Biochemical Approach to Poly(Lactide)-Copper Composite-Impact on Blood Coagulation Processes.

The paper presents the investigation of the biological properties of Poly(Lactide)-Copper composite material obtained by sputter deposition of copper onto Poly(lactide) melt-blown nonwoven fabrics. The functionalized composite material was subjected to microbial activity tests against colonies of Gram-positive (Staphylococcus aureus), Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria, Chaetomium globosum and Candida albicans fungal mold species and biochemical-hematological tests including the evaluation of the Activated Partial Thromboplastin Time, Prothrombin Time, Thrombin Time and electron microscopy fibrin network imaging. The substantial antimicrobial and antifungal activities of the Poly(Lactide)-Copper composite suggests potential applications as an antibacterial/antifungal material. The unmodified Poly(Lactide) fabric showed accelerated human blood plasma clotting in the intrinsic pathway, while copper plating abolished this effect. Unmodified PLA itself could be used for the preparation of wound dressing materials, accelerating coagulation in the case of hemorrhages, and its modifications with the use of various metals might be applied as new customized materials where blood coagulation process could be well controlled, yielding additional anti-pathogen effects.

Flavonol and A-type procyanidin-rich extracts of Prunus spinosa L. flower exhibit anticoagulant activity through direct thrombin inhibition, but do not affect platelet aggregation in vitro.

Background: Blackthorn flower (Prunus spinosa L.) is a traditional herbal remedy recommended for treating cardiovascular diseases (CVDs). Aim: This in vitro study investigates the effects of flavonol and A-type procyanidin-rich blackthorn flower extracts on the hemostatic system, including the blood plasma coagulation cascade and platelet aggregation. Methods: Six distinct extracts, characterized through various techniques, including LC-MS/MS, were assessed at in vivo-relevant levels (1-50 µg/mL) for their antithrombotic activity. The thrombin, prothrombin, and activated partial thromboplastin times were measured. Additionally, the thrombin enzymatic activity was tested using the chromogenic substrate S-2238 and fibrinogen as the physiological substrate of the enzyme. To gain insights into the mechanism of action, the interactions between the primary extracts' constituents, their potential metabolites, and thrombin were examined in silico. The computational analyses were complemented by in vitro experiments and circular dichroism spectroscopy. The platelet aggregation in human platelet-rich plasma was assessed after ADP or collagen stimulation. Furthermore, the extracts' biocompatibility was tested on human peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs). Results: The extracts slightly prolonged the prothrombin and thrombin times and effectively inhibited the thrombin's enzymatic activity, reducing its amidolytic and proteolytic functions at 50 µg/mL by 91.2% and 74.8%, respectively. In silico molecular docking demonstrated a strong binding affinity of the examined polyphenols and their metabolites to thrombin. Most analytes bound exclusively within the enzyme active site; however, afzelin, kaempferitrin, and procyanidin A2 revealed the affinity to additional binding sites, including exosite I. The structure-activity relationship of flavonols as thrombin inhibitors was studied in vitro. Circular dichroism spectroscopy confirmed that the interactions between thrombin and the compounds (even at 1 µg/mL) induce alterations within the α-helices' secondary structure, resulting in noticeable changes in the enzyme's CD spectrum. On the other hand, the extracts did not influence platelet aggregation. Eventually, their cellular biocompatibility with PBMCs and RBCs was confirmed. Conclusion: The extracts directly inhibit thrombin, a critical serine protease in hemostasis and a prime anticoagulant drug target, and do not exhibit antiplatelet effects. This study enhances the knowledge of the biological activity of blackthorn flowers and supports their traditional use in CVDs.

Effects of Rheum rhaponticum and Rheum rhabarbarum extracts on haemostatic activity of blood plasma components and endothelial cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicine recommends the use of Rheum rhaponticum L. and R. rhabarbarum L. to treat over thirty complaints, including disorders related to the cardiovascular system such as heartache, pains in the pericardium, epistaxis and other types of haemorrhage, blood purification as well as disorders of venous circulation. AIM OF THE STUDY: This work was dedicated to examining for the first time the effects of extracts from petioles and roots of R. rhaponticum and R. rhabarbarum, as well as two stilbene compounds (rhapontigenin and rhaponticin) on the haemostatic activity of endothelial cells and functionality of blood plasma components of the haemostatic system. MATERIALS AND METHODS: The study was based on three main experimental modules, including the activity of proteins of the human blood plasma coagulation cascade and the fibrinolytic system as well as analyses of the haemostatic activity of human vascular endothelial cells. Additionally, interactions of the main components of the rhubarb extracts with crucial serine proteases of the coagulation cascade and fibrinolysis (i.e. thrombin, the coagulation factor Xa and plasmin) were analyzed in silico. RESULTS: The examined extracts displayed anticoagulant properties and significantly reduced the tissue factor-induced clotting of human blood plasma (by about 40%). Inhibitory effects of the tested extracts on thrombin and the coagulation factor Xa (FXa) were found as well. For the extracts, the IC50 was ranging from 20.26 to 48.11 µg/ml. Modulatory effects on the haemostatic response of endothelial cells, including the release of von Willebrand factor, tissue-type plasminogen activator and the plasminogen activator inhibitor-1, have been also found. CONCLUSIONS: Our results indicated for the first time that the examined Rheum extracts influenced the haemostatic properties of blood plasma proteins and endothelial cells, with the prevalence of the anticoagulant action. The anticoagulant effect of the investigated extracts may be partly attributed to the inhibition of the FXa and thrombin activities, the key serine proteases of the blood coagulation cascade.

Rheum rhaponticum and Rheum rhabarbarum Extracts as Modulators of Endothelial Cell Inflammatory Response.

BACKGROUND: Inflammation, endothelial dysfunction, and alterations in blood physiology are key factors contributing to atherosclerosis and other cardiovascular disorders. Hence, modulation of endothelial function and reducing its pro-inflammatory and pro-thrombotic activity is considered one of the most important cardioprotective strategies. This study aimed to evaluate the anti-inflammatory potential of rhubarb extracts isolated from petioles and underground organs of Rheum rhabarbarum L. (garden rhubarb) and R. rhaponticum L. (rhapontic rhubarb) as well as two stilbenoids, typically found in these plants, i.e., rhapontigenin (RHPG) and its glycoside, rhaponticin (RHPT). METHODS: Analysis of the anti-inflammatory effects of the indicated rhubarb-derived substances involved different aspects of the endothelial cells' (HUVECs) response: release of the inflammatory mediators; cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX) expression as well as the recruitment of leukocytes to the activated HUVECs. The ability of the rhubarb-derived extracts to inhibit COX-2 and 5-LOX activities was examined as well. The study was supplemented with the in silico analysis of major components of the analyzed extracts' interactions with COX-2 and 5-LOX. RESULTS: The obtained results indicated that the examined plant extracts and stilbenes possess anti-inflammatory properties and influence the inflammatory response of endothelial cells. Biochemical and in silico tests revealed significant inhibition of COX-2, with special importance of rhaponticin, as a compound abundant in both plant species. In addition to the reduction in COX-2 gene expression and enzyme activity, a decrease in the cytokine level and leukocyte influx was observed. Biochemical tests and computational analyses indicate that some components of rhubarb extracts may act as COX-2 inhibitors, with marginal inhibitory effect on 5-LOX.

High Molecular Weight Kininogen: A Review of the Structural Literature.

Kininogens are multidomain glycoproteins found in the blood of most vertebrates. High molecular weight kininogen demonstrate both carrier and co-factor activity as part of the intrinsic pathway of coagulation, leading to thrombin generation. Kininogens are the source of the vasoactive nonapeptide bradykinin. To date, attempts to crystallize kininogen have failed, and very little is known about the shape of kininogen at an atomic level. New advancements in the field of cryo-electron microscopy (cryoEM) have enabled researchers to crack the structure of proteins that has been refractory to traditional crystallography techniques. High molecular weight kininogen is a good candidate for structural investigation by cryoEM. The goal of this review is to summarize the findings of kininogen structural studies.

Finding inhibitors for PCSK9 using computational methods.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is one of the key targets for atherosclerosis drug development as its binding with low-density lipoprotein receptor leads to atherosclerosis. The protein-ligand interaction helps to understand the actual mechanism for the pharmacological action. This research aims to discover the best inhibitory candidates targeting PCSK9. To start with, reported ACE inhibitors were incorporated into pharmacophore designing using PharmaGist to produce pharmacophore models. Selected models were later screened against the ZINC database using ZINCPHARMER to define potential drug candidates that were docked with the target protein to understand their interactions. Molecular docking revealed the top 10 drug candidates against PCSK9, with binding energies ranging from -9.8 kcal·mol-1 to -8.2 kcal·mol-1, which were analyzed for their pharmacokinetic properties and oral bioavailability. Some compounds were identified as plant-derived compounds like (S)-canadine, hesperetin or labetalol (an antihypertensive drug). Molecular dynamics results showed that these substances formed stable protein-ligand complexes. (S)-canadine-PCSK9 complex was the most stable with the lowest RMSD. It was concluded that (S)-canadine may act as a potential inhibitor against atherosclerosis for the development of new PCSK9 inhibitory drugs in future in vitro research.

Pulmonaria obscura and Pulmonaria officinalis Extracts as Mitigators of Peroxynitrite-Induced Oxidative Stress and Cyclooxygenase-2 Inhibitors-In Vitro and In Silico Studies.

The Pulmonaria species (lungwort) are edible plants and traditional remedies for different disorders of the respiratory system. Our work covers a comparative study on biological actions in human blood plasma and cyclooxygenase-2 (COX-2) -inhibitory properties of plant extracts (i.e., phenolic-rich fractions) originated from aerial parts of P. obscura Dumort. and P. officinalis L. Phytochemical profiling demonstrated the abundance of phenolic acids and their derivatives (over 80% of the isolated fractions). Danshensu conjugates with caffeic acid, i.e., rosmarinic, lithospermic, salvianolic, monardic, shimobashiric and yunnaneic acids were identified as predominant components. The examined extracts (1-100 µg/mL) partly prevented harmful effects of the peroxynitrite-induced oxidative stress in blood plasma (decreased oxidative damage to blood plasma components and improved its non-enzymatic antioxidant capacity). The cellular safety of the extracts was confirmed in experimental models of blood platelets and peripheral blood mononuclear cells. COX-2 inhibitor screening evidently suggested a stronger activity of P. officinalis (IC50 of 13.28 and 7.24 µg/mL, in reaction with synthetic chromogen and physiological substrate (arachidonic acid), respectively). In silico studies on interactions of main components of the Pulmonaria extracts with the COX-2 demonstrated the abilities of ten compounds to bind with the enzyme, including rosmarinic acid, menisdaurin, globoidnan A and salvianolic acid H.

The evolution of factor XI and the kallikrein-kinin system.

Factor XI (FXI) is the zymogen of a plasma protease (FXIa) that contributes to hemostasis by activating factor IX (FIX). In the original cascade model of coagulation, FXI is converted to FXIa by factor XIIa (FXIIa), a component, along with prekallikrein and high-molecular-weight kininogen (HK), of the plasma kallikrein-kinin system (KKS). More recent coagulation models emphasize thrombin as a FXI activator, bypassing the need for FXIIa and the KKS. We took an evolutionary approach to better understand the relationship of FXI to the KKS and thrombin generation. BLAST searches were conducted for FXI, FXII, prekallikrein, and HK using genomes for multiple vertebrate species. The analysis shows the KKS appeared in lobe-finned fish, the ancestors of all land vertebrates. FXI arose later from a duplication of the prekallikrein gene early in mammalian evolution. Features of FXI that facilitate efficient FIX activation are present in all living mammals, including primitive egg-laying monotremes, and may represent enhancement of FIX-activating activity inherent in prekallikrein. FXI activation by thrombin is a more recent acquisition, appearing in placental mammals. These findings suggest FXI activation by FXIIa may be more important to hemostasis in primitive mammals than in placental mammals. FXI activation by thrombin places FXI partially under control of the vitamin K-dependent coagulation mechanism, reducing the importance of the KKS in blood coagulation. This would explain why humans with FXI deficiency have a bleeding abnormality, whereas those lacking components of the KKS do not.

Antagonists of Vitamin K-Popular Coumarin Drugs and New Synthetic and Natural Coumarin Derivatives.

Many natural coumarins and their chemically synthesized analogs and derivatives exert diverse properties, such as anticancer, antioxidant, anti-inflammatory, or anticoagulant, with the latter being of the utmost importance. The widely used warfarin, acenocoumarol, and phenprocoumon exert anticoagulant properties by inhibiting the vitamin K epoxide reductase complex. In this interdisciplinary review, we present biochemical principles of the coagulation processes and possible methods for their tuning based on the use of coumarins. We also summarize chemical methods of synthesis of coumarins and discuss structures and properties of those that have been used for a long time, as well as newly synthesized compounds. Brief information on the clinical use of coumarins and other anticoagulant drugs is given, including the severe effects of overdosing and methods for reversing their action.

Interaction of Arylidenechromanone/Flavanone Derivatives with Biological Macromolecules Studied as Human Serum Albumin Binding, Cytotoxic Effect, Biocompatibility Towards Red Blood Cells.

The aim of this study was to determine the cytotoxic effect of 3-arylidenechromanone (1) and 3arylideneflavanone (2) on HL-60 and NALM-6 cell lines (two human leukemia cell lines) and a WM-115 melanoma cell line. Both compounds exhibited high cytotoxic activity with higher cytotoxicity exerted by compound 2, for which IC50 values below 10 µM were found for each cell line. For compound 1, the IC50 values were higher than 10 µM for HL-60 and WM-115 cell lines, but IC50 < 10 µM was found for the NALM-6 cell line. Both compounds, at the concentrations close to IC50 (concentration range: 5⁻24 µM/L for compound 1 and 6⁻10 µM/L for compound 2), are not toxic towards red blood cells. The synthesized compounds were characterized using spectroscopic methods ¹H- and 13C-NMR, IR, MS, elemental analysis, and X-ray diffraction. The lipophilicity of both synthesized compounds was determined using an RP-TLC method and the logP values found were compared with the theoretical ones taken from the Molinspiration Cheminformatics (miLogP) software package. The mode of binding of both compounds to human serum albumin was assessed using molecular docking methods.

A biological, fluorescence and computational examination of synthetic coumarin derivatives with antithrombotic potential.

BACKGROUND: Scientists still look for new drugs, which have anticoagulant properties. This is so important because existing anticoagulant drugs give many side effects, for example major bleeding. In this study we examined nine coumarin derivatives - candidates to be future antithrombotic drugs, which were synthetized and crystallized in our previous paper. METHODS: Here we show the fluorescence and fluorescence quenching of coumarin derivatives with di- or trimethoxybenzylamine moieties in C-3 position. All nine compounds were checked by lactate dehydrogenase assay to examine their cytotoxic activity on hepatic cells. We also investigated the other biological properties (bioactivity, drug-likeness and blind docking) using computational tools. Lipophilicity coefficient logP of all obtained compounds was determined using by RP-TLC and compared to theoretical predictions. RESULTS: The obtained coumarins exhibited low lipophilic character. The substances bound with HSA and did not demonstrate cytotoxicity against isolated liver cells. The most interesting compound (3b) possessed two methoxy- group in 2- and 4-position in benzene ring, ability to interact with two HSA binding sites and probably smaller steric hindrance in comparison to other synthesized derivatives. CONCLUSIONS: Our present study shows that after examination of fluorescence, cytotoxic activity, lipophilicity, theoretical bioactivity, drug-likeness and blind docking of our synthesized compounds they have potential as antithrombotic medicines and may be candidates to be drugs after further studies.

Bufadienolides from Kalanchoe daigremontiana modulate the enzymatic activity of plasmin - In vitro and in silico analyses.

Plasmin (EC 3.4.21.7) is a key enzyme of the fibrinolytic system, responsible for the degradation of fibrin clot and maintaining blood fluidity. Hence, alterations of the fibrinolytic capacity of blood plasma may contribute to thrombotic or bleeding complications. The aim of this study was to determine effects of a bufadienolide-rich fraction, isolated from roots of Kalanchoe daigremontiana (0.05-50 µg/ml) on enzymatic properties of plasmin. Hydrolysis of a synthetic substrate S-2251 (H-D-Valyl-l-leucyl-l-lysine-p-nitroaniline dihydrochloride) by plasmin revealed that the bufadienolide-rich fraction had a diverse effect on this enzyme, dependently on the concentration range. While the lower concentrations of the examined fraction (0.05-2.5 µg/ml) significantly enhanced the amidolytic activity of plasmin, at 25-50 µg/ml concentrations, the enzyme was evidently inhibited (by about 60%). The Lineweaver-Burk plot indicated on an uncompetitive inhibition of plasmin. Inhibitory effects (up to 80%) were also found in the streptokinase-induced plasminogen activation to plasmin. Docking results suggest that only some of compounds (mostly bersaldegenin 1-acetate (10), bryotoxin (13) and hovetrichoside C (17)) were bound to plasminogen/plasmin, depending on the presence or absence of the substrate in the active site. The obtained findings suggest allosteric regulation of plasminogen activation and plasmin activity by components of the examined fraction.

Bufadienolides from Kalanchoe daigremontiana as thrombin inhibitors-In vitro and in silico study.

Thrombin is an active plasma coagulation factor II, critical for the formation of fibrin clot during blood coagulation. For that reason, this protein is also a crucial target for different anti-thrombotic therapies. The work is based on in vitro evaluation of the inhibitory effect of bufadienolide-rich fraction, isolated from roots of Kalanchoe daigremontiana (1-50µg/ml) on enzymatic properties of a serine proteinase - thrombin. The efficacy of the inhibition of amidolytic activity of thrombin (measured as a hydrolysis of the chromogenic substrate S-2238, Chromogenix) attained about 10 and 66%, respectively. The IC50, established for the examined bufadienolide fraction was 2.79µg/ml, while the IC50 calculated for argatroban (reference compound) was 0.78µg/ml. Linearization conducted using Lineweaver-Burk plot indicated that the K. daigremontiana fraction contains compounds that are uncompetitive inhibitors of thrombin. K. daigremontiana fraction was also able to reduce the proteolytic activity of thrombin towards its physiological substrate, i.e. fibrinogen. Additionally, this study is supported by in silico analysis of interactions of the most common compounds, identified in the examined in Kalanchoe extract to crystal structure of this enzyme.

Radical scavenging and antioxidant effects of Matricaria chamomilla polyphenolic-polysaccharide conjugates.

Matricaria chamomilla L. (MC), a member of the Asteraceae family, is one of the oldest medicinal plants, widely used worldwide for a variety of healing applications. Its recommendations, derived from both traditional and modern medicine, include numerous disorders such as inflammation, ulcers, wounds, gastrointestinal disorders, stomach ache, pharyngitis, rheumatic pain, as well as the other ailments. This work is focused on another aspect of the biological activity of chamomile polyphenolic-polysaccharide conjugates--their antioxidant properties in the protection of blood plasma components against in vitro oxidative stress. Measurements of DPPH and ABTS radical scavenging indicated considerable anti-free radical action of MC. Pre-incubation of blood plasma with MC considerably diminished the extent of ONOO(-)-induced oxidative modifications such as protein carbonyl groups, SH groups, 3-nitrotyrosine, as well as the formation of lipid hydroperoxides. The analysis of the FRAP assay result shows a considerable increase of ferric reducing ability of blood plasma in the presence of MC. The results obtained in this study indicate that polyphenolic-polysaccharide conjugates isolated from M. chamomilla substances possess antioxidant properties. The M. chamomilla macromolecular glycoconjugates may be useful in the creation of new natural-based medications or dietary supplements, helpful in the prevention and treatment of oxidative stress-mediated disorders.

[The formation, metabolism and the evolution of blood platelets]. / Powstawanie, metabolizm i ewolucja plytek krwi.

Platelets are the smallest, depleted of nucleus blood cells which contain a typical cellular organelles including the mitochondria, so that have active metabolism. Platelets possess the highly organized cytoskeleton, specific secretory granules and unique membrane receptors system responsible for their high reactivity. The key role of blood platelets is to maintain normal hemostasis, but they also play important roles in inflammation, immune processes and the cancer progression. The anucleated, small platelets occur in representatives of all clusters of mammals, so it seems to be an adaptation feature. In other vertebrates similar hemostatic functions are played by large nucleated platelets, which are much more weakly reactive. Small, reactive platelets, appearing in the evolution of mammals, allowed the formation of clots faster and slower blood loss in case of injury, but also increased the risk of thromboembolic and cardiovascular diseases. Daily the human body forms about 1x10¹¹ platelets, which are produced by a process of differentiation, maturation and fragmentation of the cytoplasm of mature megakaryocytes. The emergence of platelets is the final stage of megakaryocyte differentiation and is followed by formation of the direct precursors called proplatelets. The anucleated platelets are regarded as terminally differentiated cells, which are not capable of further cell division. However, despite the absence of a nucleus, in blood platelets the synthesis and transcription of mitochondrial DNA and protein synthesis occurring on the basis of mRNA from megakaryocytes has been confirmed. However, recent studies published in 2012 show that the platelets are capable not only of the process of protein synthesis, but also of generation of new cells, which are functionally and structurally similar to the parent platelets.

The role of fibrinogen, fibrin and fibrin(ogen) degradation products (FDPs) in tumor progression.

Participation of fibrin, fibrinogen, and their degradation products in pathogenesis and progression of cancer may lead to complications of thromboembolic events. The tumor may be a source of fibrinogen. Fibrinogen inside the tumor is one of the factors of its growth and metastasis. Fibrinogen, fibrin and their degradation products possess proinflammatory activity. They indirectly stimulate endothelium to secrete von Willebrand factor, leading to activation of platelets accompanying neoplastic disorders. Fragments E and D are the end products of fibrin(ogen) degradation and E and DD are the end products of stabilized fibrin. E stimulates proliferation, migration and differentiation of endothelial cells, contributing to tumor vasculature. Increased levels of DD are observed in malignant neoplasms, such as breast, lung, colon and ovary cancers. In breast cancers DD correlates with progression of disease and metastasis. The role of fibrinogen and the products of its degradation in the progression of various tumors is not sufficiently understood.

Aronia melanocarpa as a protector against nitration of fibrinogen.

Fibrinogen (Fg) also known as coagulation factor I represents about 4% of the total human plasma proteins. The main function of Fg is its involvement in last phase of blood coagulation cascade, when thrombin-induced conversion of dissolved plasma fibrinogen into an insoluble fibrin clot occurs. The reaction of fibrinogen with peroxynitrite causes both structural modifications and changes of the biological properties of this plasma glycoprotein. Recently, there is an increased interest in the screening of natural products present in fruits, vegetables and herbs for their possible antioxidative activities. Therefore, the aim of our study was to estimate the effect of extract from berries of Aronia melanocarpa against nitrative and oxidative damage induced by peroxynitrite. The extract from A. melanocarpa (0.5-50 µg/ml) added to Fg 10 min before peroxynitrite (100 µM) significantly inhibited both the formation of the high molecular weight protein aggregates and nitration of Fg molecule. The extract also abolished peroxynitrite-induced inhibition of fibrinogen polymerization (by 95% at 50 µg/ml). The obtained results indicate that natural extract from berries of A. melanocarpa has protective effects against peroxynitrite-induced nitrative damage of plasma fibrinogen, and therefore may contribute in the prevention of peroxynitrite-related cardiovascular or inflammatory diseases.

Evolution of thrombin and other hemostatic proteases by survey of protochordate, hemichordate, and echinoderm genomes.

Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications.

Protective effects of (-)-epicatechin against nitrative modifications of fibrinogen.

Fibrinogen appears to be particularly sensitive to toxic action of peroxynitrite; a potent oxidizing and nitrating species. An increased nitration of fibrinogen has been reported in cardiovascular diseases. The defense mechanisms against PN are crucial for complex hemostasis process. Flavonoids have antioxidative properties and could protect biomolecules against action of peroxynitrite. The aim of our studies was to establish, if (-)-epicatechin may in vitro protect fibrinogen molecule against peroxynitrite-induced nitration of tyrosines and change its thrombin-catalyzed polymerization. The exposure of purified fibrinogen (6 µM) to peroxynitrite (1-100 µM) resulted in both structural modifications and clotting ability of this glycoprotein. Peroxynitrite at the concentration of 1 µM increased maximum velocity of Fg polymerization, whereas exposure to 100 µM PN resulted in a significant decrease of Vmax. (-)-Epicatechin (1-100 µM) caused a dose-dependent inhibition of 3-nitrotyrosine formation in fibrinogen treated with peroxynitrite (100 µM) in both Western blot assays and C-ELISA assays. At the highest concentration of (-)-epicatechin (100 µM) the level of 3-NT in fibrinogen reached the control values. At lower doses (-)-epicatechin reduced tyrosine nitration by approx. 23% and 40% at the concentration of 1 µM and 10 µM, respectively. (-)-Epicatechin also abolished the pro-thrombotic effect of peroxynitrite on fibrinogen clotting. The presented in vitro results demonstrated for the first time that (-)-epicatechin might have protective effects against the impairment of structure and properties of Fg, caused by action of the strong biologic oxidant/nitration and inflammatory mediators.

Oxidative modification of patient's plasma proteins and its role in pathogenesis of multiple sclerosis.

BACKGROUND: Oxidative stress plays an important role in multiple sclerosis (MS). OBJECTIVE AND METHODS: The present study was designed to evaluate the modifications of plasma proteins by estimation markers of oxidative/nitrosative stress: carbonyl groups and 3-nitrotyrosines (3-NT) levels in relapsing-remitting (RR) (n=10) and secondary progressive (SP) (n=10) clinical course of multiple sclerosis. Moreover, we estimated the level of uric acid (UA) in plasma of MS patients. RESULTS: Compared to controls (n=10), the levels of carbonyl groups in plasma proteins were elevated (P<0.0001) as well in RRMS as in SPMS. The highest concentration of 3-NT was observed in plasma proteins obtained from SPMS patients (P<0.0005). The level of uric acid in plasma was significantly lower in RRMS (P<0.0001) than SPMS. CONCLUSION: This is the first report which presented differences between SPMS and RRMS patients in 3-NT and protein carbonyl groups in plasma proteins.