A novel natural mutation AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function

2014 ◽  
Vol 111 (01) ◽  
pp. 79-87 ◽  
Author(s):  
Zuzana Riedelová-Reicheltová ◽  
Roman Kotlín ◽  
Jiří Suttnar ◽  
Věra Geierová ◽  
Tomáš Riedel ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Coiled Coil ◽  
Fibrin Clot ◽  
Coiled Coil Region ◽  
Platelet Aggregometry ◽  
Sds Page ◽  
Lysis Rate ◽  
Thrombotic Complications ◽  
Natural Mutation ◽  
And Function

SummaryThe aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95–98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss.

Two cases of congenital dysfibrinogenemia associated with thrombosis – Fibrinogen Praha III and Fibrinogen Plzeň

2009 ◽  
Vol 102 (09) ◽  
pp. 479-486 ◽  
Author(s):  
Zuzana Reicheltová ◽  
Martin Malý ◽  
Jiří Suttnar ◽  
Alžbòta Sobotková ◽  
Peter Salaj ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Coiled Coil ◽  
Helical Conformation ◽  
Side Chain ◽  
Test Results ◽  
Aromatic Side Chain ◽  
Sds Page ◽  
History Of ◽  
Asparagine Residue ◽  
Fibrinogen Molecule

SummaryCongenital dysfibrinogenemia is a rare disease characterised by inherited abnormality in the fibrinogen molecule, resulting in functional defects. Two patients, a 26-year-old woman and a 61-year-old man, both with history of thrombotic events, had abnormal coagulation test results. DNA sequencing showed the heterozygous γY363N mutation (Fibrinogen Praha III) and the heterozygous Aα N106D mutation (Fibrinogen Plzeň), respectively. Fibrin polymerisation, after addition of either thrombin or reptilase, showed remarkably delayed polymerisation in both cases. Fibrinolysis experiments showed slower tPA initiated lysis of clots. SDS-PAGE did not show any difference between normal and Praha III and Plzeň fibrinogens. Both mutations had a significant effect on platelet aggregation. In the presence of either ADP or TRAP, both mutations caused the decrease of platelet aggregation. SEM revealed abnormal clot morphology, with a large number of free ends and narrower fibres of both fibrin Praha III and Plzeň. Praha III mutation was situated in the polymerisation pocket “a”. The replacement of the bulky aromatic side chain of tyrosine by the polar uncharged small side chain of asparagine may lead to a conformational change, possibly altering the conformation of the polymerisation pocket. The Plzeň mutation is situated in the coiled-coil connector and this replacement of polar uncharged asparagine residue by polar acidic aspartate changes the alpha-helical conformation of the coiled-coil connector;and may destabilise hydrogen bonds in its neighborhood. Although both mutations are situated in different regions of the molecule, both mutations have a very similar effect on fibrinogen functions and both are connected with thromboses.

TRANSIENT AGGREGATION RESISTANCE OF HUMAN PLATELET-RICH PLASMA; A NEGLECTED IN VITRO PHENOMENON WITH PHYSIOLOGICAL IMPACT

1987 ◽  
Author(s):  
M R Hardeman ◽  
J Vreeken
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Platelet Aggregometry ◽  
Wide Range ◽  
Plasma Factor ◽  
Thrombotic Complications ◽  
Fresh Plasma ◽  
Physiological Impact ◽  
Spontaneous Platelet Aggregation

Due to instability in the first in vitro period, platelet-aggregometry is usually deliberately postponed until ca. 1 hour after venepuncture (VP) . At that time aggregability is fairly constant for 1 hour or more. Investigation of the period immediately followed VP, hcwever, revealed a high aggregation resistance - measured as the threshold ADP-concentration which the platelets just could resist before they aggregate maximally and irreversibly - which subsequently decreased exponentially with time. This “Transient Aggregation Resistance” (TAR) appeared to be superimposed on a stable, so called Baseline Aggregation Resistance (BAR) .The latter, measurable 60 min or more after VP, yields the “classical” threshold ADP-concentration.Parallel aggregation-studies started 6 min after VP, subsequent studies were performed every 4 min. pH was controlled during storage of PRP at rocmtenperature. Extrapolation of the TAR-curve to t=0 (i.e. time of VP) yields the maximal value:TARmax Coefficients of variation for TARmax-method: 9.4% (n=6) ; intraindividial 15% (n=15, over 3 yrs); interindividual: 51% (n=16,wide range).This TAR-phencmenon which is proven to be caused by a plasma-factor, can be influenced by dietary n-3 fattty acids and can be also inhibited by ASA,suggesting a prostanoid nature. The physiological significance of TAIfoax can be illustrated by the following findings:1. Patients with myocardial infarction, hyperlipoproteinemia, sickle cell anemia (i.e. diseases with a high risk for thrombotic complications) have low TARmax-values. 2.Individuals with “spontaneous platelet aggregation” in vitro,but asymptomatic, have positive TARmax-values. 3.There is a clear,reciproke age-dependency of TARmax It is concluded that a technique is available measuring the effect of circulating,labile platelet-aggregation influencing plasma factor(s). Furthermore, using this technique,it was found that normal fresh plasma contains a labile aggregation-inhibiting factor which is several orders of magnitude more potent than other stabile factors either present in plasma or associated with platelets. This factor is probably of prostanoid nature and might have significance as a reflection of the antithrcmbotic potential of the endothelium.

scholarly journals Roles of fibrin α- and γ-chain specific cross-linking by FXIIIa in fibrin structure and function

2014 ◽  
Vol 111 (05) ◽  
pp. 842-850 ◽  
Author(s):  
Cédric Duval ◽  
Peter Allan ◽  
Simon D. A. Connell ◽  
Victoria C. Ridger ◽  
Helen Philippou ◽  
...  
Keyword(s):  
Potential Role ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Cross Linking ◽  
Appearance Time ◽  
Lysis Rate ◽  
Α Chain ◽  
And Function ◽  
Clot Structure

SummaryFactor XIII is responsible for the cross-linking of fibrin γ-chains in the early stages of clot formation, whilst α-chain cross-linking occurs at a slower rate. Although γ- and α-chain cross-linking was previously shown to contribute to clot stiffness, the role of cross-linking of both chains in determining clot structure is currently unknown. Therefore, the aim of this study was to determine the role of individual α- and γ-chain cross-linking during clot formation, and its effects on clot structure. We made use of a recombinant fibrinogen (γQ398N/Q399N/K406R), which does not allow for y-chain cross-linking. In the absence of cross-linking, intact D-D interface was shown to play a potential role in fibre appearance time, clot stiffness and elasticity. Cross-linking of the fibrin α-chain played a role in the thickening of the fibrin fibres over time, and decreased lysis rate in the absence of α2-antiplasmin. We also showed that α-chain cross-linking played a role in the timing of fibre appearance, straightening fibres, increasing clot stiffness and reducing clot deformation. Cross-linking of the γ-chain played a role in fibrin fibre appearance time and fibre density. Our results show that α- and γ-chain cross-linking play independent and specific roles in fibrin clot formation and structure.

scholarly journals The MLKL kinase-like domain dimerization is an indispensable step of mammalian MLKL activation in necroptosis signaling

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Yu Zhang ◽  
Jia Liu ◽  
Dandan Yu ◽  
Xinxin Zhu ◽  
Xiaoyan Liu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Self Assembly ◽  
Mammalian Species ◽  
Coiled Coil ◽  
Direct Consequence ◽  
Activation Process ◽  
Sequential Process ◽  
Coiled Coil Region ◽  
And Function ◽  
General Step

AbstractMLKL phosphorylation by RIP3 is the commitment step of necroptosis execution, which could induce MLKL activation featured as MLKL monomer-oligomer transition. Here, we reported that the dimerization of the MLKL kinase-like domain was the direct consequence of RIP3 triggered MLKL-phosphorylation. Two inter-dimer interfaces were found in the crystal structure of human MLKL. Mutations destroying both interfaces could prevent RIP3-induced MLKL oligomerization and necroptosis efficiently. Moreover, we confirmed MLKL self-assembly by the internal coiled-coil region is necessary for MLKL oligomerization and function. The mutations disrupting coiled-coil self-assembly repressed necroptosis, but it did not prevent RIP3-induced dimerization of the MLKL kinase-like domain. So that, MLKL activation is a sequential process, which begins with kinase-like domain dimerization, and followed by internal coiled-coil region self-assembly to form a proper MLKL oligomer. Besides human MLKL, structural and functional analysis showed the kinase-like domain dimerization was conserved among mammalian species, suggesting it is a general step of the RIP3-induced MLKL activation process.

Abnormal Fibrin Clot Architecture in Nephrotic Patients Is Related to Hypofibrinolysis: Influence of Plasma Biochemical Modifications

1999 ◽  
Vol 82 (11) ◽  
pp. 1482-1489 ◽  
Author(s):  
Zohar Mishal ◽  
Claude Lesty ◽  
Manoucher Mirshahi ◽  
Jacqueline Peynet ◽  
Alain Baumelou ◽  
...  
Keyword(s):  
Fibrin Clot ◽  
Albumin Level ◽  
Morphological Properties ◽  
Plasma Modification ◽  
Fibrin Gels ◽  
Pharmacological Prevention ◽  
Fibrin Network ◽  
Lysis Rate ◽  
Thrombotic Complications ◽  
Fibrin Fiber

SummaryPorosity, viscoelasticity and morphological properties of plasma fibrin from 16 nephrotic patients and 16 healthy volunteers were compared. Nephrotic patients were characterized by formation of tight and rigid plasma fibrin gels which resulted in a slower rate of fibrin lysis studied either under pressure-driven permeation or diffusional transport of fibrinolytic agents. These latter findings indicated that both abnormal fibrin network conformation and abnormal fibrin fiber structure were involved in hypofibrinolysis. Albumin supplementation up to 40 mg/ml partially restored normal fibrin architecture and increased the rate of fibrinolysis in these patients. Multiparametric analysis showed that nephrotic patients were mainly characterized by a low plasma albumin level (R = -0.85), a low albumin to fibrinogen ratio (R = -0.89) and a high resistance to lysis (R = -0.82). High triglycerides level was the only plasma modification related to the slower fibrin lysis rate (R = -0.54). High fibrin rigidity (G’) was the only fibrin parameter simultaneously related to the nephrotic state (R = 0.75) and the lysis resistance (R = -0.71). After eliminating the effects of age, albumin and fibrinogen levels, low fibrin porosity (Ks) and low fiber mass-length ratio (μ) were the main features of the nephrotic state. These findings are discussed in relation to both the pathophysiology of thrombotic complications in nephrotic syndrome and their pharmacological prevention.

scholarly journals Microtubule-bundling protein Spef1 enables mammalian ciliary central apparatus formation

2018 ◽  
Vol 11 (1) ◽  
pp. 67-77 ◽  
Author(s):  
Jianqun Zheng ◽  
Hao Liu ◽  
Lei Zhu ◽  
Yawen Chen ◽  
Huijie Zhao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Cell Movement ◽  
Ependymal Cells ◽  
Coiled Coil Region ◽  
Evolutionarily Conserved ◽  
Flow Through ◽  
Ependymal Cilia ◽  
And Function ◽  
Signal Transductions

Abstract Cilia are cellular protrusions containing nine microtubule (MT) doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions. Cilia require the central pair (CP) apparatus, consisting of two CP MTs covered with projections of CP proteins, for planar strokes. How the CP MTs of such ‘9 + 2’ cilia are constructed, however, remains unknown. Here we identify Spef1, an evolutionarily conserved microtubule-bundling protein, as a core CP MT regulator in mammalian cilia. Spef1 was selectively expressed in mammalian cells with 9 + 2 cilia and specifically localized along the CP. Its depletion in multiciliated mouse ependymal cells by RNAi completely abolished the CP MTs and markedly attenuated ciliary localizations of CP proteins such as Hydin and Spag6, resulting in rotational beat of the ependymal cilia. Spef1, which binds to MTs through its N-terminal calponin-homologous domain, formed homodimers through its C-terminal coiled coil region to bundle and stabilize MTs. Disruption of either the MT-binding or the dimerization activity abolished the ability of exogenous Spef1 to restore the structure and functions of the CP apparatus. We propose that Spef1 bundles and stabilizes central MTs to enable the assembly and functions of the CP apparatus.

Increased Response to Arachidonic Acid and U-46619 and Resistance to Inhibitory Prostaglandins in Patients with Chronic Myeloproliferative Disorders

1988 ◽  
Vol 59 (01) ◽  
pp. 073-076 ◽  
Author(s):  
Sergio Cortelazzo ◽  
Monica Galli ◽  
Donatella Castagna ◽  
Piera Viero ◽  
Giovanni de Gaetano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Myeloproliferative Disorders ◽  
Bone Marrow Stem Cell ◽  
Healthy Controls ◽  
Aggregation Response ◽  
Thrombotic Complications ◽  
Cyclic Endoperoxide ◽  
Marrow Stem Cell

SummaryIn patients with myeloproliferative disorders (MPD) a group of related diseases of the bone marrow stem cell and recurrent haemorrhagic and/or thrombotic complications, the production of aggregating prostaglandins (PGs) may be normal or slightly reduced, while PGI2 production is normal. However, MPD platelet sensitivity to antiaggregatory PGs is still unknown.We studied the potency of PGD2, PGI2 and PGEi as inhibitors of platelet aggregation induced by threshold aggregating concentrations of arachidonic acid and U-46619-analogue of the cyclic endoperoxide PGH2 in 20 patients with MPD in comparison with healthy controls, with the aim of evaluating the sensitivity of MPD platelets to antiaggregatory PGs. In these patients platelet prostanoid metabolism was normal. However, the functional response of platelets to aggregating and antiaggregating prostanoids was shifted towards potentially increased platelet aggregation response. These findings could have a clinical relevance in view of the haemostatic and thrombotic complications so frequent in MPD.

The Effect of Mitomycin C on Platelet Aggregation and Adenosine 3′, 5′-Monophosphate Metabolism

1978 ◽  
Vol 39 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Mitomycin C ◽  
Blood Platelets ◽  
Adenosine Diphosphate ◽  
Cellular Membrane ◽  
Adenyl Cyclase ◽  
Cyclic Amp Phosphodiesterase ◽  
Membrane Structure And Function ◽  
And Function

SummaryThe effect of Mitomycin C on aggregation, adenosine 3′, 5′-monophosphate (cyclic AMP) metabolism and reactions induced by thrombin was studied in rabbit platelets. Mitomycin C inhibited the platelet aggregation induced by adenosine diphosphate or thrombin. The level of radioactive cyclic AMP derived from 8-14C adenine or 8-14C adenosine increased after incubating intact platelets with Mitomycin G. Formation of radioactive adenosine triphosphate also increased though mitochondrial oxidation was not stimulated. Similar effect was observed also in rabbit liver. Mitomycin C failed to stimulate platelet adenyl cyclase but inhibited cyclic AMP phosphodiesterase in the absence of theophylline. In the platelets preincubated with Mitomycin C, thrombin-induced inhibition of adenyl cyclase, stimulation of membrane-bound cyclic AMP phosphodiesterase, and release of 250,000 dalton protein from platelet membranes were prevented. These results suggest that Mitomycin C will affect cellular membrane structure and function, and this extranuclear effect of Mitomycin C will lead to inhibition of aggregation in blood platelets.

Properties of Fibrinogen Cleaved by Jararhagin, a Metalloproteinase from the Venom of Bothrops jararaca

1994 ◽  
Vol 72 (02) ◽  
pp. 244-249 ◽  
Author(s):  
Aura S Kamiguti ◽  
Joseph R Slupsky ◽  
Mirko Zuzel ◽  
Charles R M Hay
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Human Fibrinogen ◽  
Activated Platelets ◽  
Bothrops Jararaca ◽  
Platelet Binding ◽  
Fibrin Monomers ◽  
Fibrinogen Molecule

SummaryHaemorrhagic metalloproteinases from Bothrops jararaca and other venoms degrade vessel-wall and plasma proteins involved in platelet plug and fibrin clot formation. These enzymes also cause proteolytic digestion of fibrinogen which has been suggested to cause defective platelet function. Fibrinogen degradation by jararhagin, a metalloproteinase from B. jararaca, and the effect of jararhagin fibrinogenolysis on both platelet aggregation and fibrin clot formation were investigated. Jararhagin was found to cleave human fibrinogen in the C-terminal region of the Aα-chain giving rise to a 285-290 kDa fibrinogen molecule lacking the Aα-chain RGD 572-574 platelet-binding site. Platelet binding and aggregation of ADP-activated platelets is unaffected by this modification. This indicates that the lost site is not essential for platelet aggregation, and that the remaining platelet binding sites located in the N-terminal portion of Aα chains (RGD 95-97) and the C-terminal of γ chains (dodecapeptide 400-411) are unaffected by jararhagin-digestion of fibrinogen. Fibrin clot formation with thrombin of this remnant fibrinogen molecule was defective, with poor polymerization of fibrin monomers but normal release of FPA. The abnormal polymerization could be explained by the loss of one of the two complementary polymerization sites required for side-by-side association of fibrin protofibrils. Jararhagin-induced inhibition of platelet function, an important cause of haemorrhage in envenomed patients, is not caused by proteolysis of fibrinogen, as had been thought, and the mechanism remains to be elucidated.

Release, Aggregation and Lysis of Human Platelets by Antilymphocyte Globulin and Antiplatelet Serum

1976 ◽  
Vol 36 (02) ◽  
pp. 411-423 ◽  
Author(s):  
Nicholas Lekas ◽  
J. C Rosenberg
Keyword(s):  
Platelet Aggregation ◽  
Gamma Globulin ◽  
Human Platelets ◽  
Plasma Medium ◽  
Release Reaction ◽  
Clinical Events ◽  
Thrombotic Complications ◽  
Platelet Release ◽  
Free Media

SummaryHuman platelets labeled with 51Cr were used to determine the contribution made by platelet lysis to the platelet release reaction and platelet aggregation induced by rabbit antihuman platelet serum (APS) and equine antihuman thymocyte globulin (ATG). Platelets were tested in both plasma (PRP) and non-plasma containing media. Antibodies directed against platelets, either as APS or ATG, induced significant amounts of platelet release and aggregation, as well as some degree of lysis, in the absence of complement. The presence of complement increased platelet lysis and aggregation, but not the release reaction. Non-immune horse gamma globulin produced different responses depending upon whether platelets were investigated in PRP or non-plasma containing media. Aggregation was seen in the latter but not the former. These differences can be explained by the presence of plasma components which prevent non-specific immune complexes from causing platelet aggregation. Since platelets in vivo are always in a plasma medium, one must be wary of utilizing data from platelet studies in synthetic plasma-free media as the basis of explaining clinical events. These observations demonstrate at least two, and possibly three, different mechanisms whereby ATG could activate platelets causing thrombotic complications and thrombocytopenia, i.e., via 1) specific and, 2) non-specific non-lytic pathways and 3) a lytic pathway.

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