Effect of methylmercuric chloride (MMC) on fibrin polymerization

Methylmercuric chloride (MMC) in concentrations 0.1–10μM reduces the amount of fibrinopeptides released from thrombinactivated human fibrinogen. However, the fibrin clot formation is not discriminated and the turbidity of the fibrin gel is even augmented. MMC does not cause such changes in the process of repolymerization of fibrin monomers. The addition of fibrinopeptides to the fibrin monomers results in a similar increase of turbidity of the repolymerizing sample in the presence of MMC as in the case of fibrinogen clotting. These experiments indicate that MMC modifies the structure of fibrin in the presence of fibrinopeptides.     

Clotting of fibrinogen. 2. Calorimetry of the reversal of the effect of calcium on clotting with thrombin and with ancrod

When clotting is effected by thrombin in the presence of calcium, the endotherm for the D nodules of fibrinogen broadens significantly and then becomes narrow again, while increasing in size. Clotting effected by the snake venom enzyme Ancrod, which releases only the A fibrinopeptides from the E nodule, shows only the broadening of the D endotherm. Accordingly, significant interactions of the D nodules of fibrinogen become possible only when the B fibrinopeptides of the E nodule are released on clotting. When calcium present during clotting is removed from the fibrin clot with ethylenediaminetetraacetic acid, the endotherm for the D nodules of fibrin shows nearly complete reversal if clotting was effected with Ancrod but appears to be divided into two endotherms if clotting was effected with thrombin. At neutral pH, new endotherms were observed for fibrinogen in the temperature range 105-140 degrees C.     

An integrated study of fibrinogen during blood coagulation.

The rate of conversion of fibrinogen (Fg) to the insoluble product fibrin (Fn) is a key factor in hemostasis. We have developed methods to quantitate fibrinopeptides (FPs) and soluble and insoluble Fg/Fn products during the tissue factor induced clotting of whole blood. Significant FPA generation (>50%) occurs prior to visible clotting (4 +/- 0.2 min) coincident with factor XIII activation. At this time Fg is mostly in solution along with high molecular weight cross-linked products. Cross-linking of gamma-chains is virtually complete (5 min) prior to the release of FPB, a process that does not occur until after clot formation. FPB is detected still attached to the beta-chain throughout the time course demonstrating release of only low levels of FPB from the clot. After release of FPB a carboxypeptidase-B-like enzyme removes the carboxyl-terminal arginine resulting exclusively in des-Arg FPB by the 20-min time point. This process is inhibited by epsilon-aminocaproic acid. These results demonstrate that transglutaminase and carboxypeptidase enzymes are activated simultaneously with Fn formation. The initial clot is a composite of Fn I and Fg already displaying gamma-gamma cross-linking prior to the formation of Fn II with Bbeta-chain remaining mostly intact followed by the selective degradation of FPB to des-Arg FPB.     

Blood coagulation studies in guineapigs (Cavia porcellus)

Blood coagulation studies were performed on 45 healthy, adult guinea pigs. Additionally thrombelastograms of 30 animals were recorded. Guineapigs revealed short partial thromboplastin times and euglobulin lysis times, but long prothrombin times and thrombin times. Fibrinogen values were within the range of human normal values. Biphasic ADP-induced aggregation of platelets, as occurs in man, was found in 29% of the animals. Short r (reaction time until the beginning of clot formation) and k times (time from the beginning of clot formation until an amplitude of 20 mm) of their thrombelastograms indicate, that whole blood clotting is enhanced in guineapigs. Higher maximum amplitudes in this species suggest a stronger clot stability than in man.     

The review of contemporary ideas about the influence of flow rate on blood clotting

Normal blood clotting is vitally important for mammals. The diffusion-convection transfer of clotting factors plays a key role in blood clot formation. Since the shear rates of blood flow are very high (up to 7000 s⁻¹), clot formation critically depends on the flow rate. The methods of study of the flow effect on clotting are indirect and the processes are rather complex; therefore, mathematical models of this process are significant for interpretation of results and understanding of the mechanisms. The review expounds the main experimental data on the effect of flow on the blood clotting cascade, some hypotheses and mathematical models explaining different regimes of the functioning of this system. The review is focused on specific problems encountered by researchers in this field. Some of the experimental works have shown that flow decreases the size of the formed fibrin clot and that the dependence of clot formation period on the flow shear rate is a threshold function. However, there are also experimental evidence that the flow can increase production of clotting factors (factor Xa), which must be expressed in the procoagulant action of the flow. Mathematical models of different aspects of clotting give no unified predictions either. Nevertheless, the combined analysis of results of detailed modeling and experiments, in our opinion, may result in understanding of the mechanisms, which determine the behavior of clotting in a flow.     

A simple peak detection and label-free quantitation algorithm for chromatography-mass spectrometry

Background

Label-free quantitation of mass spectrometric data is one of the simplest and least expensive methods for differential expression profiling of proteins and metabolites. The need for high accuracy and performance computational label-free quantitation methods is still high in the biomarker and drug discovery research field. However, recent most advanced types of LC-MS generate huge amounts of analytical data with high scan speed, high accuracy and resolution, which is often impossible to interpret manually. Moreover, there are still issues to be improved for recent label-free methods, such as how to reduce false positive/negatives of the candidate peaks, how to expand scalability and how to enhance and automate data processing. AB3D (A simple label-free quantitation algorithm for Biomarker Discovery in Diagnostics and Drug discovery using LC-MS) has addressed these issues and has the capability to perform label-free quantitation using MS1 for proteomics study.

Results

We developed an algorithm called AB3D, a label free peak detection and quantitative algorithm using MS1 spectral data. To test our algorithm, practical applications of AB3D for LC-MS data sets were evaluated using 3 datasets. Comparisons were then carried out between widely used software tools such as MZmine 2, MSight, SuperHirn, OpenMS and our algorithm AB3D, using the same LC-MS datasets. All quantitative results were confirmed manually, and we found that AB3D could properly identify and quantify known peptides with fewer false positives and false negatives compared to four other existing software tools using either the standard peptide mixture or the real complex biological samples of Bartonella quintana (strain JK31). Moreover, AB3D showed the best reliability by comparing the variability between two technical replicates using a complex peptide mixture of HeLa and BSA samples. For performance, the AB3D algorithm is about 1.2 - 15 times faster than the four other existing software tools.

Conclusions

AB3D is a simple and fast algorithm for label-free quantitation using MS1 mass spectrometry data for large scale LC-MS data analysis with higher true positive and reasonable false positive rates. Furthermore, AB3D demonstrated the best reproducibility and is about 1.2- 15 times faster than those of existing 4 software tools.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0376-0) contains supplementary material, which is available to authorized users.     

A kinetic model for the alpha-thrombin-catalyzed conversion of plasma levels of fibrinogen to fibrin in the presence of antithrombin III

In this study we report a kinetic model for the alpha-thrombin-catalyzed production of fibrin I and fibrin II at pH 7.4, 37 degrees C, gamma/2 0.17. The fibrin is produced by the action of human alpha-thrombin on plasma levels of human fibrinogen in the presence of the major inhibitor of alpha-thrombin in plasma, antithrombin III (AT). This model quantitatively accounts for the time dependence of alpha-thrombin-catalyzed release of fibrinopeptides A and B concurrent with the inactivation of alpha-thrombin by AT and delineates the concerted interactions of alpha-thrombin, fibrin(ogen), and AT during the production of a fibrin clot. The model also provides a method for estimating the concentration of alpha-thrombin required to produce a clot of known composition and predicts a direct relationship between the plasma concentration of fibrinogen and the amount of fibrin produced by a bolus of alpha-thrombin. The predicted relationship between the concentration of fibrinogen and the amount of fibrin produced in plasma provides a plausible explanation for the observed linkage between plasma concentrations of fibrinogen and the risk for ischemic heart disease.     

Systematic discovery of ectopic pregnancy serum biomarkers using 3-D protein profiling coupled with label-free quantitation

Ectopic pregnancy (EP) and normal intrauterine pregnancy (IUP) serum proteomes were quantitatively compared to systematically identify candidate biomarkers. A 3-D biomarker discovery strategy consisting of abundant protein immunodepletion, SDS gels, LC-MS/MS, and label-free quantitation of MS signal intensities identified 70 candidate biomarkers with differences between groups greater than 2.5-fold. Further statistical analyses of peptide quantities were used to select the most promising 12 biomarkers for further study, which included known EP biomarkers, novel EP biomarkers (ADAM12 and ISM2), and five specific isoforms of the pregnancy specific beta-1-glycoprotein family. Technical replicates showed good reproducibility and protein intensities from the label-free discovery analysis compared favorably with reported abundance levels of several known reference serum proteins over at least 3 orders of magnitude. Similarly, relative abundances of candidate biomarkers from the label-free discovery analysis were consistent with relative abundances from pilot validation assays performed for five of the 12 most promising biomarkers using label-free multiple reaction monitoring of both the patient serum pools used for discovery and the individual samples that constituted these pools. These results demonstrate robust, reproducible, in-depth 3-D serum proteome discovery, and subsequent pilot-scale validation studies can be achieved readily using label-free quantitation strategies.     

Mechanism of Coagulation in Gecarcinus lateralis

Agglutination of cells, degranulation, and loss of cellularmembranes compose the major form of coagulation in the hemolymphof Gecarcinus lateralis. It is only after agglutination of theformed elements of the hemolymph that fibrin-like strands appear.Sodium citrate, in a concentration of 10% or more to preventcoagulation, is always inadequate to prevent cell agglutination. Multiple studies by protein electrophoresis failed to revealany differences between plasma and serum, nor did they allowus to identify a soluble protein in plasma that did not appearin serum. Crab hemolymph changes in its capacity for clottingduring the molt cycle, with the most rapid clotting occurringin the premolt period. A new protein appears in the premoltperiod, but its relation to the whole clotting mechanism isunknown. In contradistinction to vertebrate systems, citrated hemolymphdoes not clot when calcium is added. There is no relationshipthat can be demonstrated between activating systems in vertebrateplasma and clotting in the crab. It would seem that, ratherthan the vertebrate coagulating system evolving from the crustaceantype of clotting system, the development of these clotting systemshas run in parallel. The crustacean cell, in addition, appearsto be more potent than vertebrate cells in clotting systems.The comparison of human lymph to crustacean hemolymph wouldindicate that, for a given amount of cells, crustacean hemolymphclots 2 to 20 times faster than human lymph. On the other hand,agglutination of cells is a fundamental initiating step in coagulationof both human blood and crustacean hemolymph.     

Spatiotemporal Characterization of a Fibrin Clot Using Quantitative Phase Imaging

Studying the dynamics of fibrin clot formation and its morphology is an important problem in biology and has significant impact for several scientific and clinical applications. We present a label-free technique based on quantitative phase imaging to address this problem. Using quantitative phase information, we characterized fibrin polymerization in real-time and present a mathematical model describing the transition from liquid to gel state. By exploiting the inherent optical sectioning capability of our instrument, we measured the three-dimensional structure of the fibrin clot. From this data, we evaluated the fractal nature of the fibrin network and extracted the fractal dimension. Our non-invasive and speckle-free approach analyzes the clotting process without the need for external contrast agents.     

Selective Light-Triggered Release of DNA from Gold Nanorods Switches Blood Clotting On and Off

Blood clotting is a precise cascade engineered to form a clot with temporal and spatial control. Current control of blood clotting is achieved predominantly by anticoagulants and thus inherently one-sided. Here we use a pair of nanorods (NRs) to provide a two-way switch for the blood clotting cascade by utilizing their ability to selectively release species on their surface under two different laser excitations. We selectively trigger release of a thrombin binding aptamer from one nanorod, inhibiting blood clotting and resulting in increased clotting time. We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting. Thus, the nanorod pair acts as an on/off switch. One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function. We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.     

Effects of exercise and conditioning on clotting and fibrinolytic activity in men

Sixty healthy men in three physical fitness categories (sedentary, on no organized fitness program; joggers, running 5-15 miles/wk; and marathoners, running greater than 50 miles/wk) were evaluated for changes in blood clotting and fibrinolytic activity before and after maximum exercise on a treadmill according to the Bruce protocol. The rate of blood clotting, as measured by prothrombin times, partial thromboplastin times and thrombin times, was accelerated by exercise (all P less than 0.005). The ability of euglobulin clots and plasma clots to lyse incorporated 125I-fibrin, termed 125I-euglobulin clot lysis (IEL) and 125I-plasma clot lysis (IPCL), were used as indexes of fibrinolytic activity. Marathoners had greater increases in fibrinolytic activity with exercise (76% compared with 63% for joggers and 55% for sedentary subjects by IEL; 427% compared with 418% for joggers and 309% for sedentary subjects by IPCL; all P less than 0.05). Fibrin degradation products increased with exercise (P less than 0.005 for the total group of 60 subjects). The absolute concentrations of alpha 2-plasmin inhibitor, alpha 2-macroglobulin, and antithrombin III increased with exercise (all P less than 0.005), but when concentrations were corrected for acute shifts of plasma water during exercise, the quantity of these inhibitors actually decreased (all P less than 0.005). The changes in clotting assays with exercise were not significantly correlated with changes in whole blood lactate, blood pyruvate, or rectal temperatures. Fibrinolytic assays before and after exercise correlated poorly to moderately with blood lactates (IEL: r = 0.441 and r = 0.425, respectively; IPCL: r = 0.294 and r = 0.544, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism

Background

It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein—fibrinogen and fibrin that forms a plasma clot.

Methods

The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC–MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results.

Results

Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC–MS/MS.

Conclusions

The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

     

Poly(carboxybetaine methacrylamide)-modified nanoparticles: a model system for studying the effect of chain chemistry on film properties, adsorbed protein conformation, and clot formation kinetics

Nonfouling polymer architectures are considered important to the successful implementation of many biomaterials. It is thought that how these polymers induce conformational changes in proteins upon adsorption may dictate the fate of the device being utilized. Herein, oxidized silicon nanoparticles (SiNP) were modified with various forms of poly(carboxybetaine methacrylamide) (PCBMA) for the express purpose of understanding how polymer chemistry affects film hydration, adsorbed protein conformation, and clot formation kinetics. To this end, carboxybetaine monomers differing in intercharge separating spacer groups were synthesized, and nitroxide-mediated free radical polymerization (NMP) was conducted using alkoxyamine initiators with hydrophobic (TEMPO) and hydrophilic (β-phosphonate) terminal groups. The physical properties (surface composition, thickness, grafting density, etc.) of the resulting polymer-SiNP conjugates were quantified using several techniques, including Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and thermogravimetric analysis (TGA). The effect of spacer group on the surface charge density was determined using zeta potential measurements. Three proteins, viz., lysozyme, bovine α-lactalbumin, and human serum albumin, were used to evaluate the effect film properties (charge, hydration, end-group) have on adsorbed protein conformation, as determined by circular dichroism (CD), fluorescence spectroscopy, and fluorescence quenching techniques. Hemocompatibility of these surfaces was observed by measuring clot formation kinetics using the plasma recalcification time assay. It was found that chain chemistry, as opposed to end-group chemistry, was a major determiner for water structure, adsorbed protein conformation, and clotting kinetics. It is thought that the systematic evaluation of how both chain (internal) and end-group (external) polymer properties affect film hydration, protein conformation, and clot formation will provide valuable insight that can be applied to all engineered surfaces for biomedical applications.     

Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga

Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.     

Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: Interference and comparison with established methods

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.     

Application and validation of the lattice Boltzmann method for modelling flow-related clotting

The purpose of this paper is to present a simple clotting model, based on residence time and shear stress distribution, that can simulate the deposition over time of enzyme-activated milk in an in vitro system. Results for the model are compared with experiments exhibiting clot deposition in the region of a sharp-edged stenosis. The milk experiments have been shown to be a valuable analogue for the experimental representation of flow-induced blood clotting, particularly in the context of separation of hydrodynamic from biochemical factors. The facility to predict the flow-induced clotting of the blood analogue, in which the chemistry reduces to what is effectively a zeroth order reaction, gives confidence in this physics-based approach to simulation of the final part of the coagulation cascade. This type of study is a necessary precursor to the development of a complex, multi-factorial, biochemical model of the process of thrombosis. In addition to the clotting simulations, comparisons are reported between the computed flow patterns prior to clot deposition and flow visualisation studies. Excellent agreement of hydrodynamic parameters is reported for a Reynolds number of 100, and qualitative agreement is seen for the complex, disturbed flow occurring at a physiologically relevant Reynolds number of 550. The explicit, time-stepping lattice Boltzmann approach may have particular merit for the transitional flow at this higher Reynolds number.     

Influence of common preanalytical variations on the metabolic profile of serum samples in biobanks

A blood pre-centrifugation delay of 24 h at room temperature influenced the proton NMR spectroscopic profiles of human serum. A blood pre-centrifugation delay of 24 h at 4°C did not influence the spectroscopic profile as compared with 4 h delays at either room temperature or 4°C. Five or ten serum freeze–thaw cycles also influenced the proton NMR spectroscopic profiles. Certain common in vitro preanalytical variations occurring in biobanks may impact the metabolic profile of human serum.