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Nobuo Okumura - One of the best experts on this subject based on the ideXlab platform.
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a novel amino acid substitution Fibrinogen bβp pro234leu associated with hypoFibrinogenemia causing impairment of Fibrinogen assembly and secretion
International Journal of Molecular Sciences, 2020Co-Authors: Takahiro Kaido, Masahiro Yoda, Tomu Kamijo, Chiaki Taira, Yumiko Higuchi, Shinpei Arai, Nobuo OkumuraAbstract:We identified a novel heterozygous variant, Bβp.Pro234Leu (Fibrinogen Tokorozawa), which was suspected to be associated with hypoFibrinogenemia. Therefore, we analyzed the assembly and secretion of this Fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of Fibrinogen of the Bβp.P234L and γp.G242E substitutions, we established recombinant variant Fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed Fibrin Polymerization using purified plasma Fibrinogen and in-silico analysis. Both Bβp.P234L and γp.G242E impaired the secretion and synthesis of Fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bβγ complex in Bβp.P234L. On the other hand, the Fibrin Polymerization of Fibrinogen Tokorozawa was similar to that of normal Fibrinogen. In-silico analysis revealed that the Bβp.P234 residue is located in the contact region between the Bβ and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bβp.P234L substitution resulted in hypoFibrinogenemia by decreasing the assembly and secretion of Fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bβ and γ chains impact the assembly and secretion of Fibrinogen.
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heterozygous variant Fibrinogen γa289v kanazawa iii was confirmed as hypodysFibrinogenemia by plasma and recombinant Fibrinogens
International Journal of Laboratory Hematology, 2020Co-Authors: Takahiro Kaido, Masahiro Yoda, Tomu Kamijo, Chiaki Taira, Yumiko Higuchi, Nobuo OkumuraAbstract:INTRODUCTION Congenital Fibrinogen disorders are classified as aFibrinogenemia, hypoFibrinogenemia, dysFibrinogenemia, and hypodysFibrinogenemia. However, difficulties are associated with discriminating between dysFibrinogenemia, hypoFibrinogenemia, and hypodysFibrinogenemia using routine analyses. We previously reported a heterozygous variant Fibrinogen (γA289V; Kanazawa III) as hypodysFibrinogenemia; however, the same variant had previously been described as hypoFibrinogenemia. To clarify the production of γA289V Fibrinogen, we expressed recombinant γA289V (r-γA289V) Fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant Fibrinogens. METHODS Target mutations were introduced into a Fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant Fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and Fibrinogen functions were analyzed using Fibrin Polymerization, plasmin protection, and FXIIIa-catalyzed Fibrinogen cross-linking. RESULTS The Fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V Fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) Fibrinogen was similar to WT, we compared r-γF290L Fibrinogen functions with WT. The Fibrin Polymerization of Kanazawa III plasma (K-III) Fibrinogen was significantly weaker than normal plasma Fibrinogen. Moreover, K-III Fibrinogen showed a markedly reduced "D:D" interaction. However, all functions of r-γF290L Fibrinogen were similar to WT. An in silico analysis confirmed the above results. CONCLUSION The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced Fibrinogen secretion. Moreover, K-III Fibrinogen showed markedly reduced Fibrin Polymerization and "D:D" interactions. γA289V Fibrinogen was confirmed as hypodysFibrinogenemia.
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2014Co-Authors: Nobuo Okumura, Fumiko Terasawa, Susan T. Lord, Oleg V. Gorkun, Nobuo Okumura Ph. DAbstract:Substitution of the γ-chain Asn 308 disturbs the D:D interface affecting Fibrin Polymerization, Fibrinopeptide B release, and FXIII catalyzed cross-linking
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functional analysis for dysFibrinogenemias toyama and adachi which have a mutation of aalpha16arg his cgt cat with aberrant Fibrinopeptide a release
The Japanese journal of clinical pathology, 2012Co-Authors: Keisuke Soya, Fumiko Terasawa, Yuka Takezawa, Nobuo OkumuraAbstract:We found and identified four heterozygous dysFibrinogenemias with AalphaR16H(CGT-->CAT) mutation in two families by coagulation tests and direct sequence analysis for PCR-amplified DNA fragments. Two dysFibrinogens were designated as Fibrinogen Toyama and Adachi, according to the place of residence of proposituses, respectively. Patients' Fibrinogen purified from plasma using immunoaffinity-chromatography was subjected to thrombin- or batroxobin-catalyzed Fibrin Polymerization, Fibrinopeptide A (FPA) release, and clottability test. AalphaR16H-Fibrinogen showed impaired thrombin or batroxobin-catalyzed Fibrin Polymerization in comparison with normal control Fibrinogen. It is interesting that the period of protofibril formation of Toyama propositus was longest in those of four affected people. The clottability of AalphaR16H-Fibrinogen was 66-70% with thrombin and higher than with batroxobin, 35-50%. In the same condition with Fibrin Polymerization, thrombin and batroxobin did not cleave the Aalpha16H-17G peptide-bonding, resulting in no release of variant FPA. From these results, we speculated that elongation of the two-stranded protofibril formation would be terminated by participation of the heterodimer Fibrinogen molecules composed with a normal and an aberrant Aalpha-chain, and it would result in a decrease in Fibrin Polymerization. We speculated that the difference in the extent of impairment of Fibrin Polymerization among the patients might be caused by the different amount of heterodimers. Moreover, we also speculated that batroxobin-induced clottability was lower than thrombin-induced clottability, because batroxobin cannot induce the so-called "B-knob-b-hole" interaction, which enhances Fibrin formation.
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Polymerization defective Fibrinogen variant gammad364a binds knob a peptide mimic
Biochemistry, 2008Co-Authors: Sheryl R. Bowley, Nobuo Okumura, Oleg V. Gorkun, Betsy K. Merenbloom, Laurie Betts, Annie Heroux, Susan T. LordAbstract:Fibrin Polymerization is supported in part by interactions called 'A:a'. Crystallographic studies revealed ?364Asp is part of hole 'a' that interacts with knob 'A' peptide mimic, GPRP. Biochemical studies have shown ?364Asp is critical to Polymerization, as Polymerization of variants ?D364A, ?D364H, and ?D364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from ?D364A. Surprisingly, the structure (rfD-?D364A+GP) showed near normal 'A:a' interactions with GPRP bound to hole 'a' and no change in the overall structure of ?D364A. Of note, inspection of the structure showed negative electrostatic potential inside hole 'a' was diminished by this substitution. We examined GPRP binding to the ?364Asp variants in solution by plasmin protection assay. We found no protection of either ?D364H or ?D364V but partial protection of ?D364A, indicating the peptide does not bind to either ?D364H or ?D364V and binds more weakly than normal to ?D364A. We also examined protection by calcium and found all variants were indistinguishable from normal, suggesting the global structures of the variants are not markedly different from normal. Our data imply that ?364Asp per se is not required for knob 'A' binding to hole 'a'; rather,more » this residue's negative charge has a critical role in the electrostatic interactions that facilitate the important first step in Fibrin Polymerization.« less
Robert A S Ariens - One of the best experts on this subject based on the ideXlab platform.
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Fibrinogen αc regions are not directly involved in Fibrin Polymerization as evidenced by a double detroit recombinant Fibrinogen mutant and knobs mimic peptides
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Cedric Duval, Julia S Gauer, Aldo Profumo, Anna Aprile, Annalisa Salis, Enrico Millo, Gianluca Damonte, Robert A S Ariens, Mattia RoccoAbstract:BACKGROUND Fibrin Polymerization, following Fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α- and β-chains N-termini (GPR, GHR; A-, B-knobs, respectively) engaging preexistent a and b pockets in other Fibrin(ogen) molecules' γ- and (B)β-chains C-terminal regions. A role for mostly disordered (A)α-chains C-terminal regions "bridging" between Fibrin molecules/fibrils has been proposed. OBJECTIVES Fibrinogen Detroit is a clinically observed mutation (AαR19 → S) with nonengaging GPS A-knobs. By analogy, a similar Bβ-chain mutation, BβR17 → S, should produce nonengaging GHS B-knobs. A homozygous "Double-Detroit" mutant (AαR19 → S, BβR17 → S; DD-FG) was developed: with A-a and B-b engagements endogenously blocked, other interactions would become apparent. METHODS DD-FG, wild-type recombinant (WT-FG), and human plasma (hp-FG) Fibrinogen self-association was studied by turbidimetry coupled with Fibrinopeptides release high-performance liquid chromatography (HPLC)/mass spectrometry analyses, and by light-scattering following size-exclusion chromatography (SE-HPLC). RESULTS In contrast to WT-FG and hp-FG, DD-FG produced no turbidity increase, irrespective of thrombin concentration. The SE-HPLC profile of concentrated DD-FG was unaffected by thrombin treatment, and light-scattering, at lower concentration, showed no intensity and hydrodynamic radius changes. Compared with hp-FG, both WT-FG and DD-FG showed no FpA cleavage difference, while ~50% FpB was not recovered. Correspondingly, SDS-PAGE/Western-blots revealed partial Bβ-chain N-terminal and Aα-chain C-terminal degradation. Nevertheless, ~70% DD-FG molecules bearing (A)αC-regions potentially able to associate were available. Higher-concentration, nearly intact hp-FG with 500-fold molar excess GPRP-NH2 /GHRP-NH2 knobs-mimics experiments confirmed these no-association findings. CONCLUSIONS (A)αC-regions interactions appear too weak to assist native Fibrin Polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered.
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evidence that Fibrinogen γ directly interferes with protofibril growth implications for Fibrin structure and clot stiffness
Journal of Thrombosis and Haemostasis, 2012Co-Authors: Peter Allan, Uitte S De Willige, Radwa H Abousaleh, Simon D Connell, Robert A S AriensAbstract:Summary. Background: Fibrinogen contains an alternatively spliced γ-chain (γ′), which mainly exists as a heterodimer with the common γA-chain (γA/γ′). Fibrinogen γ′ has been reported to inhibit thrombin and modulate Fibrin structure, but the underlying mechanisms are unknown. Objective: We aimed to investigate the molecular mechanism underpinning the influence of γ′ on Fibrin Polymerization, structure and viscoelasticity. Methods: γA/γA and γA/γ′ Fibrinogens were separated using anion exchange chromatography. Cross-linking was controlled with purified FXIIIa and a synthetic inhibitor. Fibrin Polymerization was analyzed by turbidity and gel-point time was measured using a coagulometer. We used atomic force microscopy (AFM) to image protofibril formation while final clot structure was assessed by confocal and scanning electron microscopy. Clot viscoelasticity was measured using a magnetic microrheometer. Results: γA/γ′ Fibrin formed shorter oligomers by AFM than γA/γA, which in addition gelled earlier. γA/γ′ clots displayed a non-homogenous arrangement of thin fibers compared with the uniform arrangements of thick fibers for γA/γA clots. These differences in clot structure were not due to thrombin inhibition as demonstrated in clots made with reptilase. Non-cross-linked γA/γA Fibrin was approximately 2.7 × stiffer than γA/γ′. Cross-linking by FXIIIa increased the stiffness of both Fibrin variants; however, the difference in stiffness increased to approximately 4.6 × (γA/γA vs. γA/γ′). Conclusions: Fibrinogen γ′ is associated with the formation of mechanically weaker, non-uniform clots composed of thin fibers. This is caused by direct disruption of protofibril formation by γ′.
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the effect of dimethylbiguanide on thrombin activity fxiii activation Fibrin Polymerization and Fibrin clot formation
Diabetes, 2002Co-Authors: Kristina F Standeven, John W Weisel, Robert A S Ariens, Paul Whitaker, Alison E Ashcroft, Peter J GrantAbstract:The antihyperglycemic drug dimethylbiguanide (DMB, also known as metformin) reduces the risk of cardiovascular complications in type 2 diabetes, although the mechanism(s) involved are unclear. DMB reduces glycosylation-related protein cross-linking, a process similar to Fibrin cross-linking catalyzed by activated factor XIII (FXIII). To investigate whether the cardioprotective effect of DMB could be related to effects on clot stabilization, we studied the effects of DMB on FXIII, thrombin activity, and cleavage of Fibrin(ogen). Activity of purified and plasma FXIII was inhibited by DMB. Analysis by mass spectrometry and FXIII-coupled magnetic particles excluded binding of DMB to FXIII. Thrombin-induced cleavage of the activation peptide from FXIII was inhibited in a dose-dependent manner, as was Fibrinopeptide cleavage from Fibrinogen. Ancrod-induced cleavage of Fibrinopeptide A was not affected. DMB prolonged clotting time of normal plasma. Fiber thickness and pore size of Fibrin clots, measured by permeation experiments and visualized by scanning electron microscopy, decreased significantly with DMB. No interactions between DMB and the active site of thrombin were found. Turbidity experiments demonstrated that DMB changed Polymerization and lateral aggregation of protofibrils. These results suggest that DMB interferes with FXIII activation and Fibrin Polymerization, but not only by binding to thrombin on a different location than the active site. In patients on DMB therapy, FXIII antigen and activity levels in vivo were reduced over a 12-week period. These findings indicate that part of the cardioprotective effect of DMB in patients with type 2 diabetes may be attributed to alterations in Fibrin structure/function.
S. V. Komisarenko - One of the best experts on this subject based on the ideXlab platform.
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Calixarene methylene bisphosphonic acids as promising effectors of biochemical processes
National Academy of Sciences of Ukraine and Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine., 2013Co-Authors: S. V. Komisarenko, S. O. Kosterin, E. V. Lugovskoy, V. I. KalchenkoAbstract:This interdisciplinary study, performed with participation of research workers of Palladin Institute of Biochemistry and Institute of Organic Chemistry of NAS of Ukraine, is devoted to analysis of biochemical effects of some calixarene methylene bisphosphonic acids (cyclic phenol oligomers) on two well-known biological phenomenons – Mg2+-dependent ATP hydrolysis (myosin subfragment-1 of myometrium smooth muscle was used as an example) and Fibrin Polymerization. Calix[4]arene С-97 (calix[4]arene methylene bisphosphonic acids) is a macrocyclic substance, which contains intramolecular highly ordered lipophilic cavity formed by four aromatic rings, one of which is functionalized at the upper rim with methylene bisphosphonic group. At concentration of 100 µM, this substance was shown to effectively inhibit ATPase activity of pig myometrium myosin subfragment-1 (inhibition coefficient І0.5 = 83 ± 7 µM). At the same time, this calix[4]arene causes significant (vs. control) increase of myosin subfragment-1 hydrodynamic diameter, which may indicate formation of an intermolecular complex between calixarene and myosin head. Computer simulation methods (docking and molecular dynamics with addition of grid technologies) enabled to elucidate the grounds of intermolecular interactions between calix[4]arene С-97 and myometrium myosin subfragment-1, that involve hydrophobic, electrostatic and π-π-stacking interactions, some of which are close to the ATPase active centre. In view of the ability of calixarenes to penetrate into the cell and their low toxicity, the results obtained may be used as a basis for further development of a new generation of supramolecular effectors (starting from the above mentioned substances, in particular calix[4]arene С-97) for regulation of smooth muscle contractile activity at the level of ATP dependent actin-myosin interaction. Calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on Fibrin Polymerization. The most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maximum rate of Fibrin Polymerization in the Fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52·10-6 M (IC50). At this concentration, the molar ratio of the compound to Fibrinogen was 1.7 : 1. For the case of desAB Fibrin Polymerization, the IC50 was 1.26·10-6 M at a molar ratio of C-192 to Fibrin monomer of 4 : 1. Dipropoxycalix[4]-arene bis-methylene-bis-phosphonic acid (C-98) inhibited Fibrin desAB Polymerization with an IC50 = 1.31·10-4 M. We hypothesized that C-192 blocks Fibrin formation by combining with Polymerization site ‘A’ (Aa17–19), which ordinarily initiates protofibril formation in a ‘knob-hole’ manner. This suggestion was confirmed by an HPLC assay, which showed a host–guest inclusion complex of C-192 with the synthetic peptide Gly-Pro-Arg-Pro, an analogue of site ‘A’. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. Calixarene C-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13·10-5 and 1.10·10-5 M, respectively. These experiments demonstrate that C-192 is a specific inhibitor of Fibrin Polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents
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USAGE OF MONOCLONAL ANTIBODIES FOR DETERMINATION OF LOCALIZATION OF ANTIGENIC DETERMINANTS AND Fibrin Polymerization SITES WITHIN FibrinOGEN AND Fibrin MOLECULES AND THEIR APPLICATION IN TEST--SYSTEMS FOR DIAGNOSTICS AND THE THREAT OF THROMBUS FORMATION
National Academy of Sciences of Ukraine and Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine., 2013Co-Authors: E. V. Lugovskoi, I N Kolesnikova, S. V. KomisarenkoAbstract:It was shown by monoclonal antibodies that B?N-region of Fibrin desA molecule (B?1-53) comprises the Polymerization site including the peptide bond B?14-15. This site participates in the second stage of Fibrin Polymerization — lateral association of protofibrils. In the B?15-53 fragment was also found the site called «C», which together with the site «A» participate in the first stage of Polymerization — the protofibrils formation. The model of the primary intermolecular interaction of Fibrin was designed. It was found by monoclonal antibodies II-4d the site («c») in the N-terminal half of ? chain of the Fibrin D-region. This site participates in the protofibrils formation and is complement to site «C» as we assume. We have discovered two neoantigenic determinants. One of these determinants exposes within the coiledcoil fragment B?126-135 of Fibrin as a result of Fibrinopeptide A splitting off from Fibrinogen by thrombin. The structural rearrangements discovered in this site of the Fibrin molecule are necessary for the following protofibrils lateral association. The second neoantigenic determinant is localized in the fragment B?134-190 of D-dimer formed after plasmin degradation of Fibrin stabilized by FXIIIa. We have obtained the Fibrin-specific monoclonal antibodie FnI-3C to the first determinant and D-dimer-specific mAb III-3b to the second one. Three monoclonal antibodies were obtained against the ?C-region of Fibrin(ogen) molecule. It has been experimentally shown by of one of them that ?C-domains is connected with the Fibrinopeptides B in Fibrinogen and Fibrin desA molecules, but removes from the core of the molecules after Fibrinopeptides B splitting off by thrombin. Two other monoclonal antibodies specifically inhibit the Fibrin Polymerization by blocking two unknown Polymerization sites within the ?C-region. The test-systems for the soluble Fibrin and D-dimer quantification in human blood plasma were designed on the basis of monoclonal antibodies FnI-3C and III-3b as «catch»-antibodies and one II-4d as a «tag»-antibody, respectively. The clinical trials of the test-systems were carried out in Ukraine. It was shown that for the prediction of postoperative thrombotic complications and monitoring the efficiency of antithrombotic therapy the simultaneous quantification of soluble Fibrin and D-dimer before the operation and at different time intervals after the operation is required. Only in this case it is possible to get information about the state of the balance between blood coagulation and Fibrinolytic systems, and determine the degree of the threat of thrombosis
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functional role of bβ chain n terminal fragment in the Fibrin Polymerization process
FEBS Journal, 2007Co-Authors: E. V. Lugovskoy, P G Gritsenko, L G Kapustianenko, I N Kolesnikova, V I Chernishov, S. V. KomisarenkoAbstract:Four mAbs of the IgG1 class to the thrombin-treated N-terminal disulfide knot of Fibrin, secreted by various hybridomas, have been selected. Epitopes for two mAbs, I-3C and III-10d, were situated in human Fibrin fragment Bβ15–26, and those for two other mAbs, I-5G and I-3B, were in fragment Bβ26–36. Three of these mAbs, I-5G, I-3B and III-10D, as well as their Fab-fragments, decreased the maximum rate of Fibrin desAA and desAABB Polymerization up to 90–95% at a molar ratio of mAb (or Fab-fragment) to Fibrin of 1 or 2. The fourth mAb, I-3C, did not influence the Fibrin desAABB Polymerization and inhibited by 50% the maximum rate of Fibrin desAA Polymerization. These results suggest that these mAb inhibitors block a longitudinal Fibrin Polymerization site. As the mAbs retard both Fibrin desAABB and Fibrin desAA Polymerization, one can conclude that the Polymerization site does not coincide with Polymerization site ‘B’ (Bβ15–17). To verify this suggestion, the Polymerization inhibitory activity of synthetic peptides BβSARGHRPLDKKREEA(12–26), BβLDKKREEA(19–26), BβAPSLRPAPPPI(26–36), BβAPSLRPAPPPISGGGYRARPA(26–46) and BβGYRARPA(40–46), which imitate the various sequences in the N-terminal region of the Fibrin Bβ-chain, have been investigated. Peptides Bβ12–26 and Bβ26–46, but not Bβ40–46, Bβ19–26, and Bβ26–36, proved to be specific inhibitors of Fibrin Polymerization. The IC50 values for Bβ12–26 and Bβ26–46 were 2.03 × 10−4 and 2.19 × 10−4 m, respectively. Turbidity and electron microscopy data showed that peptides Bβ12–26 and Bβ26–46 inhibited the Fibrin protofibril formation stage of Fibrin Polymerization. The conclusion was drawn that Fibrin fragment Bβ12–46 took part in Fibrin protofibril formation simultaneously with site ‘A’ (Aα17–19) prior to removal of Fibrinopeptide B. A model of the intermolecular connection between fragment Bβ12–46 of one Fibrin desAA molecule and the D-domain of another has been constructed.
John W Weisel - One of the best experts on this subject based on the ideXlab platform.
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mechanisms of Fibrin Polymerization and clinical implications
Blood, 2013Co-Authors: John W Weisel, Rustem I LitvinovAbstract:Research on all stages of Fibrin Polymerization, using a variety of approaches including naturally occurring and recombinant variants of Fibrinogen, x-ray crystallography, electron and light microscopy, and other biophysical approaches, has revealed aspects of the molecular mechanisms involved. The ordered sequence of Fibrinopeptide release is essential for the knob-hole interactions that initiate oligomer formation and the subsequent formation of 2-stranded protofibrils. Calcium ions bound both strongly and weakly to Fibrin(ogen) have been localized, and some aspects of their roles are beginning to be discovered. Much less is known about the mechanisms of the lateral aggregation of protofibrils and the subsequent branching to yield a 3-dimensional network, although the αC region and B:b knob-hole binding seem to enhance lateral aggregation. Much information now exists about variations in clot structure and properties because of genetic and acquired molecular variants, environmental factors, effects of various intravascular and extravascular cells, hydrodynamic flow, and some functional consequences. The mechanical and chemical stability of clots and thrombi are affected by both the structure of the Fibrin network and cross-linking by plasma transglutaminase. There are important clinical consequences to all of these new findings that are relevant for the pathogenesis of diseases, prophylaxis, diagnosis, and treatment.
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visualization of the dynamics of Fibrin clot growth 1 molecule at a time by total internal reflection fluorescence microscopy
Blood, 2013Co-Authors: Alina Hategan, Kathryn C Gersh, Daniel Safer, John W WeiselAbstract:Individual fluorescently labeled Fibrin(ogen) molecules and their assembly to make a clot were observed by total internal reflection fluorescence microscopy (TIRFM). We used the bleaching of the fluorescent labels to determine the number of active fluorophores attached nonspecifically to each molecule. From the total intensity of bleaching steps, as single-molecule signature events, and the distribution of active labeling, we developed a new single-molecule intensity calibration, which accounts for all molecules, including those “not seen.” Live observation of Fibrin Polymerization in TIRFM by diffusive mixing of thrombin and plasma revealed the real-time growth kinetics of individual Fibrin fibers quantitatively at the molecular level. Some fibers thickened in time to thousands of molecules across, equivalent to hundreds of nanometers in diameter, whereas others reached an early stationary state at smaller diameters. This new approach to determine the molecular dynamics of fiber growth provides information important for understanding clotting mechanisms and the associated clinical implications.
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the effect of dimethylbiguanide on thrombin activity fxiii activation Fibrin Polymerization and Fibrin clot formation
Diabetes, 2002Co-Authors: Kristina F Standeven, John W Weisel, Robert A S Ariens, Paul Whitaker, Alison E Ashcroft, Peter J GrantAbstract:The antihyperglycemic drug dimethylbiguanide (DMB, also known as metformin) reduces the risk of cardiovascular complications in type 2 diabetes, although the mechanism(s) involved are unclear. DMB reduces glycosylation-related protein cross-linking, a process similar to Fibrin cross-linking catalyzed by activated factor XIII (FXIII). To investigate whether the cardioprotective effect of DMB could be related to effects on clot stabilization, we studied the effects of DMB on FXIII, thrombin activity, and cleavage of Fibrin(ogen). Activity of purified and plasma FXIII was inhibited by DMB. Analysis by mass spectrometry and FXIII-coupled magnetic particles excluded binding of DMB to FXIII. Thrombin-induced cleavage of the activation peptide from FXIII was inhibited in a dose-dependent manner, as was Fibrinopeptide cleavage from Fibrinogen. Ancrod-induced cleavage of Fibrinopeptide A was not affected. DMB prolonged clotting time of normal plasma. Fiber thickness and pore size of Fibrin clots, measured by permeation experiments and visualized by scanning electron microscopy, decreased significantly with DMB. No interactions between DMB and the active site of thrombin were found. Turbidity experiments demonstrated that DMB changed Polymerization and lateral aggregation of protofibrils. These results suggest that DMB interferes with FXIII activation and Fibrin Polymerization, but not only by binding to thrombin on a different location than the active site. In patients on DMB therapy, FXIII antigen and activity levels in vivo were reduced over a 12-week period. These findings indicate that part of the cardioprotective effect of DMB in patients with type 2 diabetes may be attributed to alterations in Fibrin structure/function.
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computer modeling of Fibrin Polymerization kinetics correlated with electron microscope and turbidity observations clot structure and assembly are kinetically controlled
Biophysical Journal, 1992Co-Authors: John W Weisel, Chandrasekaran NagaswamiAbstract:Although much is known about Fibrin Polymerization, because it is complex, the effects of various modifications are not intuitively obvious and many experimental observations remain unexplained. A kinetic model presented here that is based on information about mechanisms of assembly accounts for most experimental observations and allows hypotheses about the effects of various factors to be tested. Differential equations describing the kinetics of Polymerization were written and then solved numerically. The results have been related to turbidity profiles and electron microscope observations. The concentrations of intermediates in Fibrin Polymerization, and fiber diameters, fiber and protofibril lengths have been calculated from these models. The simplest model considered has three steps; Fibrinopeptide A cleavage, protofibril formation, and lateral aggregation of protofibrils to form fibers. The average number of protofibrils per fiber, which is directly related to turbidity, can be calculated and plotted as a function of time. The lag period observed in turbidity profiles cannot be accurately simulated by such a model, but can be simulated by modifying the model such that oligomers must reach a minimum length before they aggregate. Many observations, reported here and elsewhere, can be accounted for by this model; the basic model may be modified to account for other experimental observations. Modeling predicts effects of changes in the rate of Fibrinopeptide cleavage consistent with electron microscope and turbidity observations. Changes only in the rate constants for initiation of fiber growth or for addition of protofibrils to fibers are sufficient to account for a wide variety of other observations, e.g., the effects of ionic strength or Fibrinopeptide B removal or thrombospondin. The effects of lateral aggregation of fibers has also been modeled: such behavior has been observed in turbidity curves and electron micrographs of clots formed in the presence of platelet factor 4. Thus, many aspects of clot structure and factors that influence structure are directly related to the rates of these steps of Polymerization, even though these effects are often not obvious. Thus, to a large extent, clot structure is kinetically determined.
F I Ataullakhanov - One of the best experts on this subject based on the ideXlab platform.
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procoagulant platelets form an α granule protein covered cap on their surface that promotes their attachment to aggregates
Journal of Biological Chemistry, 2013Co-Authors: Anastasia A Abaeva, Matthias Canault, Yana N Kotova, Sergey I Obydennyy, Alena O Yakimenko, Nadezhda A Podoplelova, Vladimir N Kolyadko, Herve Chambost, Aleksei V Mazurov, F I AtaullakhanovAbstract:Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbβ3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that Fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without Fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface Fibrin(ogen) was strongly decreased in the presence of a Fibrin Polymerization inhibitor GPRP and also in platelets from a patient with dysFibrinogenemia and a Fibrinogen Polymerization defect. In contrast, a Fibrinogen-clotting protease ancistron increased the amount of Fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in Fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of Fibrinogen and αIIbβ3. These data show that the Fibrin(ogen)-covered cap, predominantly formed as a result of Fibrin Polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.