Metabolomic profile of prostate cancer-specific survival among 1812 Finnish men.

BACKGROUND: Abnormal metabolism and perturbations in metabolic pathways play significant roles in the development and progression of prostate cancer; however, comprehensive metabolomic analyses of human data are lacking and needed to elucidate the interrelationships. METHODS: We examined the serum metabolome in relation to prostate cancer survival in a cohort of 1812 cases in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. Using an ultrahigh-performance LC-MS/MS platform, we identified 961 known metabolites in prospectively collected serum. Median survival time from diagnosis to prostate cancer-specific death (N=472) was 6.6 years (interquartile range=2.9-11.1 years). Cox proportional hazards regression models estimated hazard ratios and 95% confidence intervals of the associations between the serum metabolites (in quartiles) and prostate cancer death, adjusted for age at baseline and diagnosis, disease stage, and Gleason sum. In order to calculate risk scores, we first randomly divided the metabolomic data into a discovery set (70%) and validated in a replication set (30%). RESULTS: Overall, 49 metabolites were associated with prostate cancer survival after Bonferroni correction. Notably, higher levels of the phospholipid choline, amino acid glutamate, long-chain polyunsaturated fatty acid (n6) arachidonate (20:4n6), and glutamyl amino acids gamma-glutamylglutamate, gamma-glutamylglycine, and gamma-glutamylleucine were associated with increased risk of prostate cancer-specific mortality (fourth versus first quartile HRs=2.07-2.14; P-values <5.2×10-5). By contrast, the ascorbate/aldarate metabolite oxalate, xenobiotics S-carboxymethyl-L-cysteine, fibrinogen cleavage peptides ADpSGEGDFXAEGGGVR and fibrinopeptide B (1-12) were related to reduced disease-specific mortality (fourth versus first quartile HRs=0.82-0.84; P-value <5.2×10-5). Further adjustment for years from blood collection to cancer diagnosis, body mass index, smoking intensity and duration, and serum total and high-density lipoprotein cholesterol did not alter the results. Participants with a higher metabolic score based on the discovery set had an elevated risk of prostate cancer-specific mortality in the replication set (fourth versus first quartile, HR=3.9, P-value for trend<0.0001). CONCLUSIONS: The metabolic traits identified in this study, including for choline, glutamate, arachidonate, gamma-glutamyl amino acids, fibrinopeptides, and endocannabinoid and redox pathways and their composite risk score, corroborate our previous analysis of fatal prostate cancer and provide novel insights and potential leads regarding the molecular basis of prostate cancer progression and mortality.

Prototyping of a Microfluidic Modulator Chip and Its Application in Heart-Cut Strong-Cation-Exchange-Reversed-Phase Liquid Chromatography Coupled to Nanoelectrospray Mass Spectrometry for Targeted Proteomics.

A novel multilayer modulator chip offering a robust miniaturized interface for multidimensional liquid chromatography has been developed. The thermoplastic microfluidic device comprises five tailor-made functional layers, and the chip is compatible with commercially available switching-valve technology. The modulator chip allows for robust ultrahigh-pressure operation up to 65 MPa. Peak-dispersion characteristics of system peaks were assessed directly at the valve outlet by monitoring fluorescein injection profiles with laser-induced fluorescence detection. Integration of a microporous monolithic mixing entity in the microchannels significantly narrows the resulting peak profile. Proof-of-concept of the applicability of the microfluidic modulator chip is demonstrated in a heart-cut multidimensional strong-cation-exchange-reversed-phase liquid chromatography proteomics analysis workflow coupled to nanoelectrospray mass spectrometry for the target analysis of Glu-1-Fibrinopeptide B spiked in a protein digest mixture of bovine serum albumin.

Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry.

Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g., ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultrafast benchtop method for radical generation for HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond time scale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g., ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that effective hydroxyl radical yields in FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be compensated on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis.

Protein structural analysis by mass spectrometry has gained significant popularity in recent years, including high-resolution protein topographical mapping by fast photochemical oxidation of proteins (FPOP). The ability to provide protein topographical information at moderate spatial resolution makes FPOP an attractive technology for the protein pharmaceutical discovery and development processes. However, current technology limits the throughput and requires significant manual sample manipulation. Similarly, as FPOP is being used on larger samples, sample flow through the capillary becomes challenging. No systematic comparison of the performance of static flash photolysis with traditional flow FPOP has been reported. Here, we evaluate a 96-well microtiter-based laser flash photolysis method for the topographical probing of proteins, which subsequently could be used to analyze higher order structure of the protein in a high-throughput fashion with minimal manual sample manipulation. We used multiple metrics to compare microtiter FPOP performance with that of traditional flow FPOP: adenine-based hydroxyl radical dosimetry, oxidation efficiency of a model peptide, and hydroxyl radical protein footprint of myoglobin. In all cases, microtiter plate FPOP performed comparably with traditional flow FPOP, requiring a small fraction of the time for exposure. This greatly reduced sample exposure time, coupled with automated sample handling in 96-well microtiter plates, makes microtiter-based FPOP an important step in achieving the throughput required to adapt hydroxyl radical protein footprinting for screening purposes.

Reduced plasminogen binding and delayed activation render γ'-fibrin more resistant to lysis than γA-fibrin.

Fibrin (Fn) clots formed from γ'-fibrinogen (γ'-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γA-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ'-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ'-Fn, and/or altered cross-linking. Clots formed from γ'-Fg lysed more slowly than those formed from γA-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ'-Fn with an association rate constant 22% lower than that to γA-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ'-Fn than with γA-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ'-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ'-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ'-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis.

Determination of [Glu1]-fibrinopeptide B purity by gas chromatography - isotope dilution mass spectrometry.

The present work assessed the purity of [Glu1]-fibrinopeptide B (GFB) as a model peptide using gas chromatography - isotope dilution mass spectrometry. GFB and various isotope-labeled amino acids were hydrolyzed in HCl and then derivatized using optimized procedures. The primary impurity in GFB was also identified and used to correct the final result. A method repeatability of 0.5% was achieved and linear calibrations were obtained for five amino acids. The LOD and LOQ were 0.041 to 0.096 µg g-1, and 0.16 to 0.56 µg g-1, respectively. The purity of GFB was found to be (0.715 ± 0.012) g g-1. This technique exhibited comparable accuracy to that obtainable from liquid chromatography - isotope dilution mass spectrometry but at lower cost. This method could be employed as a reference technique or in fields such as clinical diagnostics or bio-pharmaceutical peptide purity analysis.

Fibrinopeptides A and B release in the process of surface fibrin formation.

Fibrinogen adsorption on a surface results in the modification of its functional characteristics. Our previous studies revealed that fibrinogen adsorbs onto surfaces essentially in 2 different orientations depending on its concentration in the solution: "side-on" at low concentrations and "end-on" at high concentrations. In the present study, we analyzed the thrombin-mediated release of fibrinopeptides A and B (FpA and FpB) from fibrinogen adsorbed in these orientations, as well as from surface-bound fibrinogen-fibrin complexes prepared by converting fibrinogen adsorbed in either orientation into fibrin and subsequently adding fibrinogen. The release of fibrinopeptides from surface-adsorbed fibrinogen and from surface-bound fibrinogen-fibrin complexes differed significantly compared with that from fibrinogen in solution. The release of FpB occurred without the delay (lag phase) characteristic of its release from fibrinogen in solution. The amount of FpB released from end-on adsorbed fibrinogen and from adsorbed fibrinogen-fibrin complexes was much higher than that of FpA. FpB is known as a potent chemoattractant, so its preferential release suggests a physiological purpose in the attraction of cells to the site of injury. The N-terminal portions of fibrin ß chains including residues Bß15-42, which are exposed after cleavage of FpB, have been implicated in many processes, including angiogenesis and inflammation.

Increased thrombosis susceptibility and altered fibrin formation in STAT5-deficient mice.

To explore the effect(s) of growth hormone signaling on thrombosis, we studied signal transduction and transcription factor 5 (STAT5)-deficient mice and found markedly reduced survival in an in vivo thrombosis model. These findings were not explained by a compensatory increase in growth hormone secretion. There was a modest increase in the activity of several procoagulant factors, but there was no difference in the rate or magnitude of thrombin generation in STAT5-deficient mice relative to control. However, thrombin-triggered clot times were markedly shorter, and fibrin polymerization occurred more rapidly in plasma from STAT5-deficient mice. Fibrinogen depletion and mixing studies indicated that the effect on fibrin polymerization was not due to intrinsic changes in fibrinogen, but resulted from changes in the concentration of a circulating plasma inhibitor. While thrombin-triggered clot times were significantly shorter in STAT5-deficient animals, reptilase-triggered clot times were unchanged. Accordingly, while the rate of thrombin-catalyzed release of fibrinopeptide A was similar, the release of fibrinopeptide B was accelerated in STAT5-deficient plasma versus control. Taken together, these studies demonstrated that the loss of STAT5 resulted in a decrease in the concentration of a plasma inhibitor affecting thrombin-triggered cleavage of fibrinopeptide B. This ultimately resulted in accelerated fibrin polymerization and greater thrombosis susceptibility in STAT5-deficient animals.

Kinetic characterization of inhibition of human thrombin with DNA aptamers by turbidimetric assay.

A sensitive turbidimetric method for detecting fibrin association was used to study the kinetics of fibrinogen hydrolysis with thrombin. The data were complemented by high-performance liquid chromatography (HPLC) measurements of the peptide products, fibrinopeptides released during hydrolysis. Atomic force microscopy (AFM) data showed that the fibril diameter is the main geometric parameter influencing the turbidity. The turbidimetric assay was validated using thrombin with the standard activity. To study thrombin inhibitors, a kinetic model that allows estimating the inhibition constants and the type of inhibition was proposed. The kinetic model was used to study the inhibitory activity of the two DNA aptamers 15-TBA (thrombin-binding aptamer) and 31-TBA, which bind to thrombin exosites. For the first time, 31-TBA was shown to possess the competitive inhibition type, whereas the shortened aptamer 15-TBA has the noncompetitive inhibition type.

Oncostatin M receptor ß and cysteine/histidine-rich 1 are biomarkers of the response to erythropoietin in hemodialysis patients.

Biomarkers that evaluate the response to erythropoietic-stimulating agents largely measure inflammation and iron availability. While these are important factors in modifying an individual's response to these agents, they do not address all aspects of a poor response. To clarify this, we isolated peptides in the serum of good and poor responders to erythropoietin in order to identify biomarkers of stimulating agent response. Ninety-one candidate biomarker targets were identified and characterized using mass spectrometry, of which tandem mass spectroscopy provided partial amino-acid sequence information of 17 different peptides for 16 peptide masses whose abundance significantly differed between poor and good responders. The analysis concluded that three peptides associated with a poor response were derived from oncostatin M receptor ß (OSMRß). The 13 serum peptides associated with a good response were derived from fibrinogen α and ß, coagulation factor XIII, complement C3, and cysteine/histidine rich 1(CYHR1). Poor response was most strongly associated with the OSMRß fragment with the largest molecular weight, while a good response was most strongly associated with CYHR1. Immunoblots found the abundance of intact OSMRß and CYHR1 significantly differed between good and poor responders. Thus, two measurable biomarkers of the response to erythropoietic-stimulating agents are present in the serum of treated patients.

Novel aspects of fibrin(ogen) fragments during inflammation.

Coagulation is fundamental for the confinement of infection and/or the inflammatory response to a limited area. Under pathological inflammatory conditions such as arthritis, multiple sclerosis or sepsis, an uncontrolled activation of the coagulation system contributes to inflammation, microvascular failure and organ dysfunction. Coagulation is initiated by the activation of thrombin, which, in turn, triggers fibrin formation by the release of fibrinopeptides. Fibrin is cleaved by plasmin, resulting in clot lysis and an accompanied generation of fibrin fragments such as D and E fragments. Various coagulation factors, including fibrinogen and/or fibrin [fibrin(ogen)] and also fibrin degradation products, modulate the inflammatory response by affecting leukocyte migration and cytokine production. Fibrin fragments are mostly proinflammatory, however, Bß15-42 in particular possesses potential antiinflammatory effects. Bß15-42 inhibits Rho-kinase activation by dissociating Fyn from Rho and, hence prevents stress-induced loss of endothelial barrier function and also leukocyte migration. This article summarizes the state-of-the-art in inflammatory modulation by fibrin(ogen) and fibrin fragments. However, further research is required to gain better understanding of the entire role fibrin fragments play during inflammation and, possibly, disease development.

The application of atmospheric pressure matrix-assisted laser desorption/ionization to the analysis of long-term cryopreserved serum peptidome.

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API-MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API-MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at -80°C for 18 months or at -20°C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API-MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.

Activation of mannan-binding lectin-associated serine proteases leads to generation of a fibrin clot.

The lectin pathway of complement is activated upon binding of mannan-binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL-and FCN-associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP-1 has thrombin-like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP-2 has factor Xa-like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L-FCN-MASPs complexes, bound from serum to N-acetylcysteine-Sepharose, or MBL-MASPs complexes, bound to mannan-agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti-D-dimer antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L-FCN-MASPs complexes captured on N-acetylcysteine-Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP-2 and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP-1 and MASP-2, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP-mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP-catalysed deposition and polymerization of fibrin on the surface of micro-organisms may be protective by limiting the dissemination of infection.

Nature of T lymphocyte recognition of macrophage-associated antigens. V. Contribution of individual peptide residues of human fibrinopeptide B to T lymphocyte responses.

Guinea pig T lymphocyte responses to a decapeptide antigen (NH1-Asp5-Ans6-Glu7-Glu8-Gly9-Phe10-Phe11-Ser12-Ala13-Arg14-OH) of human fibrinopeptide B (hFPB) were examined using various synthetic peptide analogues containing single residue substitutions. Each analogue was examined for antigenicity as determined by an in vitro proliferative responses of hFPN-immune strain 2 guinae pig T cells. In addition, both strain 2 and strain 13 animals were immunized with each analogue and immunogenicity assessed by in vitro T cell-proliferative responses with the homologous immunizing analogue and the parent peptide. Replacement of arginine14 with lysine formed an immunogenic analogue which showed no antigenic cross-reactivity with the native peptide in strain 2 T cell responses. In addition, substitution of arginine14 with blocked lysine again produced a unique immunogenic analogue that showed little or no antigenic identity with the intact lysine analogue or the native peptide. In similar fashion, substitution of resideu phenylalanie10 with tyrosine or Phe(4-NO2) created unique immunogenic analogues with little or no antigenic identity to the native peptide with strain 2 T cells. By contrast, replacement of phenylalanine11 with either tyrosine or Phe(4-NO2) resulted in analogues with a total loss of immunogenicity and antigenicity in strain 2 T cell responses. An analogue in which glutamic acid7,8 were replaced with glutamine retained a small degree of antigenicity with hFPB-immune T cells, but T cells from strain 2 animals immunized with the Gln analogue responded only marginally to the Gln analogue while producing good proliferative responses with the native peptide. On the other hand, an analogue in which asparatic acid5 was replaced with asparagine retained most of the antigenic identity with hFPB for strain 2 T cell responses. None of thee analogues were immunogenic for strain 13 guinea pigs. These observations are discussed with respect to the contribution of each substituted residue to T cell respones, mechanism of Ir gene function, and a model for T cell recognition of small peptide antigens.

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen.

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.

Protein ladder sequencing.

A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost.

Fibrinopeptide B and aggregation of fibrinogen.

Removal of fibrinopeptide B from human fibrinogen by reaction with the procoagulant enzyme from copperhead snake venom below 25 degrees C resulted in tight aggregation of the fibrinogen, which, in turn, progressively blocked a concomitant but sluggish release of fibrinopeptide A by the enzyme. When the clots obtained at less than 25 degrees C were warmed, they dissociated into soluble aggregates and monomers. Release of fibrinopeptide A then resumed, and a secondary coagulation followed. The aggregation induced by release of fibrinopeptide B itself involves a plasmin-susceptible segment located just distal to B in the B beta chain of fibrinogen, a segment previously shown to be of little importance in the aggregation induced by release of fibrinopeptide A.

Nanostructured diamond-like carbon on digital versatile disc as a matrix-free target for laser desorption/ionization mass spectrometry.

A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.

Altered structure and function of fibrinogen after cleavage by Factor VII Activating Protease (FSAP).

Factor VII Activating Protease (FSAP) is a plasma protease affecting both coagulation and fibrinolysis. Although a role in hemostasis is still unclear, the identification of additional physiologic substrates will help to elucidate its role in this context. FSAP has been reported to cleave fibrinogen, but the functional consequences of this are not known. We have therefore undertaken this study to determine the implications of this cleavage for fibrin-clot formation and its lysis. Treatment of human fibrinogen with FSAP released an N-terminal peptide from the Bß chain (Bß1-53) and subsequently the fibrinopeptide B; within the Aα chain a partial truncation of the αC-region by multiple cleavages was seen. The truncated fibrinogen showed a delayed thrombin-catalyzed polymerization and formed fibrin clots of reduced turbidity, indicative of thinner fibrin fibers. Confocal laser scanning and scanning electron microscopy of these clots revealed a less coarse fibrin network with thinner fibers and a smaller pore size. A lower pore size was also seen in permeability studies. Unexpectedly, FSAP-treated fibrinogen or plasma exhibited a significantly faster tPA-driven lysis, which correlated exclusively with cleavage of fibrinogen and not with activation of plasminogen activators. Similar observations were also made in plasma after activation of endogenous zymogen FSAP, but not in plasma of carrier of the rare Marburg I single nucleotide polymorphism. In conclusion, altering fibrin clot properties by fibrinogenolysis is a novel function of FSAP in the vasculature, which facilitates clot lysis and may in vivo contribute to reduced fibrin deposition during thrombosis.

Label-free quantitative proteomics reveals fibrinopeptide B and heparin cofactor II as potential serum biomarkers in respiratory syncytial virus-infected mice treated with Qingfei oral liquid formula.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections. Qingfei oral liquid (QFOL), a traditional Chinese medicine, is widely used in clinical treatment for RSV-induced pneumonia. The present study was designed to reveal the potential targets and mechanism of action for QFOL by exploring its influence on the host cellular network following RSV infection. We investigated the serum proteomic changes and potential biomarkers in an RSV-infected mouse pneumonia model treated with QFOL. Eighteen BALB/c mice were randomly divided into three groups: RSV pneumonia model group (M), QFOL-treated group (Q) and the control group (C). Serum proteomes were analyzed and compared using a label-free quantitative LC-MS/MS approach. A total of 172 protein groups, 1009 proteins, and 1073 unique peptides were successfully identified. 51 differentially expressed proteins (DEPs) were identified (15 DEPs when M/C and 43 DEPs when Q/M; 7 DEPs in common). Classification and interaction network showed that these proteins participated in various biological processes including immune response, blood coagulation, complement activation, and so forth. Particularly, fibrinopeptide B (FpB) and heparin cofactor II (HCII) were evaluated as important nodes in the interaction network, which was closely involved in coagulation and inflammation. Further, the FpB level was increased in Group M but decreased in Group Q, while the HCII level exhibited the opposite trend. These findings not only indicated FpB and HCII as potential biomarkers and targets of QFOL in the treatment of RSV pneumonia, but also suggested a regulatory role of QFOL in the RSV-induced disturbance of coagulation and inflammation-coagulation interactions.