Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA.

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with an Fga truncation mutation.

Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga-/- mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5'-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Fibrinogen Columbus II: A novel c.1075G>T mutation in the FGG gene causing hypodysfibrinogenemia and thrombosis in an adolescent male.

Hypodysfibrinogenemia, the least frequently reported congenital fibrinogen disorder is characterized by low circulating levels of a dysfunctional protein, and is associated with phenotypic features of both hypo- and dysfibrinogenemia. Herein, we report an adolescent male with unprovoked venous thromboembolism and hypodysfibrinogenemia. Patient had recurrent, progressive thrombosis despite therapeutic anticoagulation with both low molecular weight heparin and warfarin. He had clinical and radiological improvement after transition to a direct thrombin inhibitor. Sequencing of the FGG gene identified a novel heterozygous mutation, c.1075G>T. Structural visualization of the identified variant was pursued and suggested that the mutation likely destabilizes the Ca2+ -binding site of fibrinogen resulting in pathogenicity.

Fibrinogen as a Pleiotropic Protein Causing Human Diseases: The Mutational Burden of Aα, Bß, and γ Chains.

Fibrinogen is a highly pleiotropic protein that is involved in the final step of the coagulation cascade, wound healing, inflammation, and angiogenesis. Heterozygous mutations in Aα, Bß, or γ fibrinogen-chain genes (FGA, FGB, FGG) have been described as being responsible for fibrinogen deficiencies (hypofibrinogenemia, hypo-dysfibrinogenemia, dysfibrinogenemia) and for more rare conditions, such as fibrinogen storage disease and hereditary renal amyloidosis. Instead, biallelic mutations have been associated with afibrinogenemia/severe hypofibrinogenemia, i.e., the severest forms of fibrinogen deficiency, affecting approximately 1-2 cases per million people. However, the "true" prevalence for these conditions on a global scale is currently not available. Here, we defined the mutational burden of the FGA, FGB, and FGG genes, and estimated the prevalence of inherited fibrinogen disorders through a systematic analysis of exome/genome data from ~140,000 individuals belonging to the genome Aggregation Database. Our analysis showed that the world-wide prevalence for recessively-inherited fibrinogen deficiencies could be 10-fold higher than that reported so far (prevalence rates vary from 1 in 106 in East Asians to 24.5 in 106 in non-Finnish Europeans). The global prevalence for autosomal-dominant fibrinogen disorders was estimated to be ~11 in 1000 individuals, with heterozygous carriers present at a frequency varying from 3 every 1000 individuals in Finns, to 1-2 every 100 individuals among non-Finnish Europeans and Africans/African Americans. Our analysis also allowed for the identification of recurrent (i.e., FGG-p.Ala108Gly, FGG-Thr47Ile) or ethnic-specific mutations (e.g., FGB-p.Gly103Arg in Admixed Americans, FGG-p.Ser245Phe in Africans/African Americans).

Fibrin(ogen) drives repair after acetaminophen-induced liver injury via leukocyte αMß2 integrin-dependent upregulation of Mmp12.

BACKGROUND & AIMS: Acetaminophen (APAP)-induced liver injury is coupled with activation of the blood coagulation cascade and fibrin(ogen) accumulation within APAP-injured livers of experimental mice. We sought to define the role of fibrin(ogen) deposition in APAP-induced liver injury and repair. METHODS: Wild-type, fibrinogen-deficient mice, mutant mice with fibrin(ogen) incapable of binding leukocyte αMß2 integrin (Fibγ390-396A mice) and matrix metalloproteinase 12 (Mmp12)-deficient mice were fasted, injected with 300mg/kg APAP i.p. and evaluated at a range of time-points. Plasma and liver tissue were analyzed. Rescue of Fibγ390-396A mice was carried out with exogenous Mmp12. To examine the effect of the allosteric leukocyte integrin αMß2 activator leukadherin-1 (LA-1), APAP-treated mice were injected with LA-1. RESULTS: In wild-type mice, APAP overdose increased intrahepatic levels of high molecular weight cross-linked fibrin(ogen). Anticoagulation reduced early APAP hepatotoxicity (6h), but increased hepatic injury at 24h, implying a protective role for coagulation at the onset of repair. Complete fibrin(ogen) deficiency delayed liver repair after APAP overdose, evidenced by a reduction of proliferating hepatocytes (24h) and unresolved hepatocellular necrosis (48 and 72h). Fibγ390-396A mice had decreased hepatocyte proliferation and increased multiple indices of liver injury, suggesting a mechanism related to fibrin(ogen)-leukocyte interaction. Induction of Mmp12, was dramatically reduced in APAP-treated Fibγ390-396A mice. Mice lacking Mmp12 displayed exacerbated APAP-induced liver injury, resembling Fibγ390-396A mice. In contrast, administration of LA-1 enhanced hepatic Mmp12 mRNA and reduced necrosis in APAP-treated mice. Further, administration of recombinant Mmp12 protein to APAP-treated Fibγ390-396A mice restored hepatocyte proliferation. CONCLUSIONS: These studies highlight a novel pathway of liver repair after APAP overdose, mediated by fibrin(ogen)-αMß2 integrin engagement, and demonstrate a protective role of Mmp12 expression after APAP overdose. LAY SUMMARY: Acetaminophen overdose leads to activation of coagulation cascade and deposition of high molecular weight cross-linked fibrin(ogen) species in the liver. Fibrin(ogen) is required for stimulating liver repair after acetaminophen overdose. The mechanism whereby fibrin(ogen) drives liver repair after acetaminophen overdose requires engagement of leukocyte αMß2 integrin and subsequent induction of matrix metalloproteinase 12.

Fibrinogen storage disease and cirrhosis associated with hypobetalipoproteinemia owing to fibrinogen Aguadilla in a Turkish child.

BACKGROUND AND AIMS: Fibrinogen gene mutations can rarely result in hepatic fibrinogen storage disease (HFSD). Herein, we report on the first Turkish family carrying the mutation p.Arg375Trp (fibrinogen Aguadilla) in the γ-chain of the fibrinogen (FGG) gene. METHODS: Clinical, laboratory and histopathological findings of the patient were documented. Molecular study of fibrinogen gene was performed in the patient and her family members. RESULTS: The proband was 5 years old girl presenting with advanced liver fibrosis of unknown origin. The child had very low plasma levels of fibrinogen and hypobetalipoproteinemia. Immunomorphologic and electron microscopic studies showed selective and exclusive accumulation of fibrinogen within the endoplasmic reticulum in liver biopsy of the patient. Patient, mother, two sisters and one brother carried p.Arg375Trp mutation (fibrinogen Aguadilla) in FGG gene. The patient was treated with ursodeoxycholic acid and carbamazepine. After 3 months, carbamazepine was suspended upon family decision and unresponsiveness of carbamazepine. CONCLUSIONS: HFSD is characterized by hypofibrinogenemia and accumulation of abnormal fibrinogen within hepatocytes. In addition, hypofibrinogenemia is associated with hypobetalipoproteinemia in Aguadilla mutation.

In vitro rescue of FGA deletion by lentiviral transduction of an afibrinogenemic patient's hepatocytes.

BACKGROUND: Congenital afibrinogenemia is a rare inherited autosomal recessive disorder in which a mutation in one of three genes coding for the fibrinogen polypeptide chains Aα, Bß and γ results in the absence of a functional coagulation protein. A patient with congenital afibrinogenemia, resulting from an FGA homozygous gene deletion, underwent an orthotopic liver transplant that resulted in complete restoration of normal hemostasis. The patient's explanted liver provided a unique opportunity to further investigate a potential novel treatment modality. OBJECTIVE: To explore a targeted gene therapy approach for patients with congenital afibrinogenemia. METHODS AND RESULTS: At the time of transplant, the patient's FGA-deficient hepatocytes were isolated and transduced with lentiviral vectors encoding the human fibrinogen Aα-chain. FGA-transduced hepatocytes produced fully functional fibrinogen in vitro. CONCLUSIONS: Orthotopic liver transplantation is a possible rescue treatment for failure of on-demand fibrinogen replacement therapy. In addition, we provide evidence that hepatocytes homozygous for a large FGA deletion can be genetically modified to restore Aα-chain protein expression and secrete a functional fibrinogen hexamer.

Autophagy-enhancing drug carbamazepine diminishes hepatocellular death in fibrinogen storage disease.

Fibrinogen storage disease (FSD) is a rare autosomal-dominant hereditary disorder characterized by hypofibrinogenemia and accumulation of fibrinogen aggregates within the hepatocellular endoplasmatic reticulum (ER). Some FSD patients present with elevated amino-transferases and fibrosis/cirrhosis similar to alpha-1-antitrypsin deficiency (ATD), also an ER storage disease. Pharmacological stimulation of autophagy has been shown to mediate clearance of protein aggregates and halt progression of liver fibrosis in in vivo models of ATD. Our aim was to evaluate the presence of autophagy and a possible response to autophagy-enhancing therapy in patients with FSD. Hepatic fibrosis was assessed by transient elastography in 2 newly identified FSD families with fibrinogen Aguadilla and Brescia mutations, encompassing 8 affected members. Available liver biopsies were assessed for autophagy. Two patients, who had had elevated alanine amino-transaminase levels (2-5 above upper limit of normal), were treated with the autophagy enhancer carbamazepine (CBZ). Transient elastography did not show evidence of significant fibrosis in any affected family members. Quantitative electron microscopy of one patient showed a 5.15-fold increase of late stage autophagocytic vacuoles compared to control livers. CBZ at low anticonvulsive treatment levels led to rapid normalization of alanine-aminotransferase and decrease of caspase-cleaved and uncleaved cytokeratin-18 fragments (M30 and M65). These effects reversed after discontinuation of treatment. Response to CBZ may be mediated by pharmacologically enhanced autophagy resulting in reduction of aggregate-related toxicity in FSD. These results suggest clinical applicability of pharmacological stimulation of autophagy in FSD, but potentially also in other related disorders.

Estudio clínico-epidemiológico de alteraciones hemorrágicas y necróticas causadas por el veneno de la serpiente de cascabel (crotalus durissus cumanensis) en pacientes venezolanos / Clinical and epidemiological study of hemorrhagic and necrotic alterations caused by the rattlesnake (crotalus durissus cumanensis) venom in venezuelan patients

Se describieron los efectos hemorrágicos, necróticos y edematosos de 135 pacientes provenientes de los estados Miranda, Aragua, Vargas y Distrito Capital, Venezuela, ocasionados por la mordedura de la serpiente cascabel común venezolana (Crotalus durissus cumanensis), durante los años 1998-2008. Los trastornos hemorrágicos, que tradicionalmente eran casi imperceptibles en los Crotalus venezolanos, hemos encontrado que hay evidencias francas de manifestaciones clínicas como: afibrinogenemia, alargamiento del tiempo de coagulación manual (TCM), tiempo parcial de tromboplastina (TTP) y tiempo de protrombina (TP), lo cual indica la presencia de estas fracciones hemorrágicas en el veneno de cascabeles nacionales. Se apreciaron diferencias entre ambos sexos, siendo predominante en el sexo masculino (82%). Sin embargo ha habido un aumento de incidencia significativa en el sexo femenino (17%). Por grupo etario, se observó predominancia entre 11 a 30 años de edad, en ambos sexos. El sitio de mordedura mayormente afectado fue el miembro superior (58,5%), con un porcentaje no menos significativo de miembros inferiores (40,7%). Estos hallazgos, permiten sugerir que el veneno de algunas serpientes cascabeles comunes en Venezuela, poseen un efecto sistémico sobre el músculo esquelético, y también efectos sobre capilares que generan edema, fenómenos hemorrágicos y necrosis, que habían pasado desapercibidos.

The bleeding, necrotic and edematous Snake bite effects from 135 patients of Miranda, Aragua, Vargas States and Capital District (Venezuela), caused by the Venezuelan common rattlesnake (Crotalus durissus cumanensis) from 1998 to 2008 were described. In bleeding disorders, which traditionally were almost imperceptible in Venezuelan Crotalus, we have found reliable evidence of clinical manifestations such as: afibrinogenemia, lengthening of the manual time of coagulation (MTC), and Partial Time of Thromboplastin (PTT) and Prothrombin time (PT), which indicates the presence of hemorrhagic fractions in the Venezuelan rattlesnake’s venoms. There were differences between the sexes, still predominant in male (82%). However, there has been an increase of significant impact on female (17%). By age, there was prevalence between 11 and 30 years old, both male and female. The mostly affected bite si te was upper limb (58,5%), with a no less significant percentage of lower limbs (40,7%). These findings, allowed us to suggest that some rattlesnake venoms have a systemic effect on skeletal muscle, and also effects on capillaries that generate swelling, hemorrhagic phenomena and necrosis.

Manipulating the quality control pathway in transfected cells: low temperature allows rescue of secretion-defective fibrinogen mutants.

BACKGROUND: Congenital afibrinogenemia is characterized by the absence of fibrinogen, a hexamer composed of two copies of three polypeptides, Aalpha. Bbeta and gamma. The disease is caused by mutations in one of the three fibrinogen-encoding genes, FGA, FGB and FGG. Among these, several mutations have been reported to specifically impair fibrinogen secretion. We previously showed that secretion-defective fibrinogen mutants are retained in a pre-Golgi compartment and demonstrated the importance of the homologous betaC and gammaC domains in secretion. Here our aim was to restore the secretion of these mutants and study the properties of the rescued mutant molecules. DESIGN AND METHODS: COS-7 cells were transfected and incubated with chemical chaperones or at low temperature. Clotting assays and plasmin digestion studies were performed to characterize secreted fibrinogen molecules. RESULTS: The secretion defect of two missense mutants but not that of late-truncating mutants could be partially corrected by incubating cells at 27 degrees C. By contrast, exposure of cells to chemical chaperones i.e. 4-phenylbutyrate, dimethyl sulfoxide or trimethylamine N-oxide had no effect. The mutants rescued at 27 degrees C were incorporated into fibrin clots and formed factor XIII-mediated gamma-gamma dimers in contrast to the dysfibrinogenemia Vlissingen/Frankfurt IV mutant, a negative control for these assays. However, plasmin digestion analyses revealed aberrant patterns for the mutants compared to normal fibrinogen. CONCLUSIONS: Low temperature can restore the secretion of a subset of mutant fibrinogen molecules demonstrating that therapeutic manipulation of the quality control pathway is feasible for afibrinogenemia even though functional assays suggested a non-native conformation for the mutant molecules analyzed.

Fibrinogen gamma375 arg-->trp mutation (fibrinogen aguadilla) causes hereditary hypofibrinogenemia, hepatic endoplasmic reticulum storage disease and cirrhosis.

Hypofibrinogenemia is a rare inherited disorder characterized by low levels of circulating fibrinogen, caused by mutations within 1 of the 3 fibrinogen genes. We report here the case of a 61-year-old man with chronic liver function test alterations. Liver biopsy examination revealed chronic hepatitis complicated by cirrhosis and weakly eosinophilic globular cytoplasmic inclusions within the hepatocytes, faintly stained with PAS-diastase. On immunohistochemistry, the inclusions reacted strongly with human antifibrinogen antibodies. Coagulation investigations of the propositus and his 2 sons showed low functional and antigenic fibrinogen concentrations that were indicative of hypofibrinogenemia. A liver biopsy performed on the 28-year-old son demonstrated the same globular cytoplasmic inclusions, albeit without associated chronic liver disease. PCR amplification followed by sequencing showed that all 3 were heterozygous for a CGG>TGG mutation at codon 375 of the fibrinogen gamma-chain gene (FGG), corresponding to an Arg>Trp substitution. This is the first in an adult male and the second published case with a discernible hepatic fibrinogen endoplasmic reticulum storage disease due to an FGG Arg375Trp (fibrinogen Aguadilla) mutation. Our results suggest that familial hypofibrinogenemia should be considered in the differential diagnosis of a progressive liver disease associated to hepatocellular intracytoplasmic globular inclusions.

Mutant fibrinogen cleared from the endoplasmic reticulum via endoplasmic reticulum-associated protein degradation and autophagy: an explanation for liver disease.

The endoplasmic reticulum (ER) quality control processes recognize and remove aberrant proteins from the secretory pathway. Several variants of the plasma protein fibrinogen are recognized as aberrant and degraded by ER-associated protein degradation (ERAD), thus leading to hypofibrinogenemia. A subset of patients with hypofibrinogenemia exhibit hepatic ER accumulation of the variant fibrinogens and develop liver cirrhosis. One such variant named Aguadilla has a substitution of Arg375 to Trp in the gamma-chain. To understand the cellular mechanisms behind clearance of the aberrant Aguadilla gamma-chain, we expressed the mutant gammaD domain in yeast and found that it was cleared from the ER via ERAD. In addition, we discovered that when ERAD was saturated, aggregated Aguadilla gammaD accumulated within the ER while a soluble form of the polypeptide transited the secretory pathway to the trans-Golgi network where it was targeted to the vacuole for degradation. Examination of Aguadilla gammaD in an autophagy-deficient yeast strain showed stabilization of the aggregated ER form, indicating that these aggregates are normally cleared from the ER via the autophagic pathway. These findings have clinical relevance in the understanding of and treatment for ER storage diseases.

Fibrinogen storage disease without hypofibrinogenaemia associated with acute infection.

AIMS: The presence of ground glass hepatocytes in a liver biopsy may be related to different conditions, including fibrinogen storage disease. Three types of fibrinogen storage disease have been described, namely types I, II and III. Type I is an hereditary hypofibrinogenaemia genetically characterized by a mutant variant of the fibrinogen molecule designated as fibrinogen Brescia, consistent with a gamma284 Gly-->Arg mutation. Only rare cases of types II and III fibrinogen storage disease have been described. The purpose of the present paper is to describe two cases of fibrinogen storage disease without associated hypofibrinogenaemia, which appeared during acute infectious diseases. METHODS AND RESULTS: Both patients were female, aged 77 and 73 years, who developed high transaminases during an infectious disease. In each case blood coagulation tests were within the normal range, and despite clinical and laboratory investigations no possible cause for liver disease could be found. Liver biopsies were performed; in both cases weakly eosinophilic cytoplasmic inclusions were observed. Using immunohistochemistry the inclusions were found to be due to fibrinogen accumulation. At ultrastructural level features corresponding to type II inclusions were observed. Molecular studies, performed in case 2, excluded the mutation typical of type I fibrinogen storage disease. Both patients also presented features of chronic hepatitis. In case 1, giant cell granulomas were additionally present. No close relatives of the patients presented any clinical or laboratory features of liver disease. In both patients altered liver function test values gradually, spontaneously, returned to within normal ranges after infectious disease was resolved. CONCLUSIONS: These cases suggest that, on rare occasions, hepatocytes may accumulate fibrinogen during an infectious disease.

Novel fibrinogen gamma375 Arg-->Trp mutation (fibrinogen aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia.

The proposita and her sister had chronically elevated liver function test results, and needle biopsy specimens showed scattered eosinophilic inclusions within the hepatocytes. On immunoperoxidase staining, the inclusions reacted strongly with anti-fibrinogen antisera; on electron-microscopic (EM) examination, the material appeared confined to the endoplasmic reticulum (ER) and was densely packed into tubular structures with a swirling fingerprint appearance. Coagulation investigations showed low functional and antigenic fibrinogen concentrations that were indicative of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous CGG-->TGG mutation at codon 375 of the fibrinogen gamma chain gene. This novel gamma375 Arg-->Trp substitution segregated with hypofibrinogenemia in 3 family members and was absent from 50 normal controls. When purified plasma fibrinogen chains were examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, reverse-phase chromatography, electrospray ionization mass spectrometry, and isoelectric focusing, only normal gamma chains were detected. In conclusion, we propose that this nonconservative mutation causes a conformational change in newly synthesized molecules and that this provokes aggregation within the ER and in turn causes the observed hypofibrinogenemia. Whereas the mutation site, gamma375, is located in the gammaD domain at the jaws of the primary E-to-D polymerization site, purified plasma fibrinogen showed normal polymerization, supporting our contention that molecules with variant chains never reach the circulation but accumulate in the ER.

A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I.

A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation.

[Resolution of Bbeta and gamma chains from normal human fibrinogen Zürich II into 2 variants with each differing sialic acid content]. / Trennung der Bbeta- und gamma-Ketten von normalem humanem Fibrinogen und von Fibrinogen Zürich II in je 2 Varianten mit verschiedenem Sialsäuregehalt

The gamma- and Bbeta-polypeptide chains of purified human fibrinogen (both pooled and single donor) have each been resolved into 2 major components: gammaL and gammaR, and BbetaL and BbetaR. They are similar in molecular weight (SDS-PAGE), but differ in sialic acid content, which approximates 2 and 1 residues per molecular of polypeptide in the L- and R-components respectively. Tryptic peptide maps of the L-and R- forms of the gamma chain showed differences within the small group of peptides containing the sialic acid residues. No differences between the peptide maps of BbetaL- and BbetaR-chains were found . A larger ratio of L:R in the gamma- and Bbeta-chains of dysfibrinogenemia fibrinogen "Zürich II" explains the higher content of sialic acid measured in the unmodified Zürich II fibrinogen molecule.