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1.
Ann N Y Acad Sci ; 936: 223-5, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11460479

RESUMO

Steric hindrance by the backbone of extra oligosaccharides at gamma-Asn 308 may cause the repulsive force to widen the junction at the D:D interface, and thus, interfere with the longitudinal elongation and lateral association of protofibrils.


Assuntos
Fibrina/química , Fibrinogênios Anormais/química , Oligossacarídeos/química , Fibrina/ultraestrutura , Humanos , Microscopia Eletrônica de Varredura
2.
Blood ; 96(12): 3779-85, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11090060

RESUMO

The authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bbeta gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient. (Blood. 2000;96:3779-3785)


Assuntos
Fibrinogênios Anormais/química , Adulto , Coagulação Sanguínea/genética , Cromatografia Líquida de Alta Pressão , Cisteína/química , Dimerização , Dissulfetos/química , Endopeptidases/metabolismo , Feminino , Fibrinogênios Anormais/genética , Fibrinogênios Anormais/ultraestrutura , Humanos , Microscopia Eletrônica de Varredura , Fragmentos de Peptídeos/química , Permeabilidade , Análise de Sequência de Proteína
3.
Blood ; 94(11): 3806-13, 1999 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-10572095

RESUMO

A novel BbetaAsn-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 BbetaAsn-158 residue. The functional abnormality was attributed to delayed lateral association of normally formed double-stranded protofibrils based on normal cross-linking of fibrin gamma-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 Bbeta-158 that is responsible for reducing the fiber thickness because of either local repulsive forces or steric hindrance in the coiled-coil region.


Assuntos
Fibrina/química , Fibrinogênio/química , Fibrinogênio/genética , Substituição de Aminoácidos , Asparagina , Feminino , Fibrina/metabolismo , Fibrinogênio/metabolismo , Glicosilação , Humanos , Pessoa de Meia-Idade , Mutação Puntual , Serina
4.
Blood ; 91(9): 3282-8, 1998 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-9558384

RESUMO

The truncated Aalpha-chain of fibrinogen Marburg is partly linked with albumin by a disulfide bond. Based on the recovery of the first six amino acid residues assigned to the subunit polypeptides of fibrinogen (the Aalpha-and gamma-chains) and albumin, 0.33 mol of albumin was estimated to be linked to 1 mol of the Marburg fibrinogen. When the Marburg fibrinogen was clotted with thrombin-factor XIIIa-Ca2+, various alpham gamman heteromultimers were produced, and part of the albumin was cross-linked to the gamma-chain. Acid-solubilized Marburg fibrin monomer failed to form large aggregates that could be detected by monitoring turbidity at A350, but it was able to enhance tissue-type plasminogen-activator-catalyzed plasmin generation, though not as avidly as the normal control, indicating that the double-stranded protofibrils had, to some extent, been constructed. This idea seems to be supported by normal factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains. However, the cross-linked Marburg fibrin, being apparently fragile and translucent, was highly resistant against plasmin, and its subunit components were considerably retained for 48 hours as noted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the exact mechanisms are still unclear, the albumin-incorporated factor XIIIa-cross-linked Marburg fibrin seems to have undergone a critical structural alteration(s) to acquire resistance against plasmin. This aquisition of plasmin resistance may be contributed to the postoperative pelvic vein thrombosis and recurrent pulmonary embolisms in the patient after caesarian section for her first delivery at the age of 20 years.


Assuntos
Fator XIII/química , Fibrina/metabolismo , Fibrinogênios Anormais/metabolismo , Albumina Sérica/metabolismo , Trombose/metabolismo , Cisteína/química , Dissulfetos/química , Ativação Enzimática , Fibrinolisina/metabolismo , Humanos , Substâncias Macromoleculares , Polímeros , Ativador de Plasminogênio Tecidual/metabolismo , alfa 2-Antiplasmina/metabolismo
5.
J Biochem ; 117(2): 244-50, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7608107

RESUMO

Upon incubation of human prothrombin with factor Xa bound to human umbilical vein endothelial cells (HUVEC) (0.5-0.6 fmol factor Xa/10(5) cells), three bonds at Arg273-Thr274, Arg286-Thr287, and Arg322-Ile323 were cleaved, yielding and releasing fragment 1-2 and a degraded form of alpha-thrombin, but not meizothrombin, into the fluid phase. The apparent Km for prothrombin and the Vmax were 0.25 +/- 0.07 microM and 210 +/- 40 fmol thrombin/min/10(5) cells, respectively. For the maximally bound factor Xa, the calculated catalytic efficiency (kcat = 6-7 s-1) was similar to those reported for the prothrombinase complex formed on the phospholipid vesicles and natural membrane surfaces. The prothrombin derivatives lacking the 10 gamma-carboxyglutamic acid (Gla) residues-containing region were not activated by the cell-bound factor Xa. The activation rate of prothrombins with Gla residues variously modified to gamma-methyleneglutamic acids was reduced in accordance with the number of modified residues. For the inhibition of prothrombin activation, intact fragment 1 was needed; the Gla-domain alone did not affect the reaction. Binding of monoclonal antibodies to the region of 1-48 or the kringle 1 region of prothrombin also interfered with the prothrombin activation. Prothrombin activation on the surface of HUVEC appeared to proceed via formation of a cellular prothrombinase complex composed of phospholipids of HUVEC membrane, endogenous factor Va, factor Xa, and prothrombin. The Gla-domain and kringle 1 regions are indispensable for the molecule to serve as an effective substrate for the cell-bound factor Xa.


Assuntos
Endotélio Vascular/metabolismo , Fator Xa/metabolismo , Protrombina/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Divisão Celular , Membrana Celular/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Ativação Enzimática , Humanos , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Especificidade por Substrato , Veias Umbilicais
6.
Thromb Res ; 37(3): 379-90, 1985 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-3992527

RESUMO

A congenital dysfibrinogenemia was found in a 32-year-old asymptomatic female and her immediate family. The propositus, apparently a heterozygote for the abnormality, characteristically showed defective release of fibrinopeptide A from half of her fibrinogen molecules. No fibrinopeptide A was cleaved off from the isolated abnormal molecule by thrombin or snake venoms (Reptilase and Ancrod) as evidenced by radioimmunoassay, high performance liquid chromatography and determination of the NH2-terminal amino acids. The abnormal fibrinogen formed a solid gel solely by the release of fibrinopeptide B upon incubation with thrombin. We provisionally designate this abnormal fibrinogen as "Fibrinogen Kawaguchi", although possible identity with other abnormal fibrinogens is not excluded.


Assuntos
Transtornos da Coagulação Sanguínea/genética , Fibrinogênio/metabolismo , Fibrinogênios Anormais , Adulto , Sequência de Aminoácidos , Transtornos da Coagulação Sanguínea/sangue , Feminino , Fibrina/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Fibrinopeptídeo A/metabolismo , Hemostasia , Humanos , Linhagem , Tempo de Trombina
7.
J Clin Invest ; 72(3): 1034-41, 1983 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-6886002

RESUMO

A hereditary dysfibrinogenemia associated with defective aggregation of fibrin monomers was found in a 39-yr-old female and in the members of her immediate family, who had all been asymptomatic. The abnormality was probably due to an impaired polymerization site exposed in the DD domain of two adjacent fibrin molecules, because plasmic fragment DD derived from the propositus' cross-linked fibrin bound far less tightly to insolubilized normal fragment E than that from the normal one. Its complementary polymerization site in the E domain of fibrin, which was exposed by thrombin cleavage, and the polymerization site in the D domain of fibrinogen, which was available without activation by thrombin, were both found to be normal. More anodal migration of the abnormal fragment DD than the normal one, as shown by immunoelectrophoresis, seemed to support our concept that the mutation most likely resides in the D domain of the abnormal fibrinogen molecule at or near a region closely related to the polymerization site that is exposed when two fibrin molecules are linearly aligned. The work of others on the polymerization of normal fibrin with different techniques yielded results consistent with our conclusions. We tentatively designate this type of abnormal fibrinogen "fibrinogen Tokyo II," but its possible identity with other abnormalities of fibrinogen reported heretofore is not excluded.


Assuntos
Transtornos da Coagulação Sanguínea/genética , Produtos de Degradação da Fibrina e do Fibrinogênio/genética , Fibrinogênio/genética , Fibrinogênios Anormais , Adulto , Sítios de Ligação , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/diagnóstico , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Imunoeletroforese , Substâncias Macromoleculares , Peso Molecular , Mutação , Tempo de Trombina
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