Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism.

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.

Peptidomic Characterization and Amino Acid Availability after Intake of Casein vs. a Casein Hydrolysate in a Pig Model.

It is known that casein hydrolysis accelerates gastrointestinal transit in comparison to intact casein, although the effect of the protein hydrolysis on the composition of the digests is not fully understood. The aim of this work is to characterize, at the peptidome level, duodenal digests from pigs, as a model of human digestion, fed with micellar casein and a previously described casein hydrolysate. In addition, in parallel experiments, plasma amino acid levels were quantified. A slower transit of nitrogen to the duodenum was found when the animals received micellar casein. Duodenal digests from casein contained a wider range of peptide sizes and a higher number of peptides above five amino acids long in comparison with the digests from the hydrolysate. The peptide profile was markedly different, and although ß-casomorphin-7 precursors were also found in hydrolysate samples, other opioid sequences were more abundant in the casein digests. Within the same substrate, the evolution of the peptide pattern at different time points showed minimal changes, suggesting that the protein degradation rate relies more on the gastrointestinal location than on digestion time. Higher plasma concentrations of methionine, valine, lysine and amino acid metabolites were found in animals fed with the hydrolysate at short times (<200 min). The duodenal peptide profiles were evaluated with discriminant analysis tools specific for peptidomics to identify sequence differences between both substrates that can be used for future human physiological and metabolic studies.

Identification of Endothelial Proteins in Plasma Associated With Cardiovascular Risk Factors.

OBJECTIVE: Endothelial cell (EC) dysfunction is a well-established response to cardiovascular disease risk factors, such as smoking and obesity. Risk factor exposure can modify EC signaling and behavior, leading to arterial and venous disease development. Here, we aimed to identify biomarker panels for the assessment of EC dysfunction, which could be useful for risk stratification or to monitor treatment response. Approach and Results: We used affinity proteomics to identify EC proteins circulating in plasma that were associated with cardiovascular disease risk factor exposure. Two hundred sixteen proteins, which we previously predicted to be EC-enriched across vascular beds, were measured in plasma samples (N=1005) from the population-based SCAPIS (Swedish Cardiopulmonary Bioimage Study) pilot. Thirty-eight of these proteins were associated with body mass index, total cholesterol, low-density lipoprotein, smoking, hypertension, or diabetes. Sex-specific analysis revealed that associations predominantly observed in female- or male-only samples were most frequently with the risk factors body mass index, or total cholesterol and smoking, respectively. We show a relationship between individual cardiovascular disease risk, calculated with the Framingham risk score, and the corresponding biomarker profiles. CONCLUSIONS: EC proteins in plasma could reflect vascular health status.

Implication of Opioid Receptors in the Antihypertensive Effect of a Bovine Casein Hydrolysate and αs1-Casein-Derived Peptides.

The antihypertensive activity of two αs1-casein-derived peptides and casein hydrolysate containing these sequences was evaluated in the presence of naloxone. The activity was abolished by this opioid antagonist at 2, 4, and 6 h post-administration. Similarly, the antihypertensive effect of the αs1-casein peptides 90RYLGY94 (-23.8 ± 2.5 mmHg) and 143AYFYPEL149 (-21.1 ± 3.2 mmHg) at 5 mg/kg of body weight was antagonized by the co-administration of naloxone. Because peptide 143AYFYPEL149 had recently shown opioid activity, a molecular dynamic simulation of this peptide with human µ-opioid receptor was performed to demonstrate its favorable structure and interaction energy, despite the presence of Ala at the N terminus. Altogether, these results revealed that the in vivo effect on systolic blood pressure of the studied αs1-casein peptides is mediated by interaction with opioid receptors and the antihypertensive activity of casein hydrolysate can be very likely ascribed to them with the possible contribution of other mechanisms.

Systematic Development of Sandwich Immunoassays for the Plasma Secretome.

The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity-free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles.

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score ≥ 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

Novel Multiomics Profiling of Human Carotid Atherosclerotic Plaques and Plasma Reveals Biliverdin Reductase B as a Marker of Intraplaque Hemorrhage.

Clinical tools to identify individuals with unstable atherosclerotic lesions are required to improve prevention of myocardial infarction and ischemic stroke. Here, a systems-based analysis of atherosclerotic plaques and plasma from patients undergoing carotid endarterectomy for stroke prevention was used to identify molecular signatures with a causal relationship to disease. Local plasma collected in the lesion proximity following clamping prior to arteriotomy was profiled together with matched peripheral plasma. This translational workflow identified biliverdin reductase B as a novel marker of intraplaque hemorrhage and unstable carotid atherosclerosis, which should be investigated as a potential predictive biomarker for cardiovascular events in larger cohorts.

PDGFB, a new candidate plasma biomarker for venous thromboembolism: results from the VEREMA affinity proteomics study.

There is a clear clinical need for high-specificity plasma biomarkers for predicting risk of venous thromboembolism (VTE), but thus far, such markers have remained elusive. Utilizing affinity reagents from the Human Protein Atlas project and multiplexed immuoassays, we extensively analyzed plasma samples from 2 individual studies to identify candidate protein markers associated with VTE risk. We screened plasma samples from 88 VTE cases and 85 matched controls, collected as part of the Swedish "Venous Thromboembolism Biomarker Study," using suspension bead arrays composed of 755 antibodies targeting 408 candidate proteins. We identified significant associations between VTE occurrence and plasma levels of human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1), von Willebrand factor (VWF), glutathione peroxidase 3 (GPX3), and platelet-derived growth factor ß (PDGFB). For replication, we profiled plasma samples of 580 cases and 589 controls from the French FARIVE study. These results confirmed the association of VWF and PDGFB with VTE after correction for multiple testing, whereas only weak trends were observed for HIVEP1 and GPX3. Although plasma levels of VWF and PDGFB correlated modestly (ρ ∼ 0.30) with each other, they were independently associated with VTE risk in a joint model in FARIVE (VWF P < .001; PDGFB P = .002). PDGFΒ was verified as the target of the capture antibody by immunocapture mass spectrometry and sandwich enzyme-linked immunosorbent assay. In conclusion, we demonstrate that high-throughput affinity plasma proteomic profiling is a valuable research strategy to identify potential candidate biomarkers for thrombosis-related disorders, and our study suggests a novel association of PDGFB plasma levels with VTE.

Role of intestinal brush border peptidases in the simulated digestion of milk proteins.

SCOPE: This study aimed to assess the impact of the "often neglected" intestinal brush border membranes (BBMs) hydrolases on dietary peptides, exploring the possibility that the disintegration of proteins progressed in the small intestine up to a "core" of intrinsically stable oligopeptides, persisting independently on the up-stream breakdown. METHODS AND RESULTS: Samples of sodium caseinate, skim milk powder, and whey protein isolate were submitted to in vitro simulated gastropancreatic digestion using two different procedures: (i) a simplified model involving the main compartmental specific proteases; (ii) a static digestion method based on a frameset of parameters inferred from in vivo. The gastroduodenal digesta were further hydrolyzed with peptidases from porcine jejunal BBM. The peptidomes arising from the two digestion models, characterized by combined HPLC and MS techniques, differed to some extent. However, only specific protein domains survived digestion, among which are potential bioactive or immunogenic (food allergy) peptides. The degree of hydrolysis (DH) after BBM digestion (70-77%) practically did not differ between the digestion models and significantly increased the DH after duodenal steps. CONCLUSION: Any in vitro digestion model should be supplemented with a jejunal phase to realistically determine the bioaccessibility and bioavailability of dietary peptides.

Bioavailability and kinetics of the antihypertensive casein-derived peptide HLPLP in rats.

The aim of this study was to investigate the oral bioavailability and kinetics of the milk casein-derived peptide HLPLP, which had previously demonstrated antihypertensive effect in spontaneously hypertensive rats. HLPLP disposition after single intravenous (4 mg/kg body weight) and oral (40 mg/kg body weight) doses was studied in rats. Plasma concentrations of HLPLP [ß-casein fragment f(134-138)], and two derived fragments found after HLPLP administration, LPLP [ß-casein fragment f(135-138)] and HLPL [ß-casein fragment f(134-137)], were determined by ultrahigh performance liquid chromatography (UPLC) coupled on line to a Q-TOF instrument. For HLPLP, the elimination half-lives (T1/2ß) were 7.95 min after intravenous and 11.7 min after oral administration. The volume of distribution at steady state (Vss = 30.8 L/kg) suggests a considerable uptake of HLPLP into tissues. HLPLP was converted to the peptides LPLP and HLPL. After HLPLP intravenous administration, the elimination half-lives (T1/2ß) for these biotransformed peptides, LPLP and HLPL, were 8.38 and 10.9 min, respectively. After oral administration, HLPLP was rapidly absorbed with an absorption half-life (T1/2a) of 2.79 min. The oral bioavailability of HLPLP was found to be 5.18%. Our study suggested that HLPLP was rapidly absorbed and eliminated after oral administration, biotransformed into smaller fragments LPLP and HLPL, and distributed throughout the body by the circulation blood. The present pharmacokinetic information from a preclinical kinetic study in rats can also play an important role in designing future kinetic studies in humans for assessing HLPLP dose-response relationship.

Peptidomic study of Spanish blue cheese (Valdeón) and changes after simulated gastrointestinal digestion.

It is increasingly evident that digestion can affect the biological activity of cheese by the release of new active peptides from their precursors or, on the contrary, giving rise to fragments without activity. The characterization of the peptidome of a Spanish blue cheese, Valdeón, has been conducted before and after gastrointestinal digestion, and the digests have been compared to those obtained from pasteurized skimmed milk powder (SMP) using a bioinformatics platform. Peptidomic profiling of digests revealed several regions that are especially resistant to digestion (among them ß-casein 60-93, 128-140, and 193-209). Some of them correspond to well-conserved regions between species (human, cow, sheep, and goat) and include peptide sequences with reported bioactivity. The great peptide homology found between both digests, cheese and SMP, suggests that the gastrointestinal digestion could bring closer the profile of products with different proteolytic state. Although most of the biologically active peptides found in cheese after digestion were also present in SMP digest, there were some exceptions that can be attributed to the absence of the relevant precursor peptide before digestion.

Novel antihypertensive lactoferrin-derived peptides produced by Kluyveromyces marxianus: gastrointestinal stability profile and in vivo angiotensin I-converting enzyme (ACE) inhibition.

Novel antihypertensive peptides released by Kluyveromyces marxianus from bovine lactoferrin (LF) have been identified. K. marxianus LF permeate was fractionated by semipreparative high performance liquid chromatography and 35 peptides contained in the angiotensin I-converting enzyme (ACE)-inhibitory fractions were identified by using an ion trap mass spectrometer. On the basis of peptide abundance and common structural features, six peptides were chemically synthesized. Four of them (DPYKLRP, PYKLRP, YKLRP, and GILRP) exerted in vitro inhibitory effects on ACE activity and effectively decreased systolic blood pressure after oral administration to spontaneously hypertensive rats (SHRs). Stability against gastrointestinal enzymes suggested that the sequence LRP could contribute to the in vivo effects of parental peptides. Finally, there were reductions in circulating ACE activity and angiotensin II level in SHRs after either DPYKLRP or LRP intake, thus confirming ACE inhibition as the in vivo mechanism for their antihypertensive effect.

Short communication: peptide profiling in cheeses packed using different technologies.

Peptides released during the shelf life of cheeses packaged using 2 different technologies, vacuum packaging (VP) and modified-atmosphere packaging (MAP), were identified by on-line reverse phase-HPLC-tandem mass spectrometry. A total of 22 peptides from the N-terminal domain of αS1-casein (CN) and 26 from ß-CN were identified, the latter more evenly distributed over the whole sequence. Peptides were monitored during the shelf life of these cheeses when stored at 4°C, revealing that the peptide profile changed significantly with the storage time. Qualitative differences between VP and MAP cheeses were only found for 3 αS1-CN peptides, which were absent in MAP cheeses. Semiquantitative analysis of peptides revealed some differences between cheeses packaged using different technologies. However, evolution of peptides during storage followed a common trend in both types of cheeses. In addition, the presence of certain peptides, which had been previously described because of their potential bioactivity, is illustrated. For instance, some of the identified peptides had been previously reported as antihypertensive peptides, such as peptide αS1-CN (1-9) or ß-CN f(201-209).