Fibrin Network Formation Research Articles

Effect of construction of TiO2 nanotubes on platelet behaviors: Structure-property relationships.

To realize optimal design and construction of biomaterials with desired properties for blood contact materials, a comprehensive understanding of structure-property relationships is required. In the existing literature, TiO2 nanotube has been reported to be a good candidate for biomedical applications. However, it is noticeable that the blood compatibility of TiO2 nanotubes (TNTs) remains obscure or even inconsistent in the previously published works. The inconsistency could derive from different research protocols, material properties or blood sources. Thus, a thorough investigation of the effect of surface properties on blood compatibility is crucial to the development of titanium based materials. In this paper, we explored the effect of surface properties on the response of platelet-rich plasma, especially surface morphology, chemistry, wettability and crystalline phase. The results indicated that crystalline phase was a dominant factor in platelet behaviors. Reduced adhesion and activation of platelets were observed on amorphous and rutile dominated TNTs, whereas anatase dominated TNTs activated the formation of fibrin network. We further proposed a hypothetical mechanism for better understanding of how surface properties affect the response of platelet-rich plasma. Therefore, this study expands the fundamental understanding of the structure-property relationships of titanium based materials.

Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor.

Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).

Functional characterization of tissue factor in von Willebrand factor-dependent thrombus formation under whole blood flow conditions.

Von Willebrand factor (VWF) plays an important role in mediating platelet adhesion and aggregation under high shear rate conditions. Such platelet aggregates are strengthened by fibrin-network formation triggered by tissue factor (TF). However, little is known about the role of TF in VWF-dependent thrombus formation under blood flow conditions. We evaluated TF in thrombus formation on immobilized VWF under whole blood flow conditions in an in vitro perfusion chamber system. Surface-immobilized TF amplified intra-thrombus fibrin generation significantly under both low and high shear flow conditions, while TF in sample blood showed no appreciable effects. Furthermore, immobilized TF enhanced VWF-dependent platelet adhesion and aggregation significantly under high shear rates. Neutrophil cathepsin G and elastase increased significantly intra-thrombus fibrin deposition on immobilized VWF-TF complex, suggesting the involvement of leukocyte inflammatory responses in VWF/TF-dependent mural thrombogenesis under these flow conditions. These results reveal a functional link between VWF and TF under whole blood flow conditions, in which surface-immobilized TF and VWF mutually contribute to mural thrombus formation, which is essential for normal hemostasis. By contrast, TF circulating in blood may be involved in systemic hypercoagulability, as seen in sepsis caused by severe microbial infection, in which neutrophil inflammatory responses may be active.

The clinical relevance of altered fibrinogen packaging in the presence of 17β-estradiol and progesterone

BackgroundThe effect of endogenous hormone concentrations, specifically 17β-estradiol and progesterone, on fibrin network formation has not been established. ObjectivesIt is essential to understand natural hormone mechanisms since these hormones are still present in circulation while hormonal contraceptives, which are associated with increased risk of venous thromboembolism, are used. MethodsDue to the fact that these hormones are known to increase hypercoagulability and the prothrombotic state scanning electron microscopy (SEM), atomic force microscopy (AFM), thromboelastography (TEG) and turbidimetry were employed to investigate the morphology, surface roughness, viscoelastic properties and formation and lysis of fibrin. Results17β-estradiol and progesterone showed hypercoagulable viscoelastic properties and decreased the diameter and surface roughness of fibrin while increasing dense matted deposit occurrence. Our results suggest that the additional burden of hormonal load, together with the presence of endogenous estrogen and progesterone, may result in a prothrombotic and hypercoagulable state in females with an inflammatory predisposition. ConclusionOur results are of clinical importance when considering hormones as either pathological agent or therapeutic intervention as will be assessed in future investigation.

Mechanical Properties of the Fibrin Network on the Macroscopic and Microscopic Scales

The scaffold of the thrombus is a three-dimensional network of fibrin, a fibrous protein that determines the mechanical properties of the whole clot and plays thereby an important role in hemostasis. With our atomic force microscope (AFM)-based force spectroscopy, called nano-thrombelastography, we followed the nanoscale changes in the viscoelastic properties during fibrin network formation and streptokinase (STK)-induced fibrinolysis in human plasma-derived clot.During nano-thrombelastography we measure the forces acting on an AFM cantilever which is submerged into a plasma or fibrinogen droplet during its cyclic vertical travel. The time-dependent evolution of force resembles well a macroscopic thrombelastogram: force increases gradually with the formation of the elastic fibrin network during coagulation. By measuring the area of the hysteresis loop formed by the cyclic force curves we could follow the changes in the viscous properties of the fibrin network. Effect of STK manifests in a rapid decrease in thrombus elasticity, whereas thrombus viscosity only decayed with a delay.For morphological experiments, clot formation was initiated by adding thrombin in 0,5 IU final concentration to fibrinogen solution on mica surface. In dried samples, scanning AFM images of mature fibrin fibers appeared as 17-nm-high and 12-196-nm-wide filaments. Upon lowering the sodium and chloride concentration of the solution, we observed fibrin networks wherein fibers had increased diameter. We carried out force mapping on native fibers with a diameter between 100-250 nm in Hepes-buffer. Force curves were recorded for each 100-µm2 area previously scanned. The local Young-modulus was calculated by using the Hertz model. The force-volume maps revealed that fibrin fibers exceeding 200 nm diameter have a dense surface layer and a loose core, suggesting that the thickening of the fibrils involves a distinct mechanical maturation.

フィブリン・ネットワーク形成のダイナミックス：コンピュータ・シミュレーションによる新しい理解

フィブリン・ネットワーク形成のダイナミックス：コンピュータ・シミュレーションによる新しい理解

62 Visualization of αC regions in fibrin network formation by high-resolution AFM

Fibrinogen is one of the key proteins in thrombosis. It has been intensively studied with transmission electron microscopy and X-ray diffraction. But until now, a complete 3D structure of the molec...

Platelets and physics: How platelets “feel” and respond to their mechanical microenvironment

During clot formation, platelets are subjected to various different signals and cues as they dynamically interact with extracellular matrix proteins such as von Willebrand factor (vWF), fibrin(ogen) and collagen. While the downstream signaling of platelet–ligand interactions is well-characterized, biophysical cues, such as hydrodynamic forces and mechanical stiffness of the underlying substrate, also mediate these interactions and affect the binding kinetics of platelets to these proteins. Recent studies have observed that, similar to nucleated cells, platelets mechanosense their microenvironment and exhibit dynamic physiologic responses to biophysical cues. This review discusses how platelet mechanosensing is affected by the hydrodynamic forces that dictate vWF–platelet interactions and fibrin polymerization and network formation. The similarities and differences in mechanosensing between platelets and nucleated cells and integrin-mediated platelet mechanosensing on both fibrin(ogen) and collagen are then reviewed. Further studies investigating how platelets interact with the mechanical microenvironment will improve our overall understanding of the hemostatic process.

The Reg3α (HIP/PAP) Lectin Suppresses Extracellular Oxidative Stress in a Murine Model of Acute Liver Failure.

Background and AimsAcute liver failure (ALF) is a rapidly progressive heterogeneous illness with high mortality rate and no widely accessible cure. A promising drug candidate according to previous preclinical studies is the Reg3α (or HIP/PAP) lectin, which alleviates ALF through its free-radical scavenging activity. Here we study the therapeutic targets of Reg3α in order to gain information on the nature of the oxidative stress associated with ALF.MethodsPrimary hepatocytes stressed with the reactive oxygen species (ROS) inducers TNFα and H2O2 were incubated with a recombinant Reg3α protein. ALF was induced in C57BL/6J mice by an anti-CD95 antibody. Livers and primary hepatocytes were harvested for deoxycholate separation of cellular and extracellular fractions, immunostaining, immunoprecipitation and malondialdehyde assays. Fibrin deposition was studied by immunofluorescence in frozen liver explants from patients with ALF.ResultsFibrin deposition occurs during experimental and clinical acute liver injuries. Reg3α bound the resulting transient fibrin network, accumulated in the inflammatory extracellular matrix (ECM), greatly reduced extracellular ROS levels, and improved cell viability. Hepatocyte treatment with ligands of death receptors, e.g. TNFα and Fas, resulted in a twofold increase of malondialdehyde (MDA) level in the deoxycholate-insoluble fractions. Reg3α treatment maintained MDA at a level similar to control cells and thereby increased hepatocyte survival by 35%. No antioxidant effect of Reg3α was noted in the deoxycholate-soluble fractions. Preventing fibrin network formation with heparin suppressed the prosurvival effect of Reg3α.ConclusionsReg3α is an ECM-targeted ROS scavenger that binds the fibrin scaffold resulting from hepatocyte death during ALF. ECM alteration is an important pathogenic factor of ALF and a relevant target for pharmacotherapy.

Visualization of fibrinogen αC regions and their arrangement during fibrin network formation by high‐resolution AFM

Fibrinogen has been intensively studied with transmission electron microscopy and x-ray diffraction. But until now, a complete 3D structure of the molecule has not yet been available because the two highly flexible αC regions could not be resolved in fibrinogen crystals. This study was aimed at determining whether the αC regions can be visualized by high-resolution atomic force microscopy. Atomic force microscopy with super high resolution was used to image single molecules of fibrinogen and fibrin associates. The key approach was to use a graphite surface modified with the monolayer of amphiphilic carbohydrate-glycine molecules and unique supersharp cantilevers with 1nm tip diameter. Fibrinogen αC regions were visualized along with the complete domain structure of the protein. In almost all molecules at pH 7.4 the D domain regions had one or two protrusions of average height 0.4± 0.1nm and length 21±6nm. The complex, formed between thrombin and fibrinogen, was also visualized. Images of growing fibrin fibers with clearly visible αCregions have been obtained. Fibrin αC regions were visible in protofibrils and large fibers; αC regions intertwined near a branchpoint and looked like a zipper. These results support the idea that αC regions are involved in the thickening of fibrin fibers. In addition, new details were revealed about the behavior of individual fibrin molecules during formation of the fibrin network. Under the diluted condition, the positioning of the αC regions could suggest their involvement in long-range interactions between fibrin but not fibrinogen molecules.

Calix[4]arene C-145 Effects on Plasma Haemostasis

Background: Calix[4]arene-methylene-bis-phosphonic acid C-192 and its sodium salt C-145 were shown to be efficient inhibitors of fibrin polymerization in vitro. In presented work we analyze the effects of Nalix[4]arene C-145 on haemostasis in vivo. Methods: Parameters of coagulation system of rabbits intravenously injected with C-145 were monitored. We measured thrombin time (TT), ecamulin time (ET) and activated partial thromboplastin time (APTT), determined the levels of fibrinogen, prothrombin, protein C, PAI-1, monitored overall haemostatic potential before and after the injection. Results: C-145 was administrated intravenously in the dose of 7.5 mg/kg of rabbit weight. 4 hours after the injection the thrombin time and activated partial thromboplastin time of rabbit’s blood plasma were prolonged 2 and 1,5 times respectively. Such prolongation was observed after 24 hours as well. However, the total fibrinogen and prothrombin levels did not change. Parameters of fibrinolytic system (PAI-1, clot lysis half-time) and anticoagulant system (protein C) remained unchanged. Conclusions: Therefore we assumed that calix[4]arene C-145’s effects on haemostasis consisted in selective inhibition of fibrin polymerization and formation of three-dimensional fibrin network.

Reduced platelet adhesion and improved corrosion resistance of superhydrophobic TiO2-nanotube-coated 316L stainless steel

Superhydrophilic and superhydrophobic TiO2 nanotube (TNT) arrays were fabricated on 316L stainless steel (SS) to improve corrosion resistance and hemocompatibility of SS. Vertically-aligned superhydrophilic amorphous TNTs were fabricated on SS by electrochemical anodization of Ti films deposited on SS. Calcination was carried out to induce anatase phase (superhydrophilic), and fluorosilanization was used to convert superhydrophilicity to superhydrophobicity. The morphology, structure and surface wettability of the samples were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and contact angle goniometry. The effects of surface wettability on corrosion resistance and platelet adhesion were investigated. The results showed that crystalline phase (anatase vs. amorphous) and wettability strongly affected corrosion resistance and platelet adhesion. The superhydrophilic amorphous TNTs failed to protect SS from corrosion whereas superhydrophobic amorphous TNTs slightly improved corrosion resistance of SS. Both superhydrophilic and superhydrophobic anatase TNTs significantly improved corrosion resistance of SS. The superhydrophilic amorphous TNTs minimized platelet adhesion and activation whereas superhydrophilic anatase TNTs activated the formation of fibrin network. On the contrary, both superhydrophobic TNTs (superhydrophobic amorphous TNTs and superhydrophobic anatase TNTs) reduced platelet adhesion significantly and improved corrosion resistance regardless of crystalline phase. Superhydrophobic anatase TNTs coating on SS surface offers the opportunity for the application of SS as a promising permanent biomaterial in blood contacting biomedical devices, where both reducing platelets adhesion/activation and improving corrosion resistance can be effectively combined.

Substrate profiling of Finegoldia magna SufA protease, inhibitor screening and application to prevent human fibrinogen degradation and bacteria growth in vitro.

SufA, which belongs to the subtilisin-like serine protease family, contains a non-canonical Asp-His-Ser catalytic triad. Under in vitro conditions, SufA is capable of human fibrinogen hydrolysis leading to inhibition of fibrin network formation, thus suggesting its important role in the development and progression of Finegoldia magna infections. In addition, it has been demonstrated that SufA can hydrolyze antibacterial peptides such as LL-37 and the chemokine MIG/CXCL 9, hence evading host defence mechanisms. Although the SufA protease from F. magna was discovered several years ago, its optimal substrate preference has not yet been identified. Considering the role of SufA, we have focused on the profiling of its substrate sequence preference spanning S1-S3 binding pockets using the FRET (fluorescence resonance energy transfer) approach. Next, based on the structure of the P1 residue of the developed substrate, we narrowed the inhibitor screening to the phosphonic analogues of amino acids containing an arginine-like side chain. Among all the compounds tested, only Cbz-6-AmNphth(P)(OPh)2 showed any inhibitory activity against SufA displaying k2/Ki value of 10,800 M(-1) s(-1). In addition, it prevented SufA-mediated human fibrinogen hydrolysis in vitro and exhibited potent antibacterial activity against F. magna, Staphylococcus aureus and Escherichia coli. Herein, we report on the substrate specificity, synthesis and kinetic evaluation of phosphonic inhibitors of SufA protease from F. magna which could help to establish its function in pathogenesis development and may lead to the elaboration of new antibacterial drugs.

A Comprehensive Mechanism of Fibrin Network Formation Involving Early Branching and Delayed Single- to Double-Strand Transition from Coupled Time-Resolved X-ray/Light-Scattering Detection

The formation of a fibrin network following fibrinogen enzymatic activation is the central event in blood coagulation and has important biomedical and biotechnological implications. A non-covalent polymerization reaction between macromolecular monomers, it consists basically of two complementary processes: elongation/branching generates an interconnected 3D scaffold of relatively thin fibrils, and cooperative lateral aggregation thickens them more than 10-fold. We have studied the early stages up to the gel point by fast fibrinogen:enzyme mixing experiments using simultaneous small-angle X-ray scattering and wide-angle, multi-angle light scattering detection. The coupled evolutions of the average molecular weight, size, and cross section of the solutes during the fibrils growth phase were thus recovered. They reveal that extended structures, thinner than those predicted by the classic half-staggered, double-stranded mechanism, must quickly form. Following extensive modeling, an initial phase is proposed in which single-bonded "Y-ladder" polymers rapidly elongate before undergoing a delayed transition to the double-stranded fibrils. Consistent with the data, this alternative mechanism can intrinsically generate frequent, random branching points in each growing fibril. The model predicts that, as a consequence, some branches in these expanding "lumps" eventually interconnect, forming the pervasive 3D network. While still growing, other branches will then undergo a Ca(2+)/length-dependent cooperative collapse on the resulting network scaffolding filaments, explaining their sudden thickening, low final density, and basic mechanical properties.

Mechanical properties and fibrin characteristics of endovascular coil–clot complexes: relevance to endovascular cerebral aneurysm repair paradigms

BackgroundAlthough coil embolization is known to prevent rebleeding from acutely ruptured cerebral aneurysms, the underlying biological and mechanical mechanisms have not been characterized. We sought to determine if microcoil-dependent interactions...

Comparative evaluation between 20% EDTA-S & ornidazole gel as root biomodification agent asem study

Background: It should be well understood that the root surface receptiveness to clot formation & initial periodontal wound healing decides the nature of the connective tissue attachments. This study was carried out to assess the initial wound healing events after the application of 20% EDTA-S & Ornidazole gel and assess the formation of fibrin network following blood. Material& Method: Thirty multi-rooted teeth indicated for extraction due to periodontal disease were selected & divided into group A (20%EDTA-S), group B (Ornidazole gel (1% W/V)), group C (7.4pH phosphate buffer saline), group D (20%EDTA-S+ Blood), group E (Ornidazole gel+ Blood), group F (7.4pH) phosphate buffer saline+ Blood). Following root planning, the root surface was cut using diamond disc under copious irrigation. Samples from each group were subjected for root conditioning agent application by passive method. Specimens were then subjected to scanning electron microscopic study. Smear layers removal were analysed by Sampaia et al index. Results: 20%EDTA-S removed the smear layer better than ornidazole gel. Fibrin network formation was seen with specimen treated with 20% EDTA-S + Blood. Conclusion: Use of 20% EDTA-S as root conditioning agent has a beneficial effect on initial wound healing events, which are important for periodontal regenerative therapies.

Coagulation Assay

Clotting times can be measured by using citrate plasma. The intrinsic pathway of coagulation is measured by the activated partial thromboplastin time (aPTT), the extrinsic pathway of coagulation, monitored by measuring the prothrombin time (PT), and thrombin-induced fibrin-network formation (thrombin clotting time; TCT)., 凝血时间可以通过使用柠檬酸盐血浆来测量。 通过测量凝血酶原时间（PT）和凝血酶诱导的纤维蛋白网络形成（凝血酶凝血时间; TCT）监测的凝血的外在途径，通过活化的部分凝血活酶时间（aPTT）测量凝固的内在途径。

Measurement of the evolution of rigid and viscoelastic mass contributions from fibrin network formation during plasma coagulation using quartz crystal microbalance

The coagulation of blood plasma and the effect of fibrinogen concentration were studied with a quartz crystal microbalance (QCM), where frequency and half-width at half-maximum (bandwidth) values measured from the conductance spectrum near resonant frequency were used. Bandwidth change is an indicator of energy dissipation, allowing for an understanding of qualitative changes occurring during fibrin clot formation. Both frequency shift (Δf) and bandwidth shift (ΔΓ) were dependent on the concentration of fibrinogen in plasma. We defined a sum of squares function α (=Δf2/1000+ΔΓ2/1000) that measures absolute changes in QCM resonant characteristics to semi-quantitatively include an overall contribution of adsorbed mass and elastic modulus components and a function β (=1−ΔΓ/Δf) that indicates qualitatively the nature of response based on its deviation from ideal Newtonian behaviour. Increasing concentration of fibrinogen resulted in an increase in the value of α, showing that a larger amount of fibrinogen results in larger amount of coupled viscoelastic mass. Changes in β indicated that the nature of changes occurring was very similar to Newtonian and that coupling of rigid-mass dominates the overall response in the early stage of coagulation and in the later stage growing elastic mass compensates some of the response.

Numerical investigation into blood clotting at the bone-dental implant interface in the presence of an electrical stimulus

The insertion of a dental implant activates a sequence of wound healing events ending with bone formation and implant osseointegration. This sequence starts with the blood coagulation process and the formation of a fibrin network that detains spilt blood. Fibrin formation can be simplified as the kinetic reaction between thrombin and fibrinogen preceding the conversion of fibrinogen into fibrin. Based on experimental observations of the electrical properties of these molecules, we present a hypothesis for the mechanism of a static electrical stimulus in controlling the formation of the blood clot. Specifically, the electrical stimulus increases the fibrin network formation in such a way that a preferential region of higher fibrin density is obtained. This hypothesis is validated by means of a numerical model for the blood clot formation at the bone-dental implant interface. Numerical results compare favorably to experimental observations for blood clotting with and without the static electrical stimulus. It is concluded that the density of the fibrin network depends on the strength of the static electrical stimulus, and that the blood clot formation has a preferential direction of formation in the presence of the electrical signal.

Molecular Mechanisms, Thermodynamics, and Dissociation Kinetics of Knob-Hole Interactions in Fibrin

Polymerization of fibrin, the primary structural protein of blood clots and thrombi, occurs through binding of knobs 'A' and 'B' in the central nodule of fibrin monomer to complementary holes 'a' and 'b' in the γ- and β-nodules, respectively, of another monomer. We characterized the A:a and B:b knob-hole interactions under varying solution conditions using molecular dynamics simulations of the structural models of fibrin(ogen) fragment D complexed with synthetic peptides GPRP (knob 'A' mimetic) and GHRP (knob 'B' mimetic). The strength of A:a and B:b knob-hole complexes was roughly equal, decreasing with pulling force; however, the dissociation kinetics were sensitive to variations in acidity (pH 5-7) and temperature (T = 25-37 °C). There were similar structural changes in holes 'a' and 'b' during forced dissociation of the knob-hole complexes: elongation of loop I, stretching of the interior region, and translocation of the moveable flap. The disruption of the knob-hole interactions was not an "all-or-none" transition as it occurred through distinct two-step or single step pathways with or without intermediate states. The knob-hole bonds were stronger, tighter, and more brittle at pH 7 than at pH 5. The B:b knob-hole bonds were weaker, looser, and more compliant than the A:a knob-hole bonds at pH 7 but stronger, tighter, and less compliant at pH 5. Surprisingly, the knob-hole bonds were stronger, not weaker, at elevated temperature (T = 37 °C) compared with T = 25 °C due to the helix-to-coil transition in loop I that helps stabilize the bonds. These results provide detailed qualitative and quantitative characteristics underlying the most significant non-covalent interactions involved in fibrin polymerization.