Fibrinogen Philadelphia, a hypodysfibrinogenemia characterized by abnormal polymerization and fibrinogen hypercatabolism due to γ S378P mutation

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3162-3168 ◽  
Author(s):  
Margaret A. Keller ◽  
Josè Martinez ◽  
Timothy C. Baradet ◽  
Chandrasekaran Nagaswami ◽  
Irina N. Chernysh ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Thrombin Time ◽  
Fibrin Polymerization ◽  
C Terminus ◽  
Transmission Electron ◽  
History Of ◽  
Proline Substitution ◽  
Lag Period ◽  
Exon 9 ◽  
Major Defect

AbstractFibrinogen Philadelphia, a hypodysfibrinogenemia described in a family with a history of bleeding, is characterized by prolonged thrombin time, abnormal fibrin polymerization, and increased catabolism of the abnormal fibrinogen. Turbidity studies of polymerization of purified fibrinogen under different ionic conditions reveal a reduced lag period and lower final turbidity, indicating more rapid initial polymerization and impaired lateral aggregation. Consistent with this, scanning and transmission electron microscopy show fibers with substantially lower average fiber diameters. DNA sequence analysis of the fibrinogen genes A, B, and G revealed a T>C transition in exon 9 resulting in a serine-to-proline substitution near the γ chain C-terminus (S378P). The S378P mutation is associated with fibrinogen Philadelphia in this kindred and was not found in 10 controls. This region of the γ chain is involved in fibrin polymerization, supporting this as the polymerization defect causing the mutation. Thus, this abnormal fibrinogen is characterized by 2 unique features: (1) abnormal polymerization probably due to a major defect in lateral aggregation and (2) hypercatabolism of the mutant protein. The location, nature, and unusual characteristics of this mutation may add to our understanding of fibrinogen protein interactions necessary for normal catabolism and fibrin formation.

Fibrinogen Matsumoto III: a Variant with γ275 Arg→Cys (CGC→TGC) – Comparison of Fibrin Polymerization Properties with those of Matsumoto I (γ364 Asp→His) and Matsumoto II (γ308 Asn→Lys)

1999 ◽  
Vol 81 (05) ◽  
pp. 763-766 ◽  
Author(s):  
Fumiko Terasawa ◽  
Yumiko Higuchi ◽  
Shinsuke Ishikawa ◽  
Minoru Tozuka ◽  
Fumihiro Ishida ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Polymerase Chain Reaction Method ◽  
Chain Gene ◽  
Thrombin Time ◽  
Fibrin Polymerization ◽  
Maximum Slope ◽  
Interaction Site ◽  
Coagulation Tests ◽  
Ca Ions ◽  
Lag Period

SummaryFibrinogen Matsumoto III (M-III) is a dysfibrinogen identified in a 66-year-old woman with rectal cancer. The fibrinogen level determined by the thrombin-time method was markedly decreased in preoperative coagulation tests of her plasma. Three fibrinogen polypeptide-chain gene fragments from the proposita were amplified by the polymerase chain reaction method, then sequenced. The triplet CGC encoding the amino acid residue γ275 was replaced by TGC, resulting in the substitution of Arg→Cys. There have been previous reports of nine families with the same alteration, nine families with an Arg→His variant and one family with an Arg→Ser variant in this residue, which has been shown to be one of the most important amino acids in the ’D:D’ interaction site. In addition, there are three silent mutations in the Aα-chain gene and two mutations in the intron of the Bβ-chain and the γ-chain gene. However, none of these mutations is thought to be the cause of the dysfunctional fibrinogen. The thrombin-catalyzed fibrin polymerization in the presence of 1 mM Ca ions was markedly delayed in purified M-III. Its lag period was longer than those of Matsumoto II (M-II; γ308Asn→Lys) and Matsumoto I (M-I; γ364Asp→His). γ364Asp is one of the most important residues in the polymerization pocket of the ’D:E’ interaction site and γ308Asn is located in the vicinity of a high affinity Ca2+ binding site in the D-domain, γ311-336. The maximum slope of the polymerization curve for M-III was about 4-fold steeper than that for M-I but less steep than that for M-II. These results may suggest that the tertiary structure of the polymerization pocket plays a more important role in the lateral aggregation of protofibrils than that of the ’D:D’ interaction site.

Hereditary Hypodysfibrinogenemia with Defective Release of Fibrinogenopeptide A (Fibrinogen Freiburg)

1979 ◽  
Author(s):  
D. Böttcher ◽  
K. Hasler ◽  
E. Köttgen ◽  
J. Maurath
Keyword(s):  
Bleeding Episode ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Thromboembolic Disease ◽  
Thrombin Time ◽  
Excessive Bleeding ◽  
Major Bleeding Episode ◽  
History Of ◽  
Fibrin Monomers ◽  
Major Defect

A new autoaomally inherited hypodysfibrinogenemia was recognized in four members in three different generations of a family. Only one patient had a major bleeding episode after trauma, the other affected members had no history of excessive bleeding or thromboembolic disease.The thrombin time and Reptilase time of plasma were greatly prolonged and partially corrected by the addition of calcium. Patient plasma prolonged the thrombin time of normal plasma. Fibrinogen levels ranged between 10 to 20 mg/100ml when measured as thrombin-clottable protein, whereas immunologically the fibrinogen levels were only slightly reduced.Functionally the major defect was impaired release of fibrinogenopeptide A upon incubation of the purified abnormal fibrinogen (94% clottable protein) with thrombin and Reptilase. The abnormal fibrinogen showed a delayed polymerisation of its purified fibrin monomers. The described abnormal fibrinogen was indistinguishable from normal fibrinogen by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate.

scholarly journals Another Inherited Abnormality in Plasma Fibrinogen

1977 ◽  
Author(s):  
N. B. Bosch ◽  
C. L. Arocha-Piñango ◽  
A. Rodriguez ◽  
A. Ojeda ◽  
Z. Carvajal
Keyword(s):  
Ionic Strength ◽  
Electrophoretic Mobility ◽  
Factor Xiii ◽  
Polymerization Rate ◽  
Plasma Fibrinogen ◽  
Fibrinogen Concentration ◽  
Thrombin Time ◽  
Fibrin Polymerization ◽  
History Of ◽  
Α Chain

A prolonged thrombin and reptilase time with normal fibrinogen concentration in a 9 — years old girl, with no history of bleeding, prompted us to study the behaviour of her fibrinogen. Thrombin and reptilase time in different conditions, immunolectrophoresis, immunodiffusion and fibrin polymerization were performed. The thrombin time was partially -corrected by calcium, ionic strength and increasing concentration of thrombin. Fibrin polymerization rate and monomer aggregation time were moderately abnormal. Immunodiffusion and immunoelectrophoresis showed lines of identity with the normal. The electrophoretic -mobility of the α β and γ fibrin chains was normal, but there was a large amount of α — chain left in the cross-linked fibrin,in the presence of factor XIII. The above results suggest the presence of another fibrinogen variant.

scholarly journals Visualization and identification of the structures formed during early stages of fibrin polymerization

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4609-4614 ◽  
Author(s):  
Irina N. Chernysh ◽  
Chandrasekaran Nagaswami ◽  
John W. Weisel
Keyword(s):  
Confocal Microscopy ◽  
Fibrin Clot ◽  
Gel Point ◽  
Fibrin Polymerization ◽  
Early Stages ◽  
Sequence Of Events ◽  
Transmission Electron ◽  
Lag Period ◽  
Slow Moving ◽  
Complementary Techniques

AbstractWe determined the sequence of events and identified and quantitatively characterized the mobility of moving structures present during the early stages of fibrin-clot formation from the beginning of polymerization to the gel point. Three complementary techniques were used in parallel: spinning-disk confocal microscopy, transmission electron microscopy, and turbidity measurements. At the beginning of polymerization the major structures were monomers, whereas at the middle of the lag period there were monomers, oligomers, protofibrils (defined as structures that consisted of more than 8 monomers), and fibers. At the end of the lag period, there were primarily monomers and fibers, giving way to mainly fibers at the gel point. Diffusion rates were calculated from 2 different results, one based on sizes and another on the velocity of the observed structures, with similar results in the range of 3.8-0.1 μm2/s. At the gel point, the diffusion coefficients corresponded to very large, slow-moving structures and individual protofibrils. The smallest moving structures visible by confocal microscopy during fibrin polymerization were identified as protofibrils with a length of approximately 0.5 μm. The sequence of early events of clotting and the structures present are important for understanding hemostasis and thrombosis.

Some early history of replica techniques for electron microscopy

1992 ◽  
Vol 50 (2) ◽  
pp. 1076-1077
Author(s):  
Robert M. Fisher
Keyword(s):  
Thin Film ◽  
Electron Microscopy ◽  
Optical Microscope ◽  
Early History ◽  
Bulk Materials ◽  
Thin Sections ◽  
Transmission Electron ◽  
Reflected Light ◽  
History Of ◽  
Electron Microscopes

By 1940, a half dozen or so commercial or home-built transmission electron microscopes were in use for studies of the ultrastructure of matter. These operated at 30-60 kV and most pioneering microscopists were preoccupied with their search for electron transparent substrates to support dispersions of particulates or bacteria for TEM examination and did not contemplate studies of bulk materials. Metallurgist H. Mahl and other physical scientists, accustomed to examining etched, deformed or machined specimens by reflected light in the optical microscope, were also highly motivated to capitalize on the superior resolution of the electron microscope. Mahl originated several methods of preparing thin oxide or lacquer impressions of surfaces that were transparent in his 50 kV TEM. The utility of replication was recognized immediately and many variations on the theme, including two-step negative-positive replicas, soon appeared. Intense development of replica techniques slowed after 1955 but important advances still occur. The availability of 100 kV instruments, advent of thin film methods for metals and ceramics and microtoming of thin sections for biological specimens largely eliminated any need to resort to replicas.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals Genetically modified human type II collagen for N- and C-terminal covalent tagging

2018 ◽  
Vol 96 (2) ◽  
pp. 204-211
Author(s):  
Andrew Wieczorek ◽  
Clara K. Chan ◽  
Suzana Kovacic ◽  
Cindy Li ◽  
Thomas Dierks ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Connective Tissue Diseases ◽  
Sulfhydryl Group ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Structural Protein ◽  
Connective Tissues ◽  
Human Type ◽  
C Terminus ◽  
Transmission Electron

Collagen is the predominant structural protein in vertebrates, where it contributes to connective tissues and the ECM; it is also widely used in biomaterials and tissue engineering. Dysfunction of this protein and its processing can lead to a wide variety of developmental disorders and connective tissue diseases. Recombinantly engineering the protein is challenging due to post-translational modifications generally required for its stability and secretion from cells. Introducing end labels into the protein is problematic, because the N- and C-termini of the physiologically relevant tropocollagen lie internal to the initially flanking N- and C-propeptide sequences. Here, we introduce mutations into human type II procollagen in a manner that addresses these concerns and purify the recombinant protein from a stably transfected HT1080 human fibrosarcoma cell line. Our approach introduces chemically addressable groups into the N- and C-telopeptide termini of tropocollagen. Simultaneous overexpression of formylglycine generating enzyme (FGE) allows the endogenous production of an aldehyde tag in a defined, substituted sequence in the N terminus of the mutated collagen, whereas the C-terminus of each chain presents a sulfhydryl group from an introduced cysteine. These modifications are designed to enable specific covalent end-labelling of collagen. We find that the doubly mutated protein folds and is secreted from cells. Higher order assembly into well-ordered collagen fibrils is demonstrated through transmission electron microscopy. Chemical tagging of thiols is successful; however, background from endogenous aldehydes present in wild-type collagen has thus far obscured the desired specific N-terminal labelling. Strategies to overcome this challenge are proposed.

scholarly journals Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

2020 ◽  
Author(s):  
Ramakrishnan B. Kumar ◽  
Pasi Purhonen ◽  
Hans Hebert ◽  
Caroline Jegerschöld
Keyword(s):  
Arachidonic Acid ◽  
Complex Formation ◽  
Calcium Ions ◽  
Dependent Manner ◽  
Present Communication ◽  
Nuclear Membranes ◽  
His Tag ◽  
C Terminus ◽  
Transmission Electron

AbstractAmong the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectroscopy. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.

scholarly journals Discovery, development and application of drugs targeting BCL-2 pro-survival proteins in cancer

2021 ◽  
Author(s):  
Erinna F. Lee ◽  
W. Douglas Fairlie
Keyword(s):  
Structural Biology ◽  
Protein Interactions ◽  
Basic Science ◽  
Great Success ◽  
Protein Protein Interactions ◽  
New Class ◽  
Success Stories ◽  
History Of ◽  
Approved Drugs ◽  
Close Relatives

The discovery of a new class of small molecule compounds that target the BCL-2 family of anti-apoptotic proteins is one of the great success stories of basic science leading to translational outcomes in the last 30 years. The eponymous BCL-2 protein was identified over 30 years ago due to its association with cancer. However, it was the unveiling of the biochemistry and structural biology behind it and its close relatives’ mechanism(s)-of-action that provided the inspiration for what are now known as ‘BH3-mimetics’, the first clinically approved drugs designed to specifically inhibit protein–protein interactions. Herein, we chart the history of how these drugs were discovered, their evolution and application in cancer treatment.

scholarly journals ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters

2004 ◽  
Vol 15 (7) ◽  
pp. 3393-3405 ◽  
Author(s):  
Markus Geisler ◽  
Marjolaine Girin ◽  
Sabine Brandt ◽  
Vincent Vincenzetti ◽  
Sonia Plaza ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Abc Transporters ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Motifs ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Domain Mapping ◽  
Tetratricopeptide Repeat Domain

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

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