Clot Architecture Research Articles

Structure, Properties and Degradation of Self-Assembled Fibrinogen Nanofiber Scaffolds.

Self-assembled fibrinogen nanofibers are promising candidates for skin tissue engineering due to their biocompatibility and ability to mimic the native blood clot architecture. Here, we studied the structure-property relationship and degradation of rehydrated fibrinogen nanofibers prepared by salt-induced self-assembly, focusing on the effect of scaffold layering, cross-linking time and freeze-drying. Optimal fiber stability was achieved with cross-linking by formaldehyde (FA) vapor, while treatment with liquid aldehydes, genipin, EDC, and transglutaminase failed to preserve the nanofibrous architecture upon rehydration. Scaffold layering did not significantly influence the mechanical properties but changed the scaffold architecture, with bulk fiber scaffolds being more compact than layered scaffolds. Freeze-drying maintained the mechanical properties and interconnected pore network with average pore diameters around 20 μm, which will enhance the storage stability of self-assembled fibrinogen scaffolds. Varying cross-linking times altered the scaffold mechanics without affecting the swelling behavior, indicating that scaffold hydration can be controlled independently of the mechanical characteristics. Cross-linking times of 240 min increased scaffold stiffness and decreased elongation, while 30 min resulted in mechanical properties similar to native skin. Cross-linking for 120 min was found to reduce scaffold degradation by various enzymes in comparison to 60 min. Overall, after 35 days of incubation, plasmin and a combination of urokinase and plasminogen exhibited the strongest degradative effect, with nanofibers being more susceptible to enzymatic degradation than planar fibrinogen due to their higher specific surface area. Based on these results, self-assembled fibrinogen fiber scaffolds show great potential for future applications in soft tissue engineering that require controlled structure-function relationships and degradation characteristics.

Analysis of fibrin networks using topological data analysis – a feasibility study

Blood clot formation, a crucial process in hemostasis and thrombosis, has garnered substantial attention for its implications in various medical conditions. Microscopic examination of blood clots provides vital insights into their composition and structure, aiding in the understanding of clot pathophysiology and the development of targeted therapeutic strategies. This study explores the use of topological data analysis (TDA) to assess plasma clot characteristics microscopically, focusing on the identification of the elements components, holes and Wasserstein distances. This approach should enable researchers to objectively classify fibrin networks based on their topologic architecture. We tested this mathematical characterization approach on plasma clots formed in static conditions from porcine and human citrated plasma samples, where the effect of dilution and direct thrombin inhibition was explored. Confocal microscopy images showing fluorescence labeled fibrin networks were analyzed. Both treatments resulted in visual differences in plasma clot architecture, which could be quantified using TDA. Significant differences between baseline and diluted samples, as well as blood anticoagulated with argatroban, were detected mathematically. Therefore, TDA could be indicative of clots with compromised stability, providing a valuable tool for thrombosis risk assessment. In conclusion, microscopic examination of plasma clots, coupled with Topological Data Analysis, offers a promising avenue for comprehensive characterization of clot microstructure. This method could contribute to a deeper understanding of clot pathophysiology and thereby refine our ability to assess clot characteristics.

Exploring the ex vivo effects of Naja mossambica venom on the ultrastructure and viscoelastic properties of human blood

BackgroundVenomous snake bites have been listed as a neglected tropical disease by the World Health Organization. The Mozambique spitting cobra (Naja mossambica) is found in Sub-Saharan African countries, and its venom has been identified to predominantly result in cytotoxic effects. However, there is limited evidence on the possible hemotoxic effects of this venom on human blood. ObjectivesIn this cross-sectional study, we investigated how Mozambique spitting cobra venom affects blood clot formation. MethodsCell morphology and clot architecture were studied by using microscopy techniques. We also studied the effects of the venom on platelets by measuring platelet activity with the global thrombosis test, followed by analyzing the viscoelasticity with thromboelastography using a 0.025 ng/μL venom concentration. ResultsThe most prominent findings indicated that the viscoelastic profile in the venom-treated blood samples formed an unstable and elastic clot. The clot architecture seen with the scanning electron microscopy analysis showed an altered fibrin network and red blood cells, confirmed by the increased axial ratios, and aggregated platelets with spreading. ConclusionThese findings may offer insights into the species-specific effects of snake venom on human blood and add value to the clinical workup in confirming envenomation. Further research is needed to correlate the 20 minute whole blood clotting test with measurable values from the thromboelastography within the context of snake envenomation. This may offer a bridge between cost, early diagnosis, and treatment of snake envenomation in resource-constrained countries.

ROS-Driven Structural and Functional Fibrinogen Modifications Are Reverted By JAK Inhibition in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are bone marrow diseases characterized by excess clonal hematopoiesis resulting in elevated peripheral blood counts. Subtypes include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They share clinical features (e.g., splenomegaly, thrombotic complications, risk of leukemic transformation), and a common molecular basis: the dysregulation of the JAK-STAT pathway, which confers a hypersensitivity of hematopoietic stem cells (HSC) to the action of growth factors and cytokines, and, as a result, an overproduction of blood cells. Genetic analysis of patients with MPNs revealed that >95% of patients with PV and about 50% of patients with ET or PMF carry an activating point mutation in the JAK2 gene (JAK2-V617F). Despite clinical evidence indicating a higher susceptibility of MPN patients to develop thrombotic disease, the underlying pathogenetic mechanisms are not fully understood. It appears that thrombosis is the result of a severe inflammatory response associated to remarkable oxidative stress in the presence of endothelial dysfunction and a procoagulant environment. Hypercoagulability is a consequence of disrupted hemostasis and fibrinolysis due to damage of the endothelium and defective fibrinolysis. Evidence suggests that overproduction of ROS and a deprived antioxidant system play a major role in the pathogenesis of MPN, as well as in the resistance to tyrosine-kinase inhibitor therapy. Moreover, systemic inflammation is thought to be the main trigger of thrombosis and seems to be mainly mediated by pro-inflammatory cytokines along with endothelial cell dysfunction. Recent studies have provided evidence that fibrinogen plays a multifaceted role in inflammatory responses and autoimmunity. The ability of fibrinogen to participate in the inflammatory response depends on its specific interaction with integrins, the leukocyte cell surface adhesion receptors expressed on neutrophils, monocytes, macrophages, and several subsets of lymphocytes. In MPN patients, endothelial cell dysfunction, increased reactive oxygen species (ROS) production, and proinflammatory cytokines have been reported, together with impaired fibrinolysis. We also investigated the potential role of oxidative stress and inflammation in the promotion of thrombosis. In order to study the mechanisms of inflammation-induced thrombosis in MPN patients, the aim of the present study was to investigate the role of oxidation-induced structural and functional fibrinogen modifications in MPN patients. The eventual counteracting effects of ruxolitinib, a first-in-class JAK inhibitor, were also assessed. 40 MPN patients and 40 age- and sex-matched healthy control subjects were included in the study. We measured specific oxidative stress markers andtheir modifications in plasma, and functional and structural parameters in purified fibrinogen fractions. Marked signs of oxidative stress in plasma (P<0.01 versus controls) and, correspondingly, an increased extent of fibrinogen oxidation (P<0.01 versus controls) were observed in patients. Furthermore, fibrinogen fractions purified from MPN patients exhibited significantly reduced clotting ability and decreased susceptibility to plasmin-induced lysis (P<0.01 versus controls). Alterations in fibrinogen secondary structure, as suggested by circular dichroism spectroscopy, and in fibrin clot architecture were also identified. Importantly, clot structure obtained by stimulated emission depletion (STED) super-resolution microscopy indicated modifications in fiber diameter and clot porosity in MPN patients (P<0.01 versus controls). In patient samples, fibrinogen clot analysis and oxidative-dependent changes reveal novel structural and functional fibrinogen modifications which may favor thrombotic complications in MPNs. In patients receiving ruxolitinib, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features, suggesting a potential impactof ruxolitinib on cardiovascular prevention in MPNs.

A Pilot Study Evaluating the Ex Vivo Effects of Varying Factor VIII Concentration on Clot Kinetics and Architecture in Patients With Haemophilia A

Background Haemophilia A (HA) is a bleeding disorder, due to a deficiency in factor VIII (FVIII). These patients are unable to produce a stable fibrin clot in the propagation phase of coagulation as they do not generate sufficient thrombin. The primary treatment for HA in South Africa remains replacement therapy with standard half-life FVIII clotting factor concentrate, aimed at reducing bleeding episodes. Objectives To evaluate the effect of varying concentrations of FVIII on whole blood (WB) clot architecture and kinetics during clot formation in patients with severe HA. Design A cross-sectional study where blood from 20 patients with HA was exposed to FVIII ex vivo and compared to a control group of 20 healthy individuals. Methods Scanning electron microscopy (SEM) was used to study WB clot architecture and thromboelastography® (TEG®) was used to quantify WB clot kinetics. Results Scanning electron microscopy studies revealed that patients with HA have sparse, disorganized fibrin networks with limited crosslinking and red blood cells (RBCs) stacked in rouleaux formation. Haemophilia A blood spiked to a 10 to 15 IU/dL FVIII concentration showed improvements in the organisation of the fibrin network with some altered RBCs. In addition, blood spiked to a 30 to 35 IU/dL FVIII concentration showed an increase in fibrin formation with normal RBCs. Thromboelastography® showed that patients with HA had an increased clot initiation time and decreased clot strength. When spiked to a 10 to 15 IU/dL FVIII concentration the clot kinetic profile showed normalization. However, an increase in FVIII concentration higher than 30 IU/dL showed altered clot architecture and kinetics. Conclusion Based on the current study, FVIII levels at 10 to 15 IU/dL improved clot kinetics but did not normalize the architecture. It may be sufficient for prevention of haemorrhages. Factor VIII levels at 30 to 35 IU/dL resulted in rapid but weaker clot formation. However, at this concentration the clot architecture was normalised which is important for haemostasis. Here it was also evident that the findings of these two modalities (TEG® and SEM) should not be separated but interpreted in conjunction with each other.

Influence of Divalent Metal Ions on the Precipitation of the Plasma Protein Fibrinogen.

Fibrinogen nanofibers are very attractive biomaterials to mimic the native blood clot architecture. Previously, we reported the self-assembly of fibrinogen nanofibers in the presence of monovalent salts and have now studied how divalent salts influence fibrinogen precipitation. Although the secondary fibrinogen structure was significantly altered with divalent metal ions, morphological analysis revealed exclusively smooth fibrinogen precipitates. In situ monitoring of the surface roughness facilitated predicting the tendency of various salts to form fibrinogen fibers or smooth films. Analysis of the chemical composition revealed that divalent salts were removed from smooth fibrinogen films upon rinsing while monovalent Na+ species were still present in fibrinogen fibers. Therefore, we assume that the decisive factor controlling the morphology of fibrinogen precipitates is direct ion-protein contact, which requires disruption of the ion-surrounding hydration shells. We conclude that in fibrinogen aggregates, this mechanism is effective only for monovalent ions, whereas divalent ions are limited to indirect fibrinogen adsorption.

Certain Associations Between Iron Biomarkers and Total and γ' Fibrinogen and Plasma Clot Properties Are Mediated by Fibrinogen Genotypes.

Introduction: Evidence for the relationship between body iron and cardiovascular disease (CVD) is inconsistent and mechanisms involved remain poorly understood. Therefore, we first investigated whether there are linear or non-linear relationships between iron status and total and γ' fibrinogen as well as plasma fibrin clot properties and, second, determined whether there are interactions with iron biomarkers and fibrinogen and FXIII single nucleotide polymorphisms (SNPs) in relation to fibrinogen concentration and functionality.Methods: In this cross-sectional analysis of 2,010 apparently healthy Black South Africans we quantified total and γ' fibrinogen, serum iron, ferritin and transferrin using standardized methods and calculated transferrin saturation (TS). Clot architecture and lysis were explored with a global analytical turbidity assay. The SNPs were determined through an Illumina BeadXpress® platform.Results: Total, but not %γ', fibrinogen negatively correlated with serum iron concentrations, although both decreased over iron tertiles. %γ' fibrinogen correlated negatively with transferrin and decreased over the transferrin tertiles. A weak negative association between total fibrinogen and TS was detected with fibrinogen decreasing over the TS tertiles and categories based on TS. Lag time correlated positively with transferrin and increased over transferrin tertiles, when adjusting for fibrinogen. Before adjusting for fibrinogen, lag time was shorter in those with adequate iron status based on TS than other iron subcategories. Clot lysis time (CLT) negatively correlated with ferritin and was longer in the first than in the third ferritin tertile. Among iron status categories based on ferritin, only CLT differed and was longer in those with adequate iron than with iron-overload. CLT negatively correlated with TS, albeit weakly, shortened over the TS tertiles and was shorter in those with adequate iron based on TS categories. Interactions were observed between FGB SNPs and some of the markers of iron status investigated, in relation to the clot properties with the most prominent associations detected in homozygous carriers of the variant alleles for whom increased iron status was more beneficial than for those harboring the wild-type alleles. Iron modulated the influence of the SNPs so that for the majority iron was beneficial in respect of clot properties, but even more so for a minority group harboring specific variant alleles.Conclusion: This is the first large-scale epidemiological study to relate fibrinogen concentration and functionality to markers of iron status and to take genetic factors into consideration. We have detected a relationship between iron biomarkers and fibrinogen as well as clot characteristics that are influenced by the genetic make-up of an individual.

Fibrinogen and Clot Structure Is Affected By Eculizumab in Patients with Paroxysmal Nocturnal Haemoglobinuria

Background Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic stem cell disorder in which an acquired somatic mutation of the PIGA gene in a haematopoietic stem cell results in the deficiency of key complement regulatory molecules, namely CD59 and CD55, from the surface of blood cells leading to an increased susceptibility of blood cells to complement-mediated activation +/- intravascular haemolysis and thrombosis. Thrombosis has been the leading cause of death in PNH. Multiple mechanisms of thrombosis in PNH have been proposed, including cross talk between the coagulation and complement system. This may lead to denser clots which are more resistant to fibrinolysis. Treatment with eculizumab, which inhibits complement factor C5 and thereby prevents terminal complement activation, reduces thromboembolic events in PNH, indicating that complement activation plays a significant role in the pathophysiology of thrombosis in this disease. Patients with other thrombotic disorders are known to form denser fibrin clots that incur greater resistance to fibrinolysis. It has not previously been explored whether changes to clot architecture contribute to thrombosis in PNH. Furthermore, the role of eculizumab in clot architecture is undocumented.Aims Characterise fibrin clot structure in patients with PNH and determine the effect of eculizumab on clot structure.Methods Informed consent was obtained from all participants. 53 patients from the PNH National Service (Leeds) were recruited. They were grouped according to the proportion of PNH granulocytes, <40% (n=18), 40> - < 90 %(n=18) and >90% (n=17) and reanalysed according to treatment with (n=19) or without (n=32) eculizumab. Plasma samples were obtained and ex vivo fibrin clot structure and function was analysed by permeation analysis of clot pore structure, turbidity analysis of clot formation and fibrinolysis, and fibrin ultrastructure by laser scanning confocal microscopy and scanning electron microscopy, according to methodology and protocols well established in our laboratory. The proportion of PNH granulocytes was measured by flow cytometry and fibrinogen levels by Clauss. P values of <0.05 were taken to indicate significance.Results The proportion of PNH granulocytes correlated with fibrinogen (r= -0.35, p=0.048), maximum absorbency (r= -0.34, p=0.046) and clot density (r= -0.36, p=0.04). Seventy per cent of patients with large proportions of PNH granulocytes were on eculizumab. Analysis of patients not on eculizumab showed a trend towards proportion of PNH granulocytes correlating positively with fibrinogen levels (r=0.42, p=ns). Patients on eculizumab had lower fibrinogen levels (3.1±0.2 v 3.6±0.1g/L, P=0.02), maximum absorbance (0.31±0.1 v 0.40±0.1, p=0.028) and fibre density/100µm (18.5±4.4 v 23.9±4.2, p=0.006), indicative of a less dense clot.Summary/Conclusions This is the first study to demonstrate that patients on eculizumab showed reduced fibrinogen, fibre thickness and fibre density. Our data indicate that the beneficial effects of eculizumab in PNH is partly due to reduced complement activation and attenuated inflammatory response, leading to lower fibrinogen levels and a less thrombotic fibrin clot structure. These novel data help to explain the impact of complement inhibition in this disease. [Display omitted] DisclosuresArnold:Alexion Pharmaceuticals, Inc.: Honoraria. Griffin:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:Janssen: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; AbbVie: Honoraria; Gilled: Honoraria. Hillmen:Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Celgene: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Richards:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Optimization of Clot Formation Under Blood Flow in a Tissue Engineered Blood Vessel

BackgroundHemostasis is a complex event relying on the intersection of coagulation and platelet plug formation at the damaged blood vessel wall. Blood flow is an important force during hemostasis that delivers and removes reactants and cells to the growing clot. Standard in vitro plasma-based clotting assays omit the influence of blood flow, blood cells, and the damaged vessel wall. To address this gap, we are developing a model of laser-induced clot formation under flow, in tissue engineered blood vessels, utilizing human proteins and cells. Our goal is to develop a system allowing the study of coagulation and platelet accumulation under physiological flow and cellular environments.MethodsBlood vessels (150 um in diameter) were formed in microfluidic devices by seeding endothelial cells in a tubular void within a collagen I matrix and culturing under flow conditions. A variety of human microvascular endothelial cells types were tested. Normal human donors provided blood with informed consent. To measure fibrin and platelet accumulation, blood was spiked with fluorescently-labelled fibrinogen and calcein-AM. Laser ablations were performed with a 532 nm pulse laser and time lapse images of clot formation were captured using widefield fluorescence microscopy.ResultsWhile most endothelial cell types tested could form blood vessels, only brain endothelial cells, with supporting brain pericytes seeded in the collagen I matrix, were able to reproducibly form blood vessels capable of supporting blood flow without leakage. Brain endothelial cell blood vessels could support blood flow for greater than 45 minutes. Upon laser ablation, and after occasional red blood cell leakage, fibrin(ogen) and platelet accumulation occurred at the site of injury. Clots began forming shortly after ablation (2.1 ± 1.2 minutes until platelet accumulation and 0.5 ± 0.4 minutes until fibrin(ogen) accumulation) and maximum platelet accumulation was reached 12.3 ± 3.2 minutes post-ablation. Overall clot architecture mirrors that reported with in vivo laser injury models where fibrin(ogen) accumulates at or outside the vessel wall and platelets form a primary plug with limited intravascular accumulation.ConclusionsTissue engineered blood vessels composed of brain endothelial cells with supporting pericytes in microfluidic devices can support blood flow and laser-induced clot formation. Clots that form have a similar architecture to clots formed in vivo . This model, which combines blood plasma and cells with blood flow and the damaged vessel wall, has the potential to facilitate investigation of the procoagulant activity of hemostatic drugs in a physiologic, all human system. DisclosuresKistanova:Bioverativ: Employment, Equity Ownership. Aleman:Bioverativ: Employment, Equity Ownership. Salas:Bioverativ: Employment, Equity Ownership.

Role of hydrogen peroxide in intra-operative wound preparation based on an in vitro fibrin clot degradation model

Three per cent hydrogen peroxide (H2O2) is widely used to irrigate acute and chronic wounds in the surgical setting and clinical experience tells us that it is more effective at removing dried-on blood than normal saline alone. We hypothesise that this is due to the effect of H2O2 on fibrin clot architecture via fibrinolysis. We investigate the mechanisms and discuss the clinical implications using an in vitro model. Coagulation assays with normal saline (NaCl), 1% and 3% concentrations of H2O2 were performed to determine the effect on fibrin clot formation. These effects were confirmed by spectrophotometry. The effects of 1%, 3% and 10% H2O2 on the macroscopic and microscopic features of fibrin clots were assessed at set time intervals and compared to a NaCl control. Quantitative analysis of fibrin networks was undertaken to determine the fibre length, diameter, branch point density and pore size. Fibrin clots immersed in 1%, 3% and 10% H2O2 demonstrated volume losses of 0.09-0.25mm3/min, whereas those immersed in the normal saline gained in volume by 0.02±0.13 mm3/min. Quantitative analysis showed that H2O2 affects the structure of the fibrin clot in a concentration-dependent manner, with the increase in fibre length, diameter and consequently pore sizes. Our results support our hypothesis that the efficacy of H2O2 in cleaning blood from wounds is enhanced by its effects on fibrin clot architecture in a concentration- and time-dependent manner. The observed changes in fibre size and branch point density suggest that H2O2 is acting on the quaternary structure of the fibrin clot, most likely via its effect on cross-linking of the fibrin monomers and may therefore be of benefit for the removal of other fibrin-dependent structures such as wound slough.

Self-assembled fibrinogen nanofibers support fibroblast adhesion and prevent E. coli infiltration

Fibrinogen nanofibers hold great potential for wound healing applications since they mimic the native blood clot architecture and offer important binding sites to support fibroblast adhesion and migration. Recently, we introduced a new method of salt-induced self-assembly to prepare nanofibrous fibrinogen scaffolds. Here, we present our results on the mechanical properties of these scaffolds and their interaction with 3T3 fibroblasts and E. coli bacteria, which we used as model systems for wound healing. Hydrated, nanofibrous fibrinogen scaffolds showed a Young's modulus of 1.3 MPa, which is close to the range of native fibrin. 3T3 fibroblasts adhered and proliferated well on nanofibrous and planar fibrinogen up to 72 h with a less pronounced actin cytoskeleton on nanofibers in comparison to planar fibrinogen. Fibroblasts on nanofibers were smaller with many short filopodia while larger cells with few long filopodia were found on planar fibrinogen. Live cell tracking revealed higher migration velocities on nanofibers in comparison to planar fibrinogen. The growth of E. coli bacteria on nanofibrous fibrinogen was significantly reduced as compared to agar controls with no bacteria migrating through the nanofibers. In summary, we conclude that self-assembled fibrinogen nanofibers could become highly attractive as future scaffolds for wound healing applications.

Principles of Fibrinogen Fiber Assembly In Vitro.

Fibrinogen nanofibers hold great potential for applications in wound healing and personalized regenerative medicine due to their ability to mimic the native blood clot architecture. Although versatile strategies exist to induce fibrillogenesis of fibrinogen in vitro, little is known about the underlying mechanisms and the associated length scales. Therefore, in this manuscript the current state of research on fibrinogen fibrillogenesis in vitro is reviewed. For the first time, the manifold factors leading to the assembly of fibrinogen molecules into fibers are categorized considering three main groups: substrate interactions, denaturing and non-denaturing buffer conditions. Based on the meta-analysis in the review it is concluded that the assembly of fibrinogen is driven by several mechanisms across different length scales. In these processes, certain buffer conditions, in particular the presence of salts, play a predominant role during fibrinogen self-assembly compared to the surface chemistry of the substrate material. Yet, to tailor fibrous fibrinogen scaffolds with defined structure-function-relationships for future tissue engineering applications, it still needs to be understood which particular role each of these factors plays during fiber assembly. Therefore, the future combination of experimental and simulation studies is proposed to understand the intermolecular interactions of fibrinogen, which induce the assembly of soluble fibrinogen into solid fibers.

Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots.

Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.

Clot composition in retrieved thrombi after mechanical thrombectomy in strokes due to carotid web

BackgroundThe association of carotid webs (CaW) and ischemic stroke is being increasingly recognized. Data on the histologic clot architecture in strokes caused by CaW has not been previously described. Understanding...

Neutrophils can promote clotting via FXI and impact clot structure via neutrophil extracellular traps in a distinctive manner in vitro

Neutrophils and neutrophil extracellular traps (NETs) have been shown to be involved in coagulation. However, the interactions between neutrophils or NETs and fibrin(ogen) in clots, and the mechanisms behind these interactions are not yet fully understood. In this in vitro study, the role of neutrophils or NETs on clot structure, formation and dissolution was studied with a combination of confocal microscopy, turbidity and permeation experiments. Factor (F)XII, FXI and FVII-deficient plasmas were used to investigate which factors may be involved in the procoagulant effects. We found both neutrophils and NETs promote clotting in plasma without the addition of other coagulation triggers, but not in purified fibrinogen, indicating that other factors mediate the interaction. The procoagulant effects of neutrophils and NETs were also observed in FXII- and FVII-deficient plasma. In FXI-deficient plasma, only the procoagulant effects of NETs were observed, but not of neutrophils. NETs increased the density of clots, particularly in the vicinity of the NETs, while neutrophils-induced clots were less stable and more porous. In conclusion, NETs accelerate clotting and contribute to the formation of a denser, more lysis resistant clot architecture. Neutrophils, or their released mediators, may induce clotting in a different manner to NETs, mediated by FXI.

Gingipain R1 and Lipopolysaccharide From Porphyromonas gingivalis Have Major Effects on Blood Clot Morphology and Mechanics.

Background: Porphyromonas gingivalis and its inflammagens are associated with a number of systemic diseases, such as cardiovascular disease and type 2 diabetes (T2DM). The proteases, gingipains, have also recently been identified in the brains of Alzheimer's disease patients and in the blood of Parkinson's disease patients. Bacterial inflammagens, including lipopolysaccharides (LPSs) and various proteases in circulation, may drive systemic inflammation.Methods: Here, we investigate the effects of the bacterial products LPS from Escherichia coli and Porphyromonas gingivalis, and also the P. gingivalis gingipain [recombinant P. gingivalis gingipain R1 (RgpA)], on clot architecture and clot formation in whole blood and plasma from healthy individuals, as well as in purified fibrinogen models. Structural analysis of clots was performed using confocal microscopy, scanning electron microscopy, and AFM-Raman imaging. We use thromboelastography® (TEG®) and rheometry to compare the static and dynamic mechanical properties of clots.Results: We found that these inflammagens may interact with fibrin(ogen) and this interaction causes anomalous blood clotting.Conclusions: These techniques, in combination, provide insight into the effects of these bacterial products on cardiovascular health, and particularly clot structure and mechanics.

Actin is associated with tissue injury in trauma patients and produces a hypercoagulable profile in vitro.

While tissue injury provokes fibrinolysis shutdown in trauma, the mechanism remains elusive. Cellular death causes release of structural proteins, including actin and myosin, which may interact with clot formation and structure. We hypothesized that tissue injury is associated with high circulating actin and that actin produces a hypercoagulable profile with decreased fibrinolysis in vitro. Blood was collected from trauma activation patients at a single Level I trauma center for thrombelastography and proteomics. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry using isotope-labeled standards for quantification of actin and its endogenous inhibitor gelsolin. Based on the results, we added physiologic concentrations of cytoskeletal G-actin to whole blood from healthy volunteers and analyzed changes in thrombelastography, as well as to plasma and examined clot architecture using confocal microscopy of fluorescently labeled fibrinogen. Overall, 108 trauma patients were included: majority (71%) men, median age of 32.7 years, 66% blunt mechanism, median New Injury Severity Score (NISS) of 41. Compared with patients without severe tissue injury (NISS < 15, n = 10), patients with severe tissue injury (NISS > 15, n = 98) had higher levels of circulating actin (0.0428 vs. 0.0301, p = 0.02). Further, there was a trend toward lower gelsolin levels in patients with fibrinolysis shutdown (0.1844 vs. 0.2052, p = 0.17) and tissue plasminogen activator resistance (0.1676 vs. 0.2188, p = 0.06).Ten healthy volunteers were included in the in vitro experiments (50% male; median age, 31.3 years). Actin significantly increased angle (40.0° to 52.9°, p = 0.002) and decreased fibrinolysis (percent clot lysis 30 minutes after reaching maximum amplitude, 4.0% to 1.6%; p = 0.002), provoking fibrinolytic shutdown in three patients. The addition of actin to control plasma decreased fiber resolvability of fibrin clots, monitored by microscopy, and decreased plasmin-mediated fibrinolysis. Actin increases clot propagation and provokes fibrinolysis shutdown in vitro, through a mechanism of plasmin inhibition. High circulating levels of actin are present in trauma patients with severe tissue injury, suggesting actin contributes to fibrinolysis shutdown in the setting of tissue injury.

Dense and dangerous: The tissue plasminogen activator-resistant fibrinolysis shutdown phenotype is due to abnormal fibrin polymerization

Both hyperfibrinolysis and fibrinolysis shutdown can occur after severe trauma. The subgroup of trauma patients with fibrinolysis shutdown resistant to tissue plasminogen activator (t-PA)-mediated fibrinolysis have increased mortality. Fibrin polymerization and structure may influence fibrinolysis subgroups in trauma, but fibrin architecture has not been characterized in acutely injured subjects. We hypothesized that fibrin polymerization measured in situ will correlate with fibrinolysis subgroups. Blood samples were collected from trauma patients and noninjured controls. We selected samples across a range of fibrinolysis phenotypes (shutdown, physiologic, hyperfibrinolysis) and t-PA sensitivities (sensitive, physiologic, resistant) determined by thrombelastography. Plasma clots were created in situ with fluorescent fibrinogen and imaged using confocal microscopy for analysis of clot architecture in three dimensions. For each clot, we quantified the fiber resolvability, a metric of fiber distinctness or clarity, by mapping the variance of fluorescence intensity relative to background fluorescence. We also determined clot porosity by measuring the size and distribution of the gaps between fibrin fibers in three-dimensional space. We compared these measures across fibrinolysis subgroups. Fiber resolvability was significantly lower in all trauma subgroups compared with controls (n = 35 and 5, respectively; p < 0.05). We observed markedly different patterns of fibrin architecture among trauma patients stratified by fibrinolysis subgroup. Subjects with t-PA-resistant fibrinolysis shutdown exhibited abnormal, densely packed fibrin clots nearly devoid of pores. Individuals with t-PA-hypersensitive fibrinolysis shutdown had highly irregular clots with pores as large as 2500 μm to 20,000 μm, versus 78 μm to 1250 μm in noninjured controls. Fiber resolvability was significantly lower in trauma patients than controls, and subgroups of fibrinolysis differ in the porosity of the fibrin clot structure. The dense fibrin network in the t-PA-resistant group may prevent access to plasmin, suggesting a mechanism for thrombotic morbidity after injury.

Is the endothelial cell responsible for the thrombus core and shell architecture?

Ischemia leading to heart attacks and strokes is the major cause of deaths in the world. This report explores the possibility that intracellular material from ruptured endothelial cells is partially responsible for the heterogeneous core-and-shell blood clot architecture, typically observed using intravital microscopy. As evidence, we present a fluid dynamic argument that platelet agonists emanating from the injury cannot activate platelets in the thrombus core, given that they would have to travel against flow of blood escaping into the extravascular. Furthermore, we demonstrate visual evidence that the core material appears to be continuous and originating from the damaged endothelium. Finally, we present a mechanism, illustrating the steps of platelet recruitment into the thrombus and sealing of the injury. If correct, the model presented herein will be beneficial to the understanding and treatment of heart attacks, strokes and hemophilia.

Microscopic evaluation of the effect of low-level laser therapy on platelet-rich fibrin: A light microscopic histological study

Background: This study evaluated and compared the concentration of platelets/leukocytes, fibrin matrix, and its distribution in nonlaser-treated platelet-rich fibrin (PRF) and laser-treated PRF. Recently, PRF has been used for regeneration of soft and hard tissue, in periodontics and oral surgery. Low-level laser therapy (LLLT) has been used for many therapeutic purpose of tissue healing. The use of PRF treated with LLLT has shown to enhance better healing potential. Materials and Methods: In this study, we have selected five healthy controls and collected 10 ml of blood from them. All blood samples were centrifuged, and from each blood sample, two PRF clots were formed. A total of ten samples obtained were divided into two groups. Each group had five samples. One group was without LLL treatment (Group I) and other was treated with LLL (Group II). All the ten samples were processed for light microscopic examination. Results: In both the groups, the PRF membrane showed a similar macroscopic structure and microscopic distribution. In the buffy coat region, the concentration of platelets–leukocyte aggregate was little higher in Group I. In Group II, the distribution pattern of fibrin clot was slightly different with that of Group I. Conclusion: The Choukroun's PRF concept leads to specific clot architecture with platelets and fibrin meshwork, especially in the buffy coat region. Changes in the PRF components with LLLT may have an affect the healing and regenerative of soft and hard tissues.