM) in a 54 year-old Caucasian man.The patient presented with a Monoclonal Gammapathy of Undetermined Significance (MGUS) (IgA lambda 15 g/L). His laboratory evaluation then revealed an activated prothrombin time (aPTT) of 37 s (normal 29–36 seconds) without any bleeding disorder. Three years later, the MGUS evolved into a stage III multiple myeloma (IgA lambda 55 g/L) and the patient presented a hemorrhage after a bone marrow biopsy. At this time, the coagulation tests showed an aPTT of 54 s, a thrombin time of 27 s (normal 20–25 seconds), a normal platelet count without any other abnormalities. Clotting factors assay revealed a fIX deficiency of 10 %. No inhibitor was found using the Bethesda method and no fIX mutation was detected in the patient DNA as well as in his sister's. After receiving chemotherapy (VAD-type chemotherapy and high-dose of Melphalan with autologous stem cells transplantation), the paraprotein concentration decreased to 9 g/L and the coagulation results returned to normal. Sixteen months later, the MM recurred (IgA lambda 78 g/L) and the patient presented hemorrhagic manifestations associated with an aPTT of 70 s and a fIX deficiency at 11 %. Factor IX deficiency and bleeding diathesis were detected in every MM relapses. He died during a severe bleeding event (epistaxis, hematuria and rectorrhagia) despite fIX concentrate treatments.The etiology of this acquired fIX deficiency is not clear. The simultaneous evolution of MM and fIX deficiency, and the absence of congenital hemophilia mutation confirm that it is an acquired deficiency. Nevertheless there is no fIX inhibitor. Until now no case of acquired fIX deficiency is described in the literature apart from Gaucher disease. Our hypothesis is that fIX can be adsorbed by the malignant plasma cells. We are currently performing immunostaining of fIX in the bone marrow of this patient to assess whether fIX is adsorbed by the medullar malignant cells.

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Thrombophilic dysfibrinogen Tokyo V with the amino acid substitution of γ Ala327Thr: formation of fragile but fibrinolysis-resistant fibrin clots and its relevance to arterial thromboembolism

AbstractThrombophilic 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 γ Ala327Thr and possibly extra glycosylation at γ 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. (Blood. 2004;103:3045-3050)

Modeling fibrin aggregation in blood flow with discrete-particles.

Excessive clotting can cause bleeding over a vast capillary area. We study the mesoscopic dynamics of clotting by using the fluid particle model. We assume that the plasma consists of fluid particles containing fibrin monomers, while the red blood cells and capillary walls are modeled with elastic mesh of "solid" particles. The fluid particles interact with each other with a short-ranged, repulsive dissipative force. The particles containing fibrin monomers have a dual character. The polymerization of fibrin monomers into hydrated fibrins is modeled by the change of the interactions between fluid particles from repulsive to attractive forces. This process occurs with a probability being an increasing function of the local density. We study the blood flow in microscopic capillary vessels about 100 microm long and with diameters in order of 10 microm. We show that the model of polymerization reflects clearly the role played by fibrins in clotting. Due to the density fluctuations caused the by the high acceleration, the fibrin chains are produced within a very short time (0.5 ms). Fibrin aggregation modifies the rheological properties of blood, slows down the incipient flow, and entraps the red blood cells, thus forming dangerous clots.

Association of fibrin monomer polymerization function, cerebrovascular risk factors and ischemic cerebrovascular disease in old people.

In order to investigate the association of fibrin monomer polymerization function (FMPF) with traditional cerebrovascular risk factors and ischemic cerebrovascular disease in old people, 1:1 paired case-control comparative study was performed for FMPF and traditional cerebrovascular risk factors on 110 cases of old ischemic cerebrovascular disease and 110 controls matched on age, sex and living condition. The results showed that cerebrovascular risk factors were more prevalent in case group than in control group. In the case group, FMPF was significantly higher than in control group. There was a significant positive correlation between hypertension and fibrin monomer polymerization velocity (FMPV), hypertension and fibrinogen (Fbg), alcohol consumption and Fbg, but no significant correlation between diabetic mellitus, smoking and FMPF was found. Among the parameters of blood lipids, there were significant positive correlations between total cholesterol (TC) and parameters of FMPF to varying degrees, triglycerides (TG) and FMPV, TG and Fbg. Our results also showed there were significant linear trends between TC and FMPV (P < 0.001), TC and Fbg (P = 0.0087), TG and FMPV/Amax (maximum absorbance) (P = 0.0143) respectively. Multiple logistic regression analysis revealed that FMPF in case group remained significantly higher than control group after adjustment of all risk factors that were significant in univariate analysis. It was concluded that there is a possible pathophysiological link between FMPF and cerebrovascular risk factors. An elevated FMPF is associated with ischemic cerebrovascular disease and an independent risk factor of this disease. In old people, detection of FMPF might be a useful screening to identify individuals at increased cerebrothrombotic risk.

The dysfibrinogenaemias.

The dysfibrinogenaemias.

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.

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.

Study of plasma fibrinogen level and fibrin monomer polymerization function in pre-eclampsia

Study of plasma fibrinogen level and fibrin monomer polymerization function in pre-eclampsia

Fibrinogen Niigata With Impaired Fibrin Assembly: An Inherited Dysfibrinogen With a Bβ Asn-160 to Ser Substitution Associated With Extra Glycosylation at Bβ Asn-158

AbstractA novel BβAsn-160 (TAA) to Ser (TGA) substitution has been identified in fibrinogen Niigata derived from a 64-year-old asymptomatic woman, who is heterozygotic for this abnormality. The mutation creates an Asn-X-Ser–type glycosylation sequence, and a partially sialylated biantennary oligosaccharide was linked to the BβAsn-158 residue. The functional abnormality was attributed to delayed lateral association of normally formed double-stranded protofibrils based on normal cross-linking of fibrin γ-chains and tissue-type plasminogen activator-catalyzed plasmin generation by polymerizing fibrin monomers. Enzymatic removal of all the N-linked oligosaccharides from fibrinogen Niigata accelerated fibrin monomer polymerization that reached the level of untreated normal fibrin monomers, but the thrombin time was prolonged from 18.2 seconds to 113 seconds (normal: 11.2 seconds to 8.9 seconds). By scanning electron micrographic analysis, Niigata fibrin fibers were found to be more curvilinear than normal fibrin fibers. After deglycosylation, Niigata fibers became straight being similar to untreated normal fibrin fibers, whereas normal deglycosylated fibrin appeared to be less-branched than untreated normal or deglycosylated Niigata fibrin. Although normal and Niigata fibrins were similar to each other in permeation and compaction studies, deglycosylated normal and Niigata fibrins had much higher permeability and compaction values, indicating that deglycosylation had brought about the formation of more porous networks. The enzymatic deglycosylation necessitates an Asn to Asp change at position Bβ-158 that is responsible for reducing the fiber thickness because of either local repulsive forces or steric hindrance in the coiled-coil region.