fibrin and ferric-chloride

The hemostatic process is a host defense mechanism to preserve the integrity of the closed high pressure circulatory system. This process must remain inactive but poised to minimize extravasation of blood from the vasculature following tissue injury. Given the complexity of the hemostatic mechanism, paradigms developed from biochemical and cell biological approaches have been revisited by studying thrombus formation in a live animal by intravital microscopy. Many of these paradigms have proven accurate, but others need to be reconsidered given the results of whole animal experiments.

Topics: Animals; Blood Coagulation Factors; Cell-Derived Microparticles; Chlorides; Collagen; Disease Models, Animal; Ferric Compounds; Fibrin; Integrin beta3; Lasers; Mesentery; Mice; Mice, Knockout; Microscopy, Fluorescence; Muscle, Skeletal; Platelet Aggregation; Protein Disulfide-Isomerases; Thromboplastin; Thrombosis

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl

Topics: Animals; Antibodies; Blood Coagulation; Blood Platelets; CD11b Antigen; Cell Communication; Chlorides; Deoxyribonuclease I; Extracellular Traps; Ferric Compounds; Fibrin; Male; Mice, Inbred C57BL; Neutrophils; P-Selectin; Platelet Aggregation; Social Defeat; Thrombosis

Topics: Animals; Boronic Acids; Carotid Artery Thrombosis; CD40 Ligand; Cell Survival; Chlorides; Drug Carriers; Drug Liberation; Endothelial Cells; Ferric Compounds; Fibrin; Fibrinolytic Agents; Fluorescent Dyes; Humans; Hydrogen Peroxide; Lipopeptides; Mice; Nanoparticles; Optical Imaging; Polymers; RAW 264.7 Cells; Theranostic Nanomedicine; Thrombosis; Tirofiban; Tumor Necrosis Factor-alpha

Iron salts are used in the treatment of iron deficiency anemia. Diabetic patients are frequently anemic and treatment includes administration of iron. Anemic patients on hemodialysis are at an increased risk of thromboembolic coronary events associated with the formation of dense fibrin clots resistant to fibrinolysis. Moreover, in chronic kidney disease patients, high labile plasma iron levels associated with iron supplementation are involved in complications found in dialyzed patients such as myocardial infarction. The aim of the present study was to investigate whether iron treatment is involved in the formation of the fibrin clots. Clotting of citrated plasma supplemented with Fe(3+) was investigated by thromboelastometry and electron microscopy. The results revealed that iron modifies coagulation in a complex manner. FeCl(3) stock solution underwent gradual chemical modification during storage and altered the coagulation profile over 29 days, suggesting that Fe(3+) interacts with both proteins of the coagulation cascade as well as the hydrolytic Fe(3+) species. Iron extends clotting of plasma by interacting with proteins of the coagulation cascade. Fe(3+) and/or its hydrolytic species interact with fibrinogen and/or fibrin changing their morphology and properties. In general FeCl(3) weakens the fibrin clot while at the same time precipitating plasma proteins immediately after application. Fe(3+) or its derivatives induced the formation of insoluble coagulums in non-enzymatic reactions including albumin and transferrin. Iron plays a role in coagulation and can precipitate plasma proteins. The formation of coagulums resistant to lysis in non‑enzymatic reactions can increase the risk of thrombosis, and extending clotting of plasma can prolong bleeding.

Topics: Adult; Blood Coagulation; Calcium; Chlorides; Female; Ferric Compounds; Fibrin; Fibrinogen; Humans; Iron; Male; Microscopy, Electron; Thrombelastography; Young Adult

C-reactive protein (CRP) promotes tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression in vitro, and an elevated plasma CRP concentration is associated with an increased risk of vein graft (VG) thrombosis after coronary artery bypass surgery. However, little is known about the effects of CRP on VG TF and PAI-1 expression in vivo, or on VG thrombosis.. We studied transgenic (Tg) mice expressing human CRP in a VG model to explore in vivo cause-and-effect relationships between CRP and TF, PAI-1, and VG thrombosis.. Vein segments from wild-type (WT) and CRP-Tg donors were transplanted into carotid arteries of WT and CRP-Tg recipients. VGs were analyzed 1-4 weeks later.. Human CRP accumulated in VGs during the first 4 weeks after surgery, but appeared to originate exclusively from systemic sources, rather than local production. Human CRP significantly increased TF gene expression, protein concentration and activity in VGs. Human CRP also increased PAI-1 concentrations in VGs, although only in vascular endothelial cells. Human CRP stimulated macrophage migration, invasion into VGs, and TF expression. Fibrin deposition was significantly greater in VGs of CRP-Tg mice than in WT controls.. CRP accumulates in VGs early after surgery, originating from systemic sources rather than local synthesis. Human CRP promotes TF and PAI-1 expression in VGs, although with different expression patterns. Human CRP stimulates macrophage invasion and fibrin deposition within VGs. These results suggest that CRP induces pathologic changes in VGs that contribute to early VG occlusion.

Topics: Animals; C-Reactive Protein; Cell Movement; Chlorides; Coronary Artery Bypass; Ferric Compounds; Fibrin; Humans; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Plasminogen Activator Inhibitor 1; Recombinant Proteins; Thromboplastin; Transgenes; Veins; Venous Thrombosis

Topics: Cell Shape; Chlorides; Diabetes Mellitus; Erythrocytes; Ferric Compounds; Ferritins; Fibrin; Humans; Iron; Microscopy, Electron, Scanning; Transferrin

Individuals with elevated prothrombin, including those with the prothrombin G20210A mutation, have increased risk of venous thrombosis. Although these individuals do not have increased circulating prothrombotic biomarkers, their plasma demonstrates increased tissue factor-dependent thrombin generation in vitro. The objectives of this study were to determine the pathological role of elevated prothrombin in venous and arterial thrombosis in vivo, and distinguish thrombogenic mechanisms in these vessels.. Prothrombin was infused into mice to raise circulating levels. Venous thrombosis was induced by electrolytic stimulus to the femoral vein or inferior vena cava ligation. Arterial thrombosis was induced by electrolytic stimulus or ferric chloride application to the carotid artery. Mice infused with prothrombin demonstrated increased tissue factor-triggered thrombin generation measured ex vivo, but did not have increased circulating prothrombotic biomarkers in the absence of vessel injury. After venous injury, elevated prothrombin increased thrombin generation and the fibrin accumulation rate and total amount of fibrin ≈ 3-fold, producing extended thrombi with increased mass. However, elevated prothrombin did not accelerate platelet accumulation, increase the fibrin accumulation rate, or shorten the vessel occlusion time after arterial injury.. These findings reconcile previously discordant findings on thrombin generation in hyperprothrombinemic individuals measured ex vivo and in vitro, and show elevated prothrombin promotes venous, but not arterial, thrombosis in vivo.

Topics: Animals; Blood Coagulation; Blood Platelets; Carotid Arteries; Chlorides; Disease Models, Animal; Femoral Vein; Ferric Compounds; Fibrin; Humans; Mice; Noxae; Prothrombin; Risk Factors; Thrombophilia; Vena Cava, Inferior; Venous Thrombosis

Blood coagulation under physiological conditions is activated by thrombin, which converts soluble plasma fibrinogen (FBG) into an insoluble clot. The structure of the enzymatically-generated clot is very characteristic being composed of thick fibrin fibers susceptible to the fibrinolytic degradation. However, in chronic degenerative diseases, such as atherosclerosis, diabetes mellitus, cancer, and neurological disorders, fibrin clots are very different forming dense matted deposits (DMD) that are not effectively removed and thus create a condition known as thrombosis. We have recently shown that trivalent iron (ferric ions) generates hydroxyl radicals, which subsequently convert FBG into abnormal fibrin clots in the form of DMDs. A characteristic feature of DMDs is their remarkable and permanent resistance to the enzymatic degradation. Therefore, in order to prevent thrombotic incidences in the degenerative diseases it is essential to inhibit the iron-induced generation of hydroxyl radicals. This can be achieved by the pretreatment with a direct free radical scavenger (e.g. salicylate), and as shown in this paper by the treatment with oxidizing agents such as hydrogen peroxide, methylene blue, and sodium selenite. Although the actual mechanism of this phenomenon is not yet known, it is possible that hydroxyl radicals are neutralized by their conversion to the molecular oxygen and water, thus inhibiting the formation of dense matted fibrin deposits in human blood.

Topics: Adult; Blood Coagulation; Chlorides; Female; Ferric Compounds; Fibrin; Fibrinogen; Humans; Hydrogen Peroxide; Hydroxyl Radical; Methylene Blue; Microscopy, Electron, Scanning; Oxidants; Oxidation-Reduction; Sodium Selenite; Thrombosis; Young Adult

The purpose of this study was to evaluate the influence of blood contamination before or after surface treatment on adhesion of 4-META/MMA-TBB resin. After bovine root dentin surfaces were contaminated with blood before or after dentin surface treatment with 10-3 solution, the contaminated surface was rinsed with water, air-dried, or re-treated with 10-3 solution. Dye leakage and microtensile bond strength (MTBS) of 4-META/MMA-TBB resin to dentin were measured after storage in water for 24 h. When blood contamination occurred before surface treatment, there was no significant difference in the leakage value and MTBS as compared with that of the uncontaminated group. When blood contamination occurred after surface treatment, the leakage value increased and MTBS significantly decreased (p<0.05) even if the blood was washed away. However, when the surface was re-treated with 10-3 solution after rinsing with water, the leakage value and MTBS were restored to those of the uncontaminated group.

Topics: Adhesiveness; Air; Animals; Blood; Boron Compounds; Carbon Compounds, Inorganic; Cattle; Caustics; Chlorides; Citric Acid; Coloring Agents; Dental Bonding; Dental Leakage; Dental Stress Analysis; Dentin; Erythrocytes; Ferric Compounds; Fibrin; Humans; Methacrylates; Methylmethacrylates; Microscopy, Electron, Scanning; Resin Cements; Rosaniline Dyes; Silicon Compounds; Stress, Mechanical; Surface Properties; Tensile Strength; Time Factors; Tooth Root; Water

Activation of coagulation pathways results in the formation of hemostatic fibrin plugs. Under normal physiologic conditions fibrin clots are gradually, albeit completely, degraded by a fibrinolytic enzyme system to ensure proper wound healing and/or blood vessel patency. Yet in pathological situations, thrombi are not effectively removed, leading to chronic thrombosis. The susceptibility of blood clots to enzymatic degradation depends on the structure and properties of fibrin fibers. Many factors have been suspected as culprits, including red blood cells (RBCs) that become transiently trapped within fibrin mesh. Here, the authors show that there is indeed a specific interaction between RBCs and fibrin-like fibers identified here as dense matted deposits (DMDs) by means of scanning electron microscopy (SEM). It is emphasized that such interactions can be observed in ischemic stroke patients, but not from healthy subjects. However, DMD/RBC aggregates can be induced in normal blood by the additions of trivalent iron ions. The plausible mechanism of the enhanced fibrin-red blood cell interaction is based on the previously described iron-induced generation of hydroxyl radicals. These radicals cause, in turn, non-enzymatic formation of fibrinogen aggregates remarkably resistant to fibrinolysis that are also similar to DMDs described in this paper. In conclusion, this relatively simple SEM analysis may become a convenient tool for diagnosing prothrombotic conditions associated with iron overload. It is suggested that future research on prevention and treatment of ischemic stroke and other thrombosis associated diseases should include testing of iron-chelating and hydroxyl radical-scavenging agents.

Topics: Blood Coagulation; Chlorides; Erythrocytes; Ferric Compounds; Fibrin; Fibrinogen; Fibrinolysis; Humans; Iron; Microscopy, Electron, Scanning

The goal of this study was to develop and validate a new fibrin-targeted imaging agent that enables high-resolution near-infrared fluorescence (NIRF) imaging of deep vein thrombosis (DVT).. NIRF imaging of fibrin could enable highly sensitive and noninvasive molecular imaging of thrombosis syndromes in vivo.. A fibrin-targeted peptide was conjugated to a near-infrared fluorophore Cy7, termed FTP11-Cy7. The NIRF peptide is based on a fibrin-specific imaging agent that has completed Phase II clinical magnetic resonance imaging trials. In vitro binding of FTP11-Cy7 to human plasma clots was assessed by using fluorescence reflectance imaging. Next, FTP11-Cy7 was intravenously injected in mice with femoral DVT induced by topical 7.5% ferric chloride treatment. Intravital fluorescence microscopy and noninvasive fluorescence molecular tomography-computed tomography were performed in 32 mice with DVT, followed by histological analyses.. In vitro human clot-binding analyses showed a 6-fold higher NIRF clot target-to-background ratio (TBR) of FTP11-Cy7 than free Cy7 (6.3 ± 0.34 vs. 1.2 ± 0.03; p < 0.0001). The thrombus TBR of acute and subacute femoral DVT with FTP11-Cy7 obtained by using intravital fluorescence microscopy was >400% higher than control free Cy7. Binding of FTP11-Cy7 to thrombi was blocked by a 100-fold excess of unlabeled competitor peptide both in vitro and in vivo (p < 0.001 for each). Histological analyses confirmed that FTP11-Cy7 specifically accumulated in thrombi. Noninvasive fluorescence molecular tomography-computed tomography imaging of fibrin in jugular DVT demonstrated strong NIRF signal in thrombi compared with sham-operated jugular veins (mean TBR 3.5 ± 0.7 vs. 1.5 ± 0.3; p < 0.05).. The fibrin-targeted NIRF agent FTP11-Cy7 was shown to avidly and specifically bind human and murine thrombi, and enable sensitive, multimodal intravital and noninvasive NIRF molecular imaging detection of acute and subacute murine DVT in vivo.

Topics: Animals; Chlorides; Disease Models, Animal; Femoral Vein; Ferric Compounds; Fibrin; Fluorescent Dyes; Half-Life; Humans; Indoles; Injections, Intravenous; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Imaging; Oligopeptides; Phlebography; Radionuclide Imaging; Spectroscopy, Near-Infrared; Tissue Distribution; Tomography, X-Ray Computed; Venous Thrombosis

The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α(2)-antiplasmin (α(2)AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α(2)AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α(2)AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.. Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H(6)NQEQVSPLTLLAG(4)Y (designated XL1); H(6)DQMMLPWAVTLG(4)Y (XL2); H(6)WQHKIDLPYNGAG(4)Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α(2)AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α(2)AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.. Fusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.

Topics: Amino Acid Motifs; Analysis of Variance; Animals; Antifibrinolytic Agents; Blood Coagulation; Chlorides; Factor XIIIa; Ferric Compounds; Fibrin; Humans; In Vitro Techniques; Mice; Molecular Sequence Data; Oligonucleotides; Pichia; Plasmids; Rabbits; Recombinant Fusion Proteins; Sequence Analysis, Protein; Serum Albumin

Activated factor XIII (FXIIIa) mediates fibrinolytic resistance and is a hallmark of newly formed thrombi. In vivo imaging of FXIIIa activity could further elucidate the role of this molecule in thrombosis and other biological processes and aid in the clinical detection of acute thrombi.. An FXIIIa-sensitive near-infrared fluorescence imaging agent (A15) was engineered by conjugating a near-infrared fluorochrome to a peptide ligand derived from the amino terminus of alpha2-antiplasmin. To evaluate the molecular specificity of A15 for FXIIIa, a control agent (C15) was also synthesized by modifying a single key glutamine residue in A15. Fluorescence imaging experiments with A15 demonstrated stronger thrombosis enhancement in human plasma clots in vitro (P<0.001 versus C15 clots and other controls). A15 was found to be highly specific for the active site of FXIIIa and was covalently bound to fibrin. In vivo murine experiments with A15 demonstrated significant signal enhancement in acute intravascular thrombi (P<0.05 versus C15 group). Minimal A15 enhancement was seen in older aged thrombi (>24 hours), consistent with an expected decline of FXIIIa activity over time. Imaging results were confirmed on correlative histopathology and fluorescence microscopy.. A15 is a novel optical imaging agent that is specifically crosslinked to fibrin by FXIIIa, permitting detection of FXIIIa activity in experimental thrombi in vivo. This agent should permit assessment of FXIIIa activity in a broad range of biological processes and could aid in the clinical diagnosis of acute thrombi.

Topics: alpha-2-Antiplasmin; Animals; Chlorides; Contrast Media; Cross-Linking Reagents; Factor XIIIa; Feasibility Studies; Ferric Compounds; Fibrin; Fluorescent Dyes; Humans; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Peptide Fragments; Protein Binding; Spectroscopy, Near-Infrared; Thrombosis; Time Factors

Platelet integrin alpha IIb beta 3 (GPIIb/IIIa) plays a central role in the initiation of arterial thrombosis, but its contribution to disseminated microvascular thrombosis is less well defined. Therefore, wild-type mice (beta 3(+/+)), beta 3-integrin-deficient mice (beta 3(-/-)), and wild-type mice treated with a hamster monoclonal antibody (1B5) that blocks murine alpha IIb beta 3 function were tested in models of large-vessel and microvascular thrombosis. In the large-vessel model, ferric chloride was used to injure the carotid artery, and the time to thrombosis was measured. In beta 3(+/+) mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the beta 3(-/-) mice tested (P <.001). Fab and F(ab')(2) fragments of 1B5 increased the median time to occlusion. To initiate systemic intravascular thrombosis, prothrombotic agents were administered intravenously, and platelet thrombus formation was monitored by the decrease in circulating platelet count. Three minutes after the injection of adenosine diphosphate (ADP), collagen + epinephrine, or tissue factor, the platelet counts in beta 3(+/+) mice decreased by 289, 424, and 429 x 10(3)/microL, respectively. beta 3(-/-) mice and wild-type mice pretreated with 1B5 Fab (1 mg/kg, IP) were nearly completely protected from the effects of ADP. In contrast, beta 3(-/-) mice were only partially protected from the effects of collagen + epinephrine and minimally protected from the effects of tissue factor. In all cases, less fibrin became deposited in the lungs of beta 3(-/-) mice than in wild-type mice. These results suggest that though alpha IIb beta 3 plays a dominant role in large-vessel thrombosis, it plays a variable role in systemic intravascular thrombosis. (Blood. 2001;98:1055-1062)

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Blood Platelets; Carotid Artery Thrombosis; Chlorides; Disease Models, Animal; Female; Ferric Compounds; Fibrin; Immunohistochemistry; Integrin beta3; Male; Mice; Mice, Knockout; Microcirculation; Microscopy, Electron; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet Membrane Glycoproteins; Receptors, Vitronectin; Thrombosis

Mutations in the gene encoding thrombomodulin (TM), a thrombin regulator, are suspected risk factors for venous and arterial thrombotic disease. We have previously described the generation of TM(Pro/Pro) mice carrying a TM gene mutation that disrupts the TM-dependent activation of protein C. Here, it is shown that inbred C57BL/6J TM(Pro/Pro) mice exhibit a hypercoagulable state and an increased susceptibility to thrombosis and sepsis. Platelet thrombus growth after FeCl(3)-induced acute endothelial injury was accelerated in mutant mice. Vascular stasis after permanent ligation of the carotid artery precipitated thrombosis in mutant but not in normal mice. Mutant mice showed increased mortality after exposure to high doses of endotoxin and demonstrated altered cytokine production in response to low-dose endotoxin. The severity of the hypercoagulable state and chronic microvascular thrombosis caused by the TM(Pro) mutation is profoundly influenced by mouse strain-specific genetic differences between C57BL/6 and 129SvPas mice. These data demonstrate that in mice, TM is a physiologically relevant regulator of platelet- and coagulation-driven large-vessel thrombosis and modifies the response to endotoxin-induced inflammation. The phenotypic penetrance of the TM(Pro) mutation is determined by as-yet-uncharacterized genetic modifiers of thrombosis other than TM.

Topics: Animals; Blood Coagulation; Carotid Artery Thrombosis; Chlorides; Cytokines; Ferric Compounds; Fibrin; Genetic Predisposition to Disease; Ligation; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Sepsis; Survival Analysis; Thrombomodulin; Thrombosis