Jan E. Dyr

Complete identification of proteins responsible for human blood plasma fouling on poly(ethylene glycol)-based surfaces.

The resistance of poly(ethylene glycol) (PEG) against protein adsorption is crucial and has been widely utilized in various biomedical applications. In this work, the complete protein composition of biofilms deposited on PEG-based surfaces from human blood plasma (BP) was identified for the first time using nanoLC-MS/MS, a powerful tool in protein analysis. The mass of deposited BP and the number of different proteins contained in the deposits on individual surfaces decreased in the order of self-assembling monolayers of oligo(ethylene glycol) alkanethiolates (SAM) > poly(ethylene glycol) end-grafted onto a SAM > poly(oligo(ethylene glycol) methacrylate) brushes prepared by surface initiated polymerization (poly(OEGMA)). The BP deposit on the poly(OEGMA) surface was composed only of apolipoprotein A-I, apolipoprotein B-100, complement C3, complement C4-A, complement C4-B, histidine-rich glycoprotein, Ig mu chain C region, fibrinogen (Fbg), and serum albumin (HSA). The total resistance of the surface to the Fbg and HSA adsorption from single protein solutions suggested that their deposition from BP was mediated by some of the other proteins. Current theories of protein resistance are not sufficient to explain the observed plasma fouling. The research focused on the identified proteins, and the experimental approach used in this work can provide the basis for the understanding and rational design of plasma-resistant surfaces.

Influence of citrate and EDTA anticoagulants on plasma malondialdehyde concentrations estimated by high-performance liquid chromatography.

Estimation of lipid peroxidation through MDA formation measured by assaying thiobarbituric acid (TBA) reactive products separated by HPLC remains the method of choice to study the development of oxidative stress in blood plasma. In this report we describe the influence of citrate and EDTA anticoagulants used for blood collection on estimation of MDA concentrations using HPLC analysis of MDA-TBA adducts. We analyzed a group of 40 blood donors (21 men and 19 women), median age 27 years, range 19-48 years. The mean MDA concentration in citrate plasma was 1.43+/-0.51 micromol/l (range: 0.61-2.57 micromol/l) and in EDTA plasma 0.36+/-0.10 micromol/l (range: 0.13-0.63 micromol/l). There was a significant difference in MDA mean concentration that we attribute to different antioxidant properties of anticoagulants used for blood collection. Consistency in the choice of anticoagulant is clearly extremely important.

Albumin and heparin multilayer coatings for blood-contacting medical devices.

Three types of covalently crosslinked assemblies consisting of multiple (1) molecular layers of human serum albumin (HSA); (2) alternating layers of HSA and unfractionated heparin; and (3) alternating layers of HSA and partly depolymerized heparin fixed with one end to HSA were prepared on various surfaces. Adsorption of fibrinogen, IgG, and antithrombin (ATIII) from human citrated plasma on coated surfaces was evaluated by ELISA. Fibrinogen adsorption on coated ELISA plates was lower than that on bare polystyrene. There was no IgG adsorption on the HSA coating alone, but considerably high IgG adsorption was detected on the heparin-containing surface. The adsorption of ATIII increased with increasing heparin on the surface. The effect of multilayer coatings on platelets was tested by incubation of modified vascular prostheses with citrated blood. The most favorable interaction with platelets was observed on the HSA assembly. The interaction of platelets with the surface bearing unfractionated heparin was higher than that of the surface covered with partly depolymerized heparin. The long-term durability of the HSA-heparin coating was proven by a 21-day implantation of coated polyurethane plates in goat heart.

Proteome changes in platelets activated by arachidonic acid, collagen, and thrombin

BackgroundPlatelets are small anucleated blood particles that play a key role in the control of bleeding. Platelets need to be activated to perform their functions and participate in hemostasis. The process of activation is accompanied by vast protein reorganization and posttranslational modifications. The goal of this study was to identify changes in proteins in platelets activated by different agonists. Platelets were activated by three different agonists - arachidonic acid, collagen, and thrombin. 2D SDS-PAGE (pI 4-7) was used to separate platelet proteins. Proteomes of activated and resting platelets were compared with each other by Progenesis SameSpots statistical software; and proteins were identified by nanoLC-MS/MS.Results190 spots were found to be significantly different. Of these, 180 spots were successfully identified and correspond to 144 different proteins. Five proteins were found that had not previously been identified in platelets: protein CDV3 homolog, protein ETHE1, protein LZIC, FGFR1 oncogene partner 2, and guanine nucleotide-binding protein subunit beta-5. Using spot expression profile analysis, we found two proteins (WD repeat-containing protein 1 and mitochondrial glycerol-3-phosphate dehydrogenase) that may be part of thrombin specific activation or signal transduction pathway(s).ConclusionsOur results, characterizing the differences within proteins in both activated (by various agonists) and resting platelets, can thus contribute to the basic knowledge of platelets and to the understanding of the function and development of new antiplatelet drugs.

Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor

We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.

Antioxidants change platelet responses to various stimulating events

The role of platelets in hemostasis may be influenced by alteration of the platelet redox state-the presence of antioxidants and the formation of reactive oxygen and nitrogen species. We investigated the effects of two antioxidants, resveratrol and trolox, on platelet activation. Trolox and resveratrol inhibited aggregation of washed platelets and platelet-rich plasma activated by ADP, collagen, and thrombin receptor-activating peptide. Resveratrol was a more effective agent in reducing platelet static and dynamic adhesion in comparison with trolox. The antioxidant capacity of resveratrol was, however, the same as that of trolox. After incubation of platelets with antioxidants, the resveratrol intraplatelet concentration was about five times lower than the intracellular concentration of trolox. Although both antioxidants comparably lowered hydroxyl radical and malondialdehyde production in platelets stimulated with collagen, TxB(2) levels were decreased by resveratrol much more effectively than by trolox. Cyclooxygenase 1 was inhibited by resveratrol and not by trolox. Our data indicate that antioxidants, apart from nonspecific redox or radical-quenching mechanisms, inhibit platelet activation also by specific interaction with target proteins. The results also show the importance of studying platelet activation under conditions of real blood flow in contact with reactive surfaces, e.g., using dynamic adhesion experiments.

Controlled preparation of thin fibrin films immobilized at solid surfaces

A technique for coating surfaces with attached fibrin structures without the formation of fibrin gel in bulk solution was developed. It is based on the catalytic effect of the surface-bound thrombin on fibrinogen stabilized with inhibitor which inhibits thrombin in solution but not the thrombin on the surface. Such an inhibitor is antithrombin, the effect of which may be enhanced with heparin. Fibrinogen is first adsorbed on the substrate surface and then incubated with thrombin. The unbound thrombin is washed out and the surface is incubated with fibrinogen solution containing antithrombin III and heparin. A fibrin gel forms at the surface by the action of surface-bound thrombin on ambient fibrinogen solution; however, the gel formation in bulk solution catalyzed by thrombin partially released from the surface is suppressed. By utilizing antithrombin-independent inhibitors or repeating thrombin binding and incubation with fibrinogen solution, the amount of surface-attached fibrin gel can be controlled. The formation of immobilized fibrin networks was observed using surface plasmon resonance and turbidity measurements and morphology was observed by TEM, SEM, and AFM. Using this technique, a porous scaffold made of polylactide fibers was coated with fibrin without filling the space between fibers with a bulk fibrin gel. The technique makes it possible to coat the inner surface of porous scaffolds with surface-attached fibrin gel while preserving free volume for cell migration into the pores.

Plasma proteome changes in cardiovascular disease patients: novel isoforms of apolipoprotein A1

BackgroundThe aim of this proteomic study was to look for changes taking place in plasma proteomes of patients with acute myocardial infarction (AMI), unstable angina pectoris (UAP), and stable angina pectoris (SAP).MethodsDepleted plasma proteins were separated by 2D SDS-PAGE (pI 4-7), and proteomes were compared using Progenesis SameSpots statistical software. Proteins were identified by nanoLC-MS/MS. Proteins were quantified using commercial kits. Apolipoprotein A1 was studied using 1D and 2D SDS-PAGE, together with western blotting.ResultsReciprocal comparison revealed 46 unique, significantly different spots; proteins in 34 spots were successfully identified and corresponded to 38 different proteins. Discrete comparisons of patient groups showed 45, 41, and 8 significantly different spots when AMI, UAP, and SAP were compared with the control group. On the basis of our proteomic data, plasma levels of two of them, alpha-1 microglobulin and vitamin D-binding protein, were determined. The data, however, failed to prove the proteins to be suitable markers or risk factors in the studied groups. The plasma level and isoform representation of apolipoprotein A1 were also estimated. Using 1D and 2D SDS-PAGE, together with western blotting, we observed extra high-molecular weight apolipoprotein A1 fractions presented only in the patient groups, indicating that the novel high-molecular weight isoforms of apolipoprotein A1 may be potential new markers or possible risk factors of cardiovascular disease.ConclusionThe reported data show plasma proteome changes in patients with AMI, UAP, and SAP. We propose some apolipoprotein A1 fractions as a possible new disease-associated marker of cardiovascular disorders.

Molecular arrangement of adsorbed fibrinogen molecules characterized by specific monoclonal antibodies and a surface plasmon resonance sensor

Abstract The exploitation of surface plasmon resonance optical sensor for the study of the accessibility of distinct domains of immobilized fibrinogen molecules and the evaluation of the fibrinogen arrangement is reported. By investigation of the interactions of sensor surface-bound fibrinogen with various specific monoclonal antibodies it has been found that density, orientation, and accessibility of the individual fibrinogen domains of surface-bound fibrinogen depend on the concentration of fibrinogen in solution during the adsorption process. At low fibrinogen surface density the majority of molecules seem to be adsorbed side-on (lying on the surface). At high fibrinogen density the molecules are adsorbed end-on (standing on the surface). The experimental data suggest that the distal ends of the (adsorbed) fibrinogen molecule (containing polymerisation sites) are oriented anti-parallel to each other, proving off-axis location of the distal end domains.

The adhesion of blood platelets on fibrinogen surface: Comparison of two biochemical microplate assays

The biocompatibility of materials is frequently assessed by blood platelet adhesion, since platelet adhesion plays a considerable role in blood interaction with artificial surfaces. Blood platelets adhesion is an essential event in haemostatic and thrombotic processes. The aim of this study was to simultaneously compare simple biochemical assays widely used for evaluation of platelet static adhesion based on the determination of enzymatic activity of either lactate dehydrogenase (LDH) or acid phosphatase (ACP) in lysates of adhered platelets. Adhesion of platelets from platelet-rich plasma and washed platelets activated by either ADP or thrombin on surfaces covered with fibrinogen and well defined fibrin was studied. The results demonstrated that the amounts of adhered platelets estimated by the LDH method were significantly lower as compared with the amount obtained by ACP method. LDH but not ACP release from platelets during adhesion was shown to take place. It suggests that the LDH method should be used rather as an assay of platelet integrity. The ACP method is much more suitable for quantitative determination of platelet adhesion especially in the development and evaluation of haemocompatibility of new biomaterials.