RESUMO
ABSTRACT: Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5'-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.
Assuntos
Plaquetas , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas , Ligação Proteica , Multimerização Proteica , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/química , Humanos , Plaquetas/metabolismo , Ligantes , Agregação Plaquetária/efeitos dos fármacos , Sítios de Ligação , Colágeno/metabolismo , Colágeno/química , Proteínas de Transporte , PeptídeosRESUMO
BACKGROUND: The glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown. OBJECTIVES: To determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure. METHODS: Procoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide. RESULTS: GPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired. CONCLUSION: Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbß3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.
Assuntos
Fibrina , Trombose , Animais , Camundongos , Plaquetas/metabolismo , Eptifibatida/farmacologia , Fibrina/química , Glicoproteínas da Membrana de Plaquetas/metabolismoRESUMO
Fibrin polymerization involves thrombin-mediated exposure of knobs on one monomer that bind to holes available on another, leading to the formation of fibers. In silico evidence has suggested that the classical A:a knob-hole interaction is enhanced by surrounding residues not directly involved in the binding pocket of hole a, via noncovalent interactions with knob A. We assessed the importance of extended knob-hole interactions by performing biochemical, biophysical, and in silico modeling studies on recombinant human fibrinogen variants with mutations at residues responsible for the extended interactions. Three single fibrinogen variants, γD297N, γE323Q, and γK356Q, and a triple variant γDEK (γD297N/γE323Q/γK356Q) were produced in a CHO (Chinese Hamster Ovary) cell expression system. Longitudinal protofibril growth probed by atomic force microscopy was disrupted for γD297N and enhanced for the γK356Q mutation. Initial polymerization rates were reduced for all variants in turbidimetric studies. Laser scanning confocal microscopy showed that γDEK and γE323Q produced denser clots, whereas γD297N and γK356Q were similar to wild type. Scanning electron microscopy and light scattering studies showed that fiber thickness and protofibril packing of the fibers were reduced for all variants. Clot viscoelastic analysis showed that only γDEK was more readily deformable. In silico modeling suggested that most variants displayed only slip-bond dissociation kinetics compared with biphasic catch-slip kinetics characteristics of wild type. These data provide new evidence for the role of extended interactions in supporting the classical knob-hole bonds involving catch-slip behavior in fibrin formation, clot structure, and clot mechanics.
Assuntos
Fibrina , Trombose , Animais , Células CHO , Cricetinae , Cricetulus , Fibrina/metabolismo , Fibrinogênio/metabolismo , Humanos , Trombina/metabolismoRESUMO
Fibrinogen is essential for blood coagulation. The C-terminus of the fibrinogen α-chain (αC-region) is composed of an αC-domain and αC-connector. Two recombinant fibrinogen variants (α390 and α220) were produced to investigate the role of subregions in modulating clot stability and resistance to lysis. The α390 variant, truncated before the αC-domain, produced clots with a denser structure and thinner fibres. In contrast, the α220 variant, truncated at the start of the αC-connector, produced clots that were porous with short, stunted fibres and visible fibre ends. These clots were mechanically weak and susceptible to lysis. Our data demonstrate differential effects for the αC-subregions in fibrin polymerisation, clot mechanical strength, and fibrinolytic susceptibility. Furthermore, we demonstrate that the αC-subregions are key for promoting longitudinal fibre growth. Together, these findings highlight critical functions of the αC-subregions in relation to clot structure and stability, with future implications for development of novel therapeutics for thrombosis.
Assuntos
Coagulação Sanguínea/fisiologia , Fibrinogênio/química , Fibrinogênio/metabolismo , Fibrinólise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Animais , Células CHO , Cricetulus , Fibrina/química , Humanos , Camundongos Knockout , Proteínas Recombinantes/químicaRESUMO
INTRODUCTION: Coronary artery disease is associated with impaired clot structure. The aim of this study was to investigate acute phase myocardial infarction (AMI) and provide detailed quantitative analysis of clot ultrastructure. MATERIALS AND METHODS: Clot formation and breakdown, pore size, fiber density, fiber radius and protofibril packing were investigated in plasma clots from AMI patients. These data were compared to those from healthy controls. RESULTS: Analysis on clot formation using turbidity showed increased lag time, suggesting changes in protofibril packing and increased fiber size for AMI patients compared to healthy controls. Additionally, increased average rate of clotting and decreased time to maximum absorbance in AMI patients suggest that clots formed more quickly. Moreover, we observed increased time from max OD to max rate of lysis. Increased fibrinogen and decreased plasminogen in AMI patients were accounted for in represented significant differences. AMI samples showed increased time to 25% and 50% lysis, but no change in 75% lysis, representative of delayed lysis onset, but expediated lysis once initiated. These data suggest that AMI patients formed less porous clots made from more densely packed fibers with decreased numbers of protofibrils, which was confirmed using decreased permeation and increased fiber density, and decreased turbidimetry. CONCLUSIONS: AMI plasma formed clots that were denser, less permeable, and lysed more slowly than healthy controls. These findings were confirmed by detailed analysis of clot ultrastructure, fiber size, and protofibril packing. Dense clot structures that are resistant to lysis may contribute to a prothrombotic milieu in AMI.
Assuntos
Infarto do Miocárdio , Trombose , Coagulação Sanguínea , Fibrina , Tempo de Lise do Coágulo de Fibrina , Fibrinólise , Humanos , Rádio (Anatomia)RESUMO
Thrombosis is a major complication of cardiovascular disease, leading to myocardial infarction, acute ischemic stroke, or venous thromboembolism. Thrombosis occurs when a thrombus forms inside blood vessels disrupting blood flow. Developments in thrombectomy to remove thrombi from vessels have provided new opportunities to study thrombus composition which may help to understand mechanisms of disease and underpin improvements in treatments. We aimed to review thrombus compositions, roles of components in thrombus formation and stability, and methods to investigate thrombi. Also, we summarize studies on thrombus structure obtained from cardiovascular patients and animal models. Thrombi are composed of fibrin, red blood cells, platelets, leukocytes, and neutrophil extracellular traps. These components have been analyzed by several techniques, including scanning electron microscopy, laser scanning confocal microscopy, histochemistry, and immunohistochemistry; however, each technique has advantages and limitations. Thrombi are heterogenous in composition, but overall, thrombi obtained from myocardial infarction are composed of mainly fibrin and other components, including platelets, red blood cells, leukocytes, and cholesterol crystals. Thrombi from patients with acute ischemic stroke are characterized by red blood cell- and platelet-rich regions. Thrombi from patients with venous thromboembolism contain mainly red blood cells and fibrin with some platelets and leukocytes. Thrombus composition from patients with myocardial infarction is influenced by ischemic time. Animal thrombosis models are crucial to gain further mechanistic information about thrombosis and thrombus structure, with thrombi being similar in composition compared with those from patients. Further studies on thrombus composition and function are key to improve treatment and clinical outcome of thrombosis.
Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Eritrócitos/metabolismo , Fibrina/metabolismo , Trombose/metabolismo , Animais , Plaquetas/patologia , Colesterol/metabolismo , Modelos Animais de Doenças , Eritrócitos/patologia , Humanos , Leucócitos/metabolismo , Trombectomia , Trombose/patologia , Trombose/terapiaRESUMO
The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.
Assuntos
Reagentes de Ligações Cruzadas/química , Fator XIIIa/metabolismo , Fibrinogênio/metabolismo , Embolia Pulmonar/etiologia , Embolia Pulmonar/patologia , Veia Cava Inferior/patologia , Trombose Venosa/complicações , Animais , Coagulação Sanguínea , Plaquetas/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Imagem Óptica , Embolia Pulmonar/sangue , Trombose Venosa/sangueRESUMO
OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Fibrinogênio/metabolismo , Fragmentos de Peptídeos/sangue , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/química , Humanos , Técnicas In Vitro , Camundongos , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Peptídeos/química , Peptídeos/metabolismo , Agregação Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Transdução de Sinais , Ressonância de Plasmônio de SuperfícieRESUMO
Vascular Dementia (VaD) is a neurocognitive disorder caused by reduced blood flow to the brain tissue, resulting in infarction, and is the second most common type of dementia. The complement and coagulation systems are evolutionary host defence mechanisms activated by acute tissue injury to induce inflammation, clot formation and lysis; recent studies have revealed that these systems are closely interlinked. Overactivation of these systems has been recognised to play a key role in the pathogenesis of neurological disorders such as Alzheimer's disease and multiple sclerosis, however their role in VaD has not yet been extensively reviewed. This review aims to bridge the gap in knowledge by collating current understanding of VaD to enable identification of complement and coagulation components involved in the pathogenesis of this disorder that may have their effects amplified or supressed by crosstalk. Exploration of these mechanisms may unveil novel therapeutic targets or biomarkers that would improve current treatment strategies for VaD.
RESUMO
BACKGROUND: Fibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α- and ß-chains N-termini (GPR, GHR; A-, B-knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' γ- and (B)ß-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 â S) with nonengaging GPS A-knobs. By analogy, a similar Bß-chain mutation, BßR17 â S, should produce nonengaging GHS B-knobs. A homozygous "Double-Detroit" mutant (AαR19 â S, BßR17 â 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 Bß-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.
Assuntos
Fibrina , Fibrinogênio , Fibrinogênio/genética , Humanos , Fragmentos de Peptídeos , Polimerização , TrombinaRESUMO
Dermatofibromas are common benign skin lesions, the etiology of which is poorly understood. We identified two unrelated pedigrees in which there was autosomal dominant transmission of multiple dermatofibromas. Whole exome sequencing revealed a rare shared heterozygous missense variant in the F13A1 gene encoding factor XIII subunit A (FXIII-A), a transglutaminase involved in hemostasis, wound healing, tumor growth, and apoptosis. The variant (p.Lys679Met) has an allele frequency of 0.0002 and is predicted to be a damaging mutation. Recombinant human Lys679Met FXIII-A demonstrated reduced fibrin crosslinking activity in vitro. Of note, the treatment of fibroblasts with media containing Lys679Met FXIII-A led to enhanced adhesion, proliferation, and type I collagen synthesis. Immunostaining revealed co-localization between FXIII-A and α4ß1 integrins, more prominently for Lys679Met FXIII-A than the wild type. In addition, both the α4ß1 inhibitors and the mutation of the FXIII-A Isoleucine-Leucine-Aspartate-Threonine (ILDT) motif prevented Lys679Met FXIII-A-dependent proliferation and collagen synthesis of fibroblasts. Our data suggest that the Lys679Met mutation may lead to a conformational change in the FXIII-A protein that enhances α4-integrin binding and provides insight into an unexpected role for FXIII-A in the pathobiology of familial dermatofibroma.
Assuntos
Fator XIII/genética , Fibrina/metabolismo , Histiocitoma Fibroso Benigno/genética , Padrões de Herança , Pele/patologia , Domínio Catalítico/genética , Proliferação de Células/genética , Colágeno Tipo I/biossíntese , Análise Mutacional de DNA , Fator XIII/metabolismo , Feminino , Fibroblastos , Células HEK293 , Histiocitoma Fibroso Benigno/patologia , Humanos , Integrina alfa4/metabolismo , Masculino , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Linhagem , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/citologia , Relação Estrutura-Atividade , Sequenciamento do ExomaRESUMO
Essentials Venous thromboembolism (VTE) recurrence leads to decreased clot elastic modulus in plasma. Recurrent VTE is not linked to changes in clot structure, fiber radius, or factor XIII activity. Other plasma components may play a role in VTE recurrence. Prospective studies should resolve if clot stiffness can be used as predictor for recurrent VTE. SUMMARY: Background Venous thromboembolism (VTE) is associated with a high risk of recurrent events after withdrawal of anticoagulation. Objectives To determine the difference in plasma clot mechanical properties between patients with recurrent VTE (rVTE) and those with non-recurrent VTE (nrVTE). Methods We previously developed a system for determining clot mechanical properties by use of an in-house magnetic tweezers system. This system was used to determine the mechanical properties of clots made from plasma of 11 patients with rVTE and 33 with nrVTE. Plasma was mixed with micrometer-sized beads, and thrombin and calcium were added to induce clotting; the mixture was then placed in small capillary tubes, and clotting was allowed to proceed overnight. Bead displacements upon manipulation with magnetic forces were analyzed to determine clot elastic and viscous moduli. Fibrin clot structure was analyzed with turbidimetry and confocal microscopy. Factor XIII was measured by pentylamine incorporation into fibrin. Results Clots from rVTE patients showed nearly two-fold less elastic and less viscous moduli than clots from nrVTE patients, regardless of male sex, unprovoked events, family history of VTE, fibrinogen concentration, or body mass index. No differences were observed in clot structure, fibrinolysis rates, or FXIII levels. Conclusion Using magnetic tweezers for the first time in patient samples, we found that plasma clots from rVTE patients showed a reduced elastic modulus and a reduced viscous modulus as compared with clots from nrVTE patients. These data indicate a possible role for fibrin clot viscoelastic properties in determining VTE recurrence.
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Fibrina/metabolismo , Hemostasia , Tromboembolia Venosa/sangue , Adolescente , Adulto , Cálcio/sangue , Módulo de Elasticidade , Fator VIII/metabolismo , Feminino , Hemorreologia , Humanos , Imãs , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Nefelometria e Turbidimetria , Recidiva , Reologia/instrumentação , Trombina/metabolismo , Tromboembolia Venosa/diagnóstico , Viscosidade , Adulto JovemAssuntos
Afibrinogenemia/sangue , Afibrinogenemia/diagnóstico , Fator XIII/metabolismo , Afibrinogenemia/etiologia , Afibrinogenemia/terapia , Biomarcadores , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Ativação Enzimática , Terapia de Reposição de Enzimas , Fator XIII/imunologia , Fibrinogênio/administração & dosagem , Fibrinogênio/efeitos adversos , Fibrinogênio/uso terapêutico , Humanos , Resultado do TratamentoRESUMO
AIM: γ' fibrinogen has been associated with thrombosis. Here the interactions between γ'γ' or γAγA fibrinogen and red blood cells (RBCs), and their role on fibrin clot properties were studied. MATERIALS & METHODS: Atomic Force microscopy (AFM)-based force spectroscopy, rheological, electron and confocal microscopy, and computational approaches were conducted for both fibrinogen variants. RESULTS & CONCLUSION: AFM shows that the recombinant human (rh)γ'γ' fibrinogen increases the binding force and the frequency of the binding to RBCs compared with rhγAγA, promoting cell aggregation. Structural changes in rhγ'γ' fibrin clots, displaying a nonuniform fibrin network were shown by microscopy approaches. The presence of RBCs decreases the fibrinolysis rate and increases viscosity of rhγ'γ' fibrin clots. The full length of the γ' chain structure, revealed by computational analysis, occupies a much wider surface and is more flexible, allowing an increase of the binding between γ' fibers, and eventually with RBCs.
Assuntos
Fibrina/metabolismo , Fibrinogênios Anormais/administração & dosagem , Tromboembolia/tratamento farmacológico , Trombose/tratamento farmacológico , Coagulação Sanguínea/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Fibrina/ultraestrutura , Fibrinogênios Anormais/química , Fibrinogênios Anormais/genética , Fibrinólise/efeitos dos fármacos , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Conformação Proteica , Reologia , Tromboembolia/patologia , Trombose/sangue , Trombose/patologia , ViscosidadeRESUMO
Hemostasis requires conversion of fibrinogen to fibrin fibers that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining clots in human and mouse models. We demonstrated that only fibrin is required for formation of the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibers. It was digested by plasmin, and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion.
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Bactérias/genética , Fenômenos Fisiológicos Bacterianos , Biofilmes , Coagulação Sanguínea , Fibrina/metabolismo , Dermatopatias Bacterianas/metabolismo , Animais , Bactérias/patogenicidade , Modelos Animais de Doenças , Humanos , Camundongos , Dermatopatias Bacterianas/microbiologiaRESUMO
BACKGROUND: Erythrocyte aggregation, a cardiovascular risk factor, is increased by high plasma fibrinogen levels. Here, the effect of different fibrinogen mutations on binding to its human erythrocyte receptor was assessed in order to identify the interaction sites. METHODS: Three fibrinogen variants were tested, specifically mutated in their putative integrin recognition sites on the Aα chain (mutants D97E, D574E and D97E/D574E) and compared with wild-type fibrinogen. RESULTS: Atomic force microscopy-based force spectroscopy measurements showed a significant decrease both on the fibrinogen-erythrocyte binding force and on its frequency for fibrinogen with the D97E mutation, indicating that the corresponding arginine-glycine-aspartate sequence (residues 95-97) is involved in this interaction, and supporting that the fibrinogen receptor on erythrocytes has a ß3 subunit. Changes in the fibrin clot network structure obtained with the D97E mutant were observed by scanning electron microscopy. CONCLUSION: These findings may lead to innovative perspectives on the development of new therapeutic approaches to overcome the risks of fibrinogen-driven erythrocyte hyperaggregation.
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Eritrócitos/metabolismo , Fibrinogênio/metabolismo , Receptores de Fibrinogênio/metabolismo , Sítios de Ligação , Fibrina/metabolismo , Fibrinogênio/genética , Humanos , Integrinas/metabolismo , Microscopia de Força Atômica , Mutação , Ligação Proteica , Trombose/metabolismoRESUMO
Fibrin is an important matrix protein that provides the backbone to the blood clot, promoting tissue repair and wound healing. Its precursor fibrinogen is one of the most heterogeneous proteins, with an estimated 1 million different forms due to alterations in glycosylation, oxidation, single nucleotide polymorphisms, splice variation and other variations. Furthermore, ligation by transglutaminase factor XIII (cross-linking) adds to the complexity of the fibrin network. The structure and function of the fibrin network is in part determined by this natural variation in the fibrinogen molecule, with major effects from splice variation and cross-linking. This mini-review will discuss the direct effects of fibrinogen αEC and fibrinogen γ' splice variation on clot structure and function and also discuss the additional role of fibrinogen γ' as thrombomodulin II. Furthermore, the effects of cross-linking on clot function will be described. Splice variation and cross-linking are major determinants of the structure and function of fibrin and may therefore impact on diseases affecting bleeding, thrombosis and tissue repair.
Assuntos
Fator XIII/metabolismo , Fibrina/metabolismo , Fibrinogênios Anormais/metabolismo , Processamento de Proteína Pós-Traducional , Trombomodulina/metabolismo , Processamento Alternativo , Coagulação Sanguínea/fisiologia , Reagentes de Ligações Cruzadas , Fator XIII/química , Fator XIII/genética , Fibrina/química , Fibrina/genética , Fibrinogênios Anormais/química , Fibrinogênios Anormais/genética , Expressão Gênica , Glicosilação , Humanos , Polimorfismo Genético , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trombomodulina/química , Trombomodulina/genética , Cicatrização/fisiologiaRESUMO
OBJECTIVE: Factor XIII (FXIII) cross-links fibrin upon activation by thrombin. Activation involves cleavage at residue 37 by thrombin, releasing an activation peptide. A common polymorphism (valine to leucine variant at residue 34, V34L), located in the activation peptide, has been associated with increased activation rates and paradoxically a protective effect in cardiovascular disease. There is, currently, no data available on the effects of V34L from in vivo models of thrombosis. We examined the effect of FXIII V34L on clot formation and cross-linking in vivo. APPROACH AND RESULTS: We generated a panel of full-length recombinant human FXIII-A2 variants with amino acid substitutions in the activation peptide to investigate the effect of these variants on activation rate, and we used wild-type, V34L, and alanine to glycine variant at residue 33 variants to study the effects of varying FXIII activation rate on thrombus formation in a murine model of FeCl3 injury. FXIII activation assay showed that residues 29, 30, 33, and 34 play a critical role in thrombin interaction. Full-length recombinant human FXIII-A2 V34L has significant effects on clot formation, structure, and lysis in vitro, using turbidity assay. This variant influenced fibrin cross-linking but not size of the thrombus in vivo. CONCLUSIONS: Mutations in the activation peptide of full-length recombinant FXIII regulate activation rates by thrombin, and V34L influences in vivo thrombus formation by increased cross-linking of the clot.
Assuntos
Coagulação Sanguínea , Fator XIIIa/metabolismo , Fibrina/metabolismo , Trombina/metabolismo , Trombose Venosa/metabolismo , Substituição de Aminoácidos , Animais , Testes de Coagulação Sanguínea , Modelos Animais de Doenças , Deficiência do Fator XIII/sangue , Deficiência do Fator XIII/genética , Deficiência do Fator XIII/metabolismo , Fator XIIIa/administração & dosagem , Fator XIIIa/genética , Genótipo , Humanos , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos CBA , Camundongos Knockout , Mutação , Fenótipo , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Trombose Venosa/sangue , Trombose Venosa/genéticaRESUMO
Previous studies have shown effects of thrombin and fibrinogen γ' on clot structure. However, structural information was obtained using electron microscopy, which requires sample dehydration. Our aim was to investigate the role of thrombin and fibrinogen γ' in modulating fibrin structure under fully hydrated conditions. Fibrin fibers were studied using turbidimetry, atomic force microscopy, electron microscopy, and magnetic tweezers in purified and plasma solutions. Increased thrombin induced a pronounced decrease in average protofibril content per fiber, with a relatively minor decrease in fiber size, leading to the formation of less compact fiber structures. Atomic force microscopy under fully hydrated conditions confirmed that fiber diameter was only marginally decreased. Decreased protofibril content of the fibers produced by high thrombin resulted in weakened clot architecture as analyzed by magnetic tweezers in purified systems and by thromboelastometry in plasma and whole blood. Fibers produced with fibrinogen γ' showed reduced protofibril packing over a range of thrombin concentrations. High-magnification electron microscopy demonstrated reduced protofibril packing in γ' fibers and unraveling of fibers into separate protofibrils. Decreased protofibril packing was confirmed in plasma for high thrombin concentrations and fibrinogen-deficient plasma reconstituted with γ' fibrinogen. These findings demonstrate that, in fully hydrated conditions, thrombin and fibrinogen γ' have dramatic effects on protofibril content and that protein density within fibers correlates with strength of the fibrin network. We conclude that regulation of protofibril content of fibers is an important mechanism by which thrombin and fibrinogen γ' modulate fibrin clot structure and strength.