Polypeptide Chains Of Fibrinogen Research Articles

Apigenin Provides Structural Protection to Human Fibrinogen against Nitrosative Stress: Biochemical and Molecular Insights.

Peroxynitrite (ONOO-) is an oxidant linked with several human pathologies. Apigenin, a natural flavonoid known for its health benefits, remains unexplored in relation to ONOO- effects. This study investigated the potential of apigenin to structurally protect fibrinogen, an essential blood clotting factor, from ONOO--induced damage. Multi-approach analyses were carried out where fibrinogen was exposed to ONOO- generation while testing the efficacy of apigenin. The role of apigenin against ONOO--induced modifications in fibrinogen was investigated using UV spectroscopy, tryptophan or tyrosine fluorescence, protein hydrophobicity, carbonylation, and electrophoretic analyses. The findings demonstrate that apigenin significantly inhibits ONOO--induced oxidative damage in fibrinogen. ONOO- caused reduced UV absorption, which was reversed by apigenin treatment. Moreover, ONOO- diminished tryptophan and tyrosine fluorescence, which was effectively restored by apigenin treatment. Apigenin also reduced the hydrophobicity of ONOO--damaged fibrinogen. Moreover, apigenin exhibited protective effects against ONOO--induced protein carbonylation. SDS-PAGE analyses revealed that ONOO-treatment eliminated bands corresponding to fibrinogen polypeptide chains Aα and γ, while apigenin preserved these changes. This study highlights, for the first time, the role of apigenin in structural protection of human fibrinogen against peroxynitrite-induced nitrosative damage. Our data indicate that apigenin offers structural protection to all three polypeptide chains (Aα, Bβ, and γ) of human fibrinogen. Specifically, apigenin prevents the dislocation or breakdown of the amino acids tryptophan, tyrosine, lysine, arginine, proline, and threonine and also prevents the exposure of hydrophobic sites in fibrinogen induced by ONOO-.

Редкий случай гиподисфибриногенемии у пациента без гематомезенхимальной дисплазии

Фибриноген и коллаген являются важнейшими протеинами межклеточного матрикса, оказывающие прямое влияние как на дифференцировку мезенхимальных стволовых клеток, так и на остеогенез. Кроме того, в клинической практике встречаются случаи протекания данного синдрома не в классическом виде, которые требуют более углубленного изучения звеньев патогенеза и разработки оптимальной коррекции заболевания. Нами проведен поиск литературных данных по ключевым словам в базах данных PubMed, UpToDate, BMJ. Представлен обзор современных научных данных и результаты наблюдения редкого клинического случая у пациента с гиподисфибриногенемией с проявлениями асептического некроза головок бедренных костей без гематомезенхимальной дисплазии. Заключение: В целом, в настоящее время подробно изучены различные аспекты клинико-биохимического патогенеза аномалий фибриногена, но остаются «белые пятна» в изучении тонких патогенетических механизмов на молекулярно-генетическом уровне, а именно: катаболизм белка, реакции раздельного синтеза отдельных полипептидных цепей фибриногена в разных тканях. Остается нерешенным вопрос о целесообразности применения корригирующих гемостаз препаратов таргетного действия при отсутствии у пациента выраженных тромботических и геморрагических проявлений. Fibrinogen and collagen are the most important intercellular matrix proteins that directly effect on the differentiation of mesenchymal stem cells and osteogenesis. In addition, there are non-classical cases of this syndrome that require indepth study of pathogenesis and optimal disease correction. We examined literature data by keywords in databases PubMed, UpToDate, BMJ. We presented a review of current scientific data and observation results of a rare clinical case in a patient with hypodysfibrinogenemia without hematomesenchymal dysplasia with manifestations of aseptic necrosis of femoral heads. Conclusions: In general, various aspects of clinical and biochemical pathogenesis of fibrinogen abnormalities are currently studied in detail, but there are “white spots” in the study of sophisticated pathogenetic mechanisms at the molecular genetic level, namely protein catabolism, the reaction of separate synthesis of individual fibrinogen polypeptide chains in different tissues. Unresolved is the question of applicability of hemostatic corrective drugs in the absence of expressed thrombotic and hemorrhagic manifestations.

Unsaturated fatty acids enhance the fibrinolytic activity of subtilisin NAT (nattokinase).

Subtilisin NAT (STN), alternatively designated nattokinase, is a serine protease with potent fibrinolytic activity. In this study, we screened several foods to enhance the fibrinolytic potential of STN and identified unsaturated fatty acid-rich ones as candidates. We isolated linoleic acid as a major active compound from one of the most active foods, red pepper. Linoleic acid promoted the STN-mediated fibrin/fibrinogen degradation at >20μg/ml. STN cleaved three of the fibrinogen polypeptide chains, among which linoleic acid accelerated Bβ-chain and γ-chain degradations, but slightly suppressed the degradation of α-chain fragments. Linoleic acid failed to affect small synthetic peptide degradation, suggesting a conformational modulation of fibrin/fibrinogen for the linoleic acid promotion of STN activity. Of the various fatty acids tested, unsaturated ones were active but saturated ones were rather inhibitory to STN-mediated fibrinolysis. Thus, our data shed new light on the dietary promotion of STN activity. PRACTICAL APPLICATIONS: Subtilisin NAT (STN) is a serine protease abundantly contained in natto, a soybean food fermented with Bacillus subtilis var. natto. The use of STN as functional foods to improve blood circulation is getting attention because STN actively degrades fibrin. Our results demonstrate that widely occurring unsaturated fatty acids such as linoleic, eicosapentaenoic, and docosahexaenoic acids enhance the fibrinolytic activity of STN. Thus, the intake of natto or STN supplements in combination with unsaturated fatty acid-containing oil can be a novel way to gain cardiovascular benefits.

Congenital structural and functional fibrinogen disorders: a primer for internists.

Congenital qualitative and quantitative fibrinogen disorders represent heterogeneous rare abnormalities caused by mutations in one of the 3 genes encoding individual fibrinogen polypeptide chains, located on chromosome 4q28. It is estimated that congenital fibrinogen disorder accounts for 8% of rare coagulation factor deficiencies. Most of congenital fibrinogen disorders are suspected in individuals with bleeding tendency or coincidentally discovered, for instance prior to surgery. Fibrinogen disorders could be also found in patients with thrombotic events, impaired wound healing, and recurrent spontaneous abortions. Afibrinogenemia manifests as mild to severe bleeding, while hypofibrinogenemia is often asymptomatic. Dysfibrinogenemia, a qualitative fibrinogen disorder, is associated with bleeding, thrombosis, or with no symptoms. Recent recommendations issued by the International Society on Thrombosis and Haemostasis in 2018 do not encourage routine evaluation of thrombin time or other coagulation tests in patients with suspected congenital fibrinogen disorders, highlighting the value of fibrinogen antigen measurement and genetic analysis, added to the key finding, that is, reduced fibrinogen concentration determined with a coagulometric assay. The current review summarizes practical issues in diagnostic workup and clinical management of patients with afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia from a perspective of internists who may encounter patients with reduced fibrinogen concentration in everyday practice. Despite the fact that hematologists are in front line for the management of patients with bleeding tendency, internists should be aware of the clinical and laboratory findings in patients with inherited fibrinogen disorders including the risk of thromboembolism and management prior to invasive procedures.

Management of a Case of Congenital Afibrinogenemia for Extradural Hematoma Evacuation with Fresh Frozen Plasma

Afibrinogenemia is a rare bleeding disorder with an estimated prevalence of 1:1,000,000. It is an autosomal recessive disease resulting from mutations in any of the 3 genes that encode the 3 polypeptide chains of fibrinogen. Spontaneous bleeding, bleeding after minor trauma and excessive bleeding during interventional procedures are the principal manifestations. Replacement therapy is the mainstay of treatment of bleeding episodes in these patients and plasma-derived fibrinogen concentrate is the agent of choice. Cryoprecipitate and fresh frozen plasma are alternative treatments that should be used only when fibrinogen concentrate is not available. We report a case of congenital afibrinogenemia, a 3 years old child, posted for emergency intracranial hematoma evacuation who was successfully managed by fresh frozen plasma as other alternatives were unavailable.

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

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, Bβ 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. To explore a targeted gene therapy approach for patients with congenital afibrinogenemia. 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. 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.

Letter to Editors Iron-induced fibrin formation may explain vascular pathology in Alzheimer’s disease

In their review article Serý et al. [6] have presented compelling arguments in favour of the role of vascular mechanisms in the development of Alzheimer’s disease (AD). These arguments are in line with a recent series of publications questioning the prevailing concept of amyloid beta-induced neurodegeneration. Already in 2009 Pimplicar published a paper reassessing the amyloid hypothesis in AD [4]. More recently another important paper was published, in which the author has indicated that therapeutic targeting Abeta brain deposits had no significant effect in AD patients [5]. In the last decade of this century, a number of papers appeared that offered alternative explanations of the molecular mechanisms of AD that are related to cardiovascular diseases (CVD). It is well known that the hallmark of CVD is the intravascular formation of persistent fibrin deposits, or thrombi. Under the conditions of normal hemostasis, fibrin is gradually albeit completely removed by a powerful blood fibrinolytic enzyme system. It is known, however, that in the pathologic situations, such as coronary and/or cerebral thrombosis, fibrin clots are refractory to the thrombolytic dissolution [2]. We have recently shown that, when compared to thrombin-generated clots, the iron-induced fibrin has a radically different structure and morphological appearance as documented by scanning electron microscopy [1]. A characteristic feature of this novel form of fibrin (parafibrin) is its complete resistance to the proteolytic degradation. Thus, it may be argued that the formation of parafibrin in the cerebral circulation may be an important factor contributing to AD pathology. Apparently, unfolding of fibrinogen polypeptide chains and its scrambled refolding induced by iron potentiates and/or mimics amyloid deposits in the brain, and in this way contributes to AD pathology. This concept is supported by the findings of Strickland and his group, who have shown that persistent fibrin deposits contribute to neuro-inflammation [3]. In conclusion, we believe that the information presented above supports arguments presented by Serý et al. [6] that deserve further attention.

Fibrinogen gene regulation

The Aα, Bβ and γ polypeptide chains of fibrinogen are encoded by a three gene cluster on human chromosome four. The fibrinogen genes (FGB-FGA-FGG) are expressed almost exclusively in hepatocytes where their output is coordinated to ensure a sufficient mRNA pool for each chain and maintain an abundant plasma fibrinogen protein level. Fibrinogen gene expression is controlled by the activity of proximal promoters which contain binding sites for hepatocyte transcription factors, including proteins which influence fibrinogen transcription in response to acute-phase inflammatory stimuli. The fibrinogen gene cluster also contains cis regulatory elements; enhancer sequences with liver activities identified by sequence conservation and functional genomics. While the transcriptional control of this gene cluster is fascinating biology, the medical impetus to understand fibrinogen gene regulation stems from the association of cardiovascular disease risk with high level circulating fibrinogen. In the general population this level varies from about 1.5 to 3.5 g/l. This variation between individuals is influenced by genotype, suggesting there are genetic variants contributing to fibrinogen levels which reside in fibrinogen regulatory loci. A complete picture of how fibrinogen genes are regulated will therefore point towards novel sources of regulatory variants. In this review we discuss regulation of the fibrinogen genes from proximal promoters and enhancers, the influence of acute-phase stimulation, post-transcriptional regulation by miRNAs and functional regulatory variants identified in genetic studies. Finally, we discuss the fibrinogen locus in light of recent advances in understanding chromosomal architecture and suggest future directions for researching the mechanisms that control fibrinogen expression.

Role of Genetic Changes in the Progression of Cardiovascular Diseases

This review aims to investigate the role of genetic changes in the development of cardiovascular diseases [CVD]. Oxidation of Low density Lipoprotein (LDL) and mutations in LDLreceptors gene are a trigger for numerous of atherogenic events. Also, endothelial nitric oxide synthase (eNOS) plays an important role in vasodilatation of blood vessels through synthesis of nitric oxide.Three single base pair changes, 786T/C, 922A/G, and 1468T/A, have been identified in the promoter region of the eNOS gene and are associated with coronary spasm. Moreover, two distinct variable nucleotide tandem repeats (VNTRs) in introns 4 and 13 have been detected. The presence of a minimum of 38 CA repeats in intron 13 has been associated with an independent 2.2-fold increase in the risk of coronary artery disease [CAD]. Plasma glutathione peroxidase (GPx-3) maintains the vascular bioavailability of nitric oxide (NO), through depletion of reactive oxygen species. Mutation(s) or polymorphism(s) in the plasma GPx-3 gene promoter may predispose to a thrombotic disorder, and constitute a genetic risk factor for thrombotic cerebrovascular disease. Hyperhomocysteinemia is another independent risk factor for atherosclerosis and arterial thrombosis. Severe hyperhomocysteinemia could be caused by cystathionine-β-synthase enzyme deficiency but it could be due to homozygosity of a common 677C/T point mutation in the coding region of the methylenetetrahydrofolate reductase (MTHFR) gene as a 3-fold increase in risk of CAD is associated with the MTHFR 677TT genotype. A second common variant in MTHFR 1298A/C is associated with decreased enzyme activity in vitro and in vivo, especially when occurring simultaneously with the 677 C/T polymorphism. Elevated fibrinogen, an essential component of the coagulation system, has been most consistently associated with arterial thrombotic disorders. Several polymorphisms (148C/T, 455G/A, and -854G/A) have been identified in the genes encoding the 3 pairs of fibrinogen polypeptide chains. The -455G/A, and -854G/A substitutions are the most physiologically relevant mutations. In addition the -455A allele has been associated with the progression of atheroma, and also with a 2.5-fold increase in risk of multiple lacunar infarcts in a cohort of elderly patients with stroke. It is concluded that genetic changes in the previously mentioned genes could play a significant role in the initiation and progression of CVD. This review provides useful information for both physicians and medical students whom are interested in human genetics which is related to cardiovascular diseases

Afibrinogenemia

Afibrinogenemia is a rare bleeding disorder with an estimated prevalence of 1:10,00,000 [1, 2]. It is an autosomal recessive disease resulting from mutations in any of the 3 genes that encodes the 3 polypeptide chains of fibrinogen and are located on the long arm of chromosome 4 3. Spontaneous bleeding, bleeding after minor trauma, and excessive bleeding during interventional procedures are the principal manifestations [2, 4]. Here we have reviewed the process of diagnosing a case of such rare disorder in Apollo Hospitals Dhaka. We have also highlighted the treatment and management plan of such a case.DOI: http://dx.doi.org/10.3329/pulse.v4i1.6964Pulse Vol.4 January 2010 p.34-35

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

IntroductionRheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.MethodsHuman fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays.ResultsIn total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the β-chain.ConclusionsA comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides.

Treatment of congenital fibrinogen deficiency: overview and recent findings

Afibrinogenemia is a rare bleeding disorder with an estimated prevalence of 1:1,000,000. It is an autosomal recessive disease resulting from mutations in any of the 3 genes that encode the 3 polypeptide chains of fibrinogen and are located on the long arm of chromosome 4. Spontaneous bleeding, bleeding after minor trauma and excessive bleeding during interventional procedures are the principal manifestations. We review the management of afibrinogenemia. Replacement therapy is the mainstay of treatment of bleeding episodes in these patients and plasma-derived fibrinogen concentrate is the agent of choice. Cryoprecipitate and fresh frozen plasma are alternative treatments that should be used only when fibrinogen concentrate is not available. Secondary prophylactic treatment may be considered after life-threatening bleeding whereas primary prophylactic treatment is not currently recommended. We also discuss alternative treatment options and the management of surgery, pregnancy and thrombosis in these patients. The development of new tests to identify higher risk patients and of safer replacement therapy will improve the management of afibrinogenemia in the future.

Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation

The mechanism of self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation has been studied by the methods of elastic and dynamic light-scattering and viscosimetry. Fibrin obtained from oxidized fibrinogen exhibits higher average fiber mass/length ratio compared with native fibrin. Fibrinogen ozonation sharply reduced the latent period preceding aggregation of protein molecules; however, the mechanism of self-assembly of ozonated and non-ozonated fibrinogen cluster was identical. In both cases flexible polymers are formed and reaching a certain critical length they form densely packed structures and aggregate. Using infrared spectroscopy, it has been shown that free radical oxidation of amino acid residues of fibrinogen polypeptide chains catalyzed by ozone results in formation of carbonyl, hydroxyl, and ether groups. It is concluded that fibrinogen peripheral D-domains are the most sensitive to ozonation, which causes local conformational changes in them. On one hand, these changes inhibit the reaction of longitudinal polymerization of monomeric fibrin molecules; on the other hand, they expose reaction centers responsible for self-assembly of fibrinogen clusters.

Preparative electrophoresis on linear polyacrylamide‐agarose composite gels

A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)).

Relationship between exons and domains in the fibrinogen molecule

The correlation between domains structure of the fibrinogen molecule and the exon/intron composition of the genes encoding the three fibrinogen polypeptide chains has been analysed. A coincidence of the borders between structural domains and corresponding exons in the N-terminal parts of the molecule was revealed, while no such correlation was found in the C-terminal parts. The analysis indicates that the domain structure of the fibrinogen molecule and the exon/intron structure of its genes evolved independently during the later stage of evolution. This analysis also allowed delineation of the exact borders between some domains and prediction of the existence of additional domains in the central part of the fibrinogen molecule.

Multicolour immuno-staining of fibrinogen polypeptide chains for identification of their derivatives in electrophoregrams

A novel electrophoretic procedure enabling multiple, direct immunoprobing of electrophoregrams without depending on Western blotting is described, and applied to the identification of the derivatives formed in the early stages of clot stabilization. Multicolour immunostainings for positive identification of cross-linked chains in partially stabilized fibrin clots indicated that the early products of alpha-chain cross-linking by factor XIII are largely hybrids of co-cross-linking of alpha- and gamma-chains rather than alpha-chain polymers suggested from previous studies employing non-specific staining of electrophoregrams. Furthermore, plasma-fibrinogen dimers were found to contain cross-linked alpha-chains with an electrophoretic mobility very near that of gamma-gamma-dyads. A similar product is produced by tissue-transglutaminase, but not by factor XIII.

Overall study of the in vitro plasma clotting system in an invertebrate, Liocarcinus puber (crustacea decapoda): Considerations on the structure of the crustacea plasma fibrinogen in relation to evolution

An overall study of the in vitro plasma coagulation system in the crab Liocarcinus puber has been carried out using various analytical methods, namely thromboelastography, spectrophotometrical examination, and a new one based on changes of the mechanical impedance of the developing clot. From the results reported here the clotting pattern in this species appears surprisingly complex for an invertebrate and unexpectedly closer to that of the vertebrates. Indirect evidences suggest that the fibrinogen polypeptide chains in this species and very likely in the other crustacean, are very different from those of the vertebrates. This would imply that crustacean and vertebrate fibrinogens would have diverged from one another in a far remote past, far beyond the individualization of the vertebrate α chain, that is, over 1.5 million years ago.

Human fibrinogen.

The structure and physical properties of human fibrinogen and fibrin are reviewed along with methods for the detection of products of their metabolism. Interactions of human fibrinogen with thrombin, factor XIII, plasminogen, glycoprotein IIb/IIIa, and other proteins are related to their relevance to thrombosis and hemostasis. To the extent information is available, the structural determinants of these interactions are delineated, and kinetic and thermodynamic parameters associated with the interactions are listed. Individual steps in the reaction pathway for the conversion of fibrinogen to cross-linked fibrin are characterized. The altered hemostatic properties of mutational variants of fibrinogen are related to their altered structure. The structures of the genes coding for the polypeptide chains of fibrinogen are discussed along with the current state of knowledge of the control and regulation of fibrinogen synthesis. Fibrinogen catabolism and fibrinolysis are also reviewed.

Endogenous forms of fibrinogen in hep g2 cells

The three polypeptide chains of fibrinogen, Aα, Bβ and γ chain, are synthesized on separate polysomes. Fully formed fibrinogen is a six chain, disulfide-linked, dimeric molecule with a molecular weight of 340kDa. Previous pulse-chase studies with L- 35 S methionine using the human hepatocellular carcinoma cell line, Hep-G2, showed that the three chains are not immediately disulfide-linked and that there exist intermediate precursors as well as pools of Aα and gg chains (J. Biol. Chem. 259, 10574-10581, 1984). In this study the endogenous levels of fibrinogen and its precursors are measured by two different methods; pulse and steady-state labelling with L- 35 S-methionine and immunoblotting. In Hep-G2 cells intracellular fibrinogen-related antigen is primarily (30–53%) composed of an Aα-γ complex and,to a smaller degree, of fully-formed fibrinogen (13–33%). Furthermore,the Hep G2 cell also contains endogenous pools of free γ chain (11–26%). Other fibrinogen precursors (namely, the Bβ-Aα, Bβ-γ complexes as well as the fibrinogen half-molecule) do not appear to accumulate intracellularly. Most,if not all,of these precursors occur as isoforms but this heterogeneiety is not due to varying degrees of glycosylation.In all the intracellular fibrinogen forms identified thus far,free sulfhydryl groups,detected by 14C-iodoacetamide incorporation, occur only in the Aα-γ complex and the free γ chains.

Purification of three gamma-chains with different molecular weights from normal human plasma fibrinogen.

Three forms of the normal human plasma fibrinogen gamma-chain which differ in molecular weight have been purified. Plasma fibrinogen was separated by ion exchange chromatography on DEAE-Sephacel into three populations of molecules, each with a unique gamma-chain composition. Following reduction and S-carboxymethylation, the fibrinogen polypeptide chains in each chromatographic peak were separated by ion exchange chromatography on DEAE-Sephacel and identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A alpha, B beta and smallest gamma-chain (gamma 50) eluted at progressively higher ionic strengths, but the elution positions of A alpha, B beta and gamma 50 chains were identical for fibrinogen from each of the three different chromatographic fractions. The unique gamma chain of fibrinogen in the second chromatographic peak (gamma 55) eluted at an ionic strength higher than that of the gamma 50 chain, while the largest gamma-chain (gamma 57.5), which was contained only in the third chromatographic peak of fibrinogen, eluted at the highest ionic strength. The higher ionic strengths needed to elute fibrinogen in the second and third peaks was paralleled by the higher ionic strengths needed to elute the gamma-chains unique to them, suggesting that the gamma-chain composition of the three fibrinogen fractions accounted for their differential binding to the ion exchange resin. Following desialation with neuraminidase, the differences in electrophoretic mobilities between the three gamma-chain forms was maintained, indicating that differential migration on SDS-polyacrylamide gel electrophoresis was not due to variation in sialic acid content.