Multiple Modes of Blood Coagulation (original) (raw)
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Fibrinogen coagulation without thrombin: Reaction with vitamin C and copper(II)
Thrombosis Research, 1985
We describe a novel method for inducing fibrinogen derived clots. The addition of vitanin C (0.1-l &I) to a solution of fibrinogen (1 mg/ml) arxl Cu(I1) (20-150 uM) results in pxotein ccmiug out of solution . This pbmanemn can be "read" by fibraneters as "clotting time".. The reaction requires Cu(I1) aud cau be prevented by a chelating agent, such as citrate, as well by a hydroxyl radical scavenger, such as mannitol. 'Ihe insoluble protein, called "neofibe", is soluble in 4 M ureaand 2 %sDs. Isoelectric focusirg andSDG_electropboretic ~isonof Mtivefibrinogenwithneofiberevedlmolecular modifications of the starting protein. This reaction is an interesting exanple of a free radical mediated transformation of soluble protein into insoluble material . Sane findings on the connection between htmostasis, vitamin C and Cu(I1) are discussed.
Prothrombin and fibrinogen carbonylation: How that can affect the blood clotting
Redox Report, 2016
The aim of the work was the development of a simple method for measuring the plasma prothrombin carbonylation and the study the impact of prothrombin and fibrinogen oxidation on the rate of plasma clotting. Methods: A new method was based on the ability of prothrombin to be adsorbed by the barium sulfate. It consists of four steps: prothrombin mixing with the water suspension of BaSO 4 ; reaction of 2,4dinitrophenylhydrazine with the BaSO 4-bound prothrombin; desorption of prothrombin-2,4dinitrophenylhydrazone complex from BaSO 4 in an alkaline medium; neutralization and reading of the optical absorbance of the complex (λ = 370 nm). The prothrombin/fibrinogen carbonylation and plasma clotting rate in vitro in the presence of reactive oxygen species (ROS)-generating agents (0.05-0.8 mM Fe 2+ /H 2 O 2) were monitored. Results: The plasma volume required for measurement of carbonylated prothrombin was 0.4 ml. High level of linearity and reproducibility was observed (r = 0.9995, P = 0.0005for the protein; r = 0.9971, P = 0.0029for carbonyls). In the intact rats, the concentration of blood plasma prothrombin was 0.355 ± 0.009 mg/ml, and that of carbonyls was 4.94 ± 0.09 nmol/mg. Discussion: Prothrombin and plasma clotting rate was not affected by low concentrations of ROS (0.05-0.2 mM Fe 2+ /H 2 O 2). The fibrinogen was susceptible to ROS-related effect over all the used range of concentration (0.05-0.8 mM Fe 2+ /H 2 O 2). Carbonylation of fibrinogen did not affect the plasma clotting activity at low ROS concentration (0.05-0.2 mM Fe 2+ /H 2 O 2), however it retarded the clotting at higher ROS (0.2-0.8 mM Fe 2+ /H 2 O 2).
Oxidation Inhibits Iron-Induced Blood Coagulation
Current Drug Targets, 2012
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.
Although blood coagulation facilitated by non-thermal plasma has been reported several years ago, the insight to the involved mechanisms is still rather limited. In this work, we report our discovery of a new mechanism for the haem-promoted blood-coagulation caused by non-thermal plasma treatment. The reason for the haem role is due to that its oxidized form, namely, hematin, can promote the dityrosine cross-linking of fibrinogen, the most important coagulation protein, to form a membrane-like layer on the surface of the treated blood with plasma exposure. Both haem and non-thermal-plasma generated hydrogen peroxide are requisite for the cross-linking process. We confirmed that fibrinogen can coordinate with the haem iron to form a protein-haem complex which shows pseudo-peroxidase activity, and in the presence of hydrogen peroxide, the complex can induce the dityrosine formation between fibrinogen molecules, leading to the fibrin network necessary for the blood coagulation. Understanding of such an underlying mechanism can be useful to guide more efficient application of non-thermal plasma in the management of hemostasis, thrombosis and etc. Blood coagulation is a physiological process which involves the coordinated activation of various blood components , such as platelets, proteins, cells and so on 1. Under the circumstances of vessel damage, the coagulation system is triggered with the formation of the platelet plug which sticks to the injured surface. Tissue factors are then activated, promoting the protein-based coagulation cascade in which thrombin is generated 2. The produced thrombin converts fibrinogen, an important serum protein with a concentration of 2–4 g/L, to fibrin, a three dimensional protein network which together with platelets forms a stable and permanent plug preventing further blood loss 3–5. However, not all bleeding can be stopped via the physiological coagulation cascade. Hemorrhage is the major cause of death for soldiers in battlefield and also for civilian patients with traumatic injury between the ages of 5 and 44 years 6,7. Management of hemostasis is vital to surgical success because inadequate control of bleeding is associated with increased mortality rates and higher costs of care. Methods to staunch bleeding include applying pressure, suturing, using thermal-based energy devices, or using hemostatic agents 8. A large variety of hemostatic products have been developed and marketed in the last few decades, including gelatin, collagen, oxidized regenerated cellulose, polysaccharide spheres, thrombin, fibrin sealant, zeolite, chitosan, starch-related products and so on 2,9–11. Although so many hemostatic methods have been developed, many of them fail to meet all the requirements for efficient blood coagulation. Therefore, the search for ideal methods still continues. An ideal hemostatic method should not only stop blood flow effectively and quickly but may also have the ability to sterilize 12. For meeting these two requirements, non-thermal plasma treatment is now emerging as an ideal option, which does not only have the ability to assist blood coagulation 13 , but also can be bactericidal 14. In 2006, Fridman et al. first
Effect of Oxidized Fibrinogen on Hemostatic System: In Vitro Study
Clinical and Applied Thrombosis/Hemostasis, 2010
Standard coagulation assays were performed with control and oxidized fibrinogen (Fg), using prothrombin time (PT; 12.5 ± 0.4 vs 25 ± 0.8 seconds, P < .001) and activated partial thromboplastin time (aPTT; 33 ± 2.5 vs 63 ± 4.7 seconds, P < .001). Fibrin clot (MA), clot formation initiation (r), and rate of clot lysis (LY30) were measured, a reflection exposure of Fg to Fe3+/ ascorbate oxidative system by thrombelastograph (TEG) analysis (0, 6, 12, 24, and 48 hours, 6.2 ± 1.3 vs 5.5 ± 1.2, 4.3 ± 1.0 [P < .01], 3.9 ± 1.6, 3.2 ± 0.8, [P < .001]). Maximum amplitude level was found to be lower than control (69.1 ± 7.2 vs 67.9 ± 12.4, 64.0 ± 11.4, 60.2 ± 21.2, 42.2 ± 15.2, P < .001). The lysis rate was changed according to oxidation time between Fg exposed to Fe3+/ascorbate and control exposed to Fe 3+/ascorbate for the same treatment time (1.9 ± 0.71 vs 7 ± 0.5, 1.6 ± 0.1, 1.2 ± 0.5, 0.9 ± 1.3, P < .001). We revealed dysregulation of hemostatic system with contribution o...
Fibrin Clot Formation under Oxidative Stress Conditions
Antioxidants, 2021
During coagulation, the soluble fibrinogen is converted into insoluble fibrin. Fibrinogen is a multifunctional plasma protein, which is essential for hemostasis. Various oxidative posttranslational modifications influence fibrinogen structure as well as interactions between various partners in the coagulation process. The aim was to examine the effects of oxidative stress conditions on fibrin clot formation in arterial atherothrombotic disorders. We studied the changes in in vitro fibrin network formation in three groups of patients—with acute coronary syndrome (ACS), with significant carotid artery stenosis (SCAS), and with acute ischemic stroke (AIS), as well as a control group. The level of oxidative stress marker malondialdehyde measured by LC-MS/MS was higher in SCAS and AIS patients compared with controls. Turbidic methods revealed a higher final optical density and a prolonged lysis time in the clots of these patients. Electron microscopy was used to visualize changes in the ...
In vitro study of the inhibition of coagulation induced by different radiocontrast molecules
Thrombosis Research, 1985
The anticoagulant activity of seven intravascular radiocontrast molecules (RCM) was evaluated in different in vitro systems using citrated human plasma. Each RCM was tested in a concentration range of 5 to 50 mM. The thrombin time and the reptilase time showed a dose-dependent lengthening of fibrinoformation, the recording of fibrinoformation exhibited a significant delay of fibrin monomer generation and polymerization although the amplitude of the fibrinoformation was not decreased. The interfering effect with fibrin clot formation impairs also global coagulation tests and monospecific coagulation tests using fibrinofonnation as the final step of the assay, but a possible interaction between RCM and some specific coagulation factors cannot be excluded. RCM potentiated the antithrombin action of heparin but the inhibition or delay of fibrinoformation is not related to an antithrombinic effect of contrast media. The thrombin amidolytic activity is not modified by RCM but the generation of FpA is delayed and decreased. The ultrastructure of the fibrin clot is not altered at the end of the polymerization.
The effect of reagents mimicking oxidative stress on fibrinogen function
2013
Fibrinogen is one of the plasma proteins most susceptible to oxidative modification. It has been suggested that modification of fibrinogen may cause thrombotic/bleeding complications associated with many pathophysiological states of organism. We exposed fibrinogen molecules to three different modification reagents-malondialdehyde, sodium hypochlorite, and peroxynitrite-that are presented to various degrees in different stages of oxidative stress. We studied the changes in fibrin network formation and platelet interactions with modified fibrinogens under flow conditions. The fastest modification of fibrinogen was caused by hypochlorite. Fibers from fibrinogen modified with either reagent were thinner in comparison with control fibers. We found that platelet dynamic adhesion was significantly lower on fibrinogen modified with malondialdehyde and significantly higher on fibrinogen modified either with hypochlorite or peroxynitrite reflecting different prothrombotic/antithrombotic properties of oxidatively modified fibrinogens. It seems that, in the complex reactions ongoing in living organisms at conditions of oxidation stress, hypochlorite modifies proteins (e.g., fibrinogen) faster and more preferentially than malondialdehyde. It suggests that the prothrombotic effects of prior fibrinogen modifications may outweigh the antithrombotic effect of malondialdehyde-modified fibrinogen in real living systems.