Czeslaw S. Cierniewski

Comparison of platelet aggregability and P-selectin surface expression on platelets isolated by different methods.

Three methods commonly used for isolation of blood platelets from plasma were compared. Platelets were isolated by: 1) a washing method; 2) a method of metrizamide-gradient centrifugation; 3) a modified method of gel-filtration. The last method employed BSA-Sepharose gel instead of routinely used Sepharose gel saturated with BSA. BSA-Sepharose gel was prepared by covalent binding of thermally deactivated BSA to CNBr-activated Sepharose 2B. In contrast to platelets isolated by the other methods, an aggregability of the gel-filtered platelets and control platelets in plasma, both activated with ADP, were comparable. When expression of P-selectin on the surface of freshly isolated platelets was examined, the gel-filtered platelets exhibited the same extent of fluorescence signal as platelets in the citrated blood, whereas platelets isolated by the other methods exhibited twice the extent of the signal. The methods involving the centrifugation process cause a low but a significant platelet activation.

Structural Rearrangements of the 10–23 DNAzyme to β3 Integrin Subunit mRNA Induced by Cations and Their Relations to the Catalytic Activity

The intracellular ability of the “10–23” DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mm Mg2+, a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10–23 DNAzyme induced by Mg2+. For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg2+ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg2+ may occur in several distinct stages. The first stage, observed at 0.5 mm Mg2+, corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mm Mg2+, flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mm Mg2+ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide.

Lumican inhibits angiogenesis by interfering with α2β1 receptor activity and downregulating MMP-14 expression

INTRODUCTIONnPrevious studies showed that lumican, a small leucine-rich proteoglycan that binds to α2 integrin I domain, is an efficient inhibitor of cell adhesion and migration. In this report, we tested its effect on angiogenesis in vitro and in vivo.nnnMATERIALS AND METHODSnEffect of lumican on angiogenesis was evaluated by in vitro capillary tube formation test performed between Fibrin II Gels or in Matrigel™ and in vivo by Matrigel(™) plug assay in BALB/c mice. Changes in matrix metalloproteinases expression caused by lumican were analyzed in endothelial cells by real-time PCR, Western immunoblotting and gelatin zymography.nnnRESULTSnIn unchallenged endothelial cells, Matrigel™ induced robust capillary morphogenesis. In contrast, tube formation was dramatically reduced by lumican, and by siRNA to β1 integrin subunit mRNA but not by control siRNA. Similarly, lumican effectively inhibited neovascularization in vivo in assays using Matrigel™ plugs formed in BALB/c mice. Interestingly, lumican significantly reduced expression of matrix metalloproteinases, particularly MMP-14 that is known to activate other MMPs in close vicinity of endothelial cell membranes.nnnCONCLUSIONSnOur results provide strong evidence that lumican affects angiogenesis both by interfering with α2β1 receptor activity and downregulating proteolytic activity associated with surface membranes of endothelial cells.

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively. We demonstrate that, upon G-actin binding, thymosin ß4 (Tß4), induces MRTF translocation to the nucleus, SRF-activation and CCN1/2 transcription. In a murine ischaemic hindlimb model, MRTF-A or Tß4 promotes neovascularization, whereas loss of MRTF-A/B or CCN1-function abrogates the Tß4 effect. We further show that, in ischaemic rabbit hindlimbs, MRTF-A as well as Tß4 induce functional neovascularization, and that this process is inhibited by angiopoietin-2, which antagonizes pericyte recruitment. Moreover, MRTF-A improves contractile function of chronic hibernating myocardium of pigs to a level comparable to that of transgenic pigs overexpressing Tß4 (Tß4tg). We conclude that MRTF-A promotes microvessel growth (via CCN1) and maturation (via CCN2), thereby enabling functional improvement of ischaemic muscle tissue.

Atrial and brain natriuretic peptide and endothelin-1 concentration in patients with idiopathic arterial hypertension: The dependence on the selected morphological parameters

Introduction. The aim of the work was to study the maintenance of atrial and brain natriuretic peptide (ANP, BNP) and endothelin-1 (ET-1) in patients with idiopathic arterial hypertension and the relationships between cardiac morphological parameters and concentrations of examined peptides in group of patients with left ventricular hypertrophy (LVH). Methods. Seventy-six patients were enrolled in the study: 21 patients with confirmed idiopathic arterial hypertension (group 1), 18 with idiopathic hypertension and eccentric hypertrophy (group 1a), 14 with idiopathic hypertension and concentric hypertrophy (group 1b), and 23 patients without arterial hypertension, organic heart disease, or chronic respiratory tract diseases (group 2—control group). All subjects were submitted for echocardiographic evaluation. Posterior wall thickness (PWT), interventricular septum thickness (IVST), left ventricular end-diastolic diameter (LVEDd), left atrium diameter (LAD), left ventricular mass index (LVMI), ejection fraction (EF), fractional shortening (FS), midwall shortening fraction (MWS), and relative wall thickness index (RWT) were studied. Concentrations of ANP(1-28), BNP, and ET-1 were determined with the use of radioimmunological kits (RIA). The obtained results were subjected to statistical analysis. Results. A considerable increase of ANP and BNP was observed in all patients with hypertension (group 1) in comparison to patients without hypertension (group 2). Significant increases of ANP were found in groups 1a and 1b in comparison to group 1 and 2, as well as considerably increase of BNP in group 1b compared to groups 1, 1a, and 2. In the group of patients with hypertension (group 1), a significant increase in the concentration of ET-1 compared to group 2 was found. However, the concentrations of ET-1 in groups 1 and 2 were not statistically different. Significant differences in concentrations of ET-1 between groups 1a, 1b, and 1 and 2 were seen. Significant correlations were found between concentrations of ANP, BNP, ET-1 and morphological parameters: PWT, IVST, LVMI and RWT. In group 1b, a correlation between concentrations of ANP, BNP, MWS, and LAD was found. The multiple regression analysis showed that RWT independently correlates with concentrations of ANP and BNP, and the concentration of BNP is in closer relation to RWT than ANP. In the case of ET-1, the multiple regression analysis did not show that LVMI or RWT had any independent influence on secretion of ET-1 in patients with idiopathic hypertension and LVH. Conclusions. Increased concentration of ANP in patients with idiopathic hypertension may point to the coexistence of complications with type of LVH. High concentration of BNP may specifically suggest concentric LVH. This is important—especially if there are difficulties in interpretations of results of other clinical examinations. However, increased concentrations of ET-1 in the plasma of patients with hypertension and LVH should not be treated as an indicator of LVH degree.

Release of calcium and P-selectin from intraplatelet granules is hampered by procaine

The inhibition of platelets by some local anaesthetics has been related to the modulation of platelet membrane lipid fluidity, and one of these compounds, procaine, has been proven to be particularly effective inhibitor. In the present study, we examined the effect of procaine on the mobilization of intracellular granule contents in isolated washed platelets. We revealed that the presence of 10 mg/ml procaine significantly hampered platelet release reaction, as demonstrated by the significant reduction in the expression of platelet P-selectin (CD62) on one hand, and significantly enhanced expression of GPIb alpha (CD42b) antigen on the other, following either 1 hour incubation of washed platelets at room temperature (%CD62: 37.1+/-6.8% of control incubated without procaine, p<<0.0001; %CD42b: 116.2+/-6.3% of control, p<0.0001) or activation of whole blood platelets with ADP, TRAP, or thrombin. Procaine, which acted as a rigidizer, significantly decreased platelet membrane fluidity (ESR h(+1)/h0 ratio of 5-DOXYL-Ste reduced down to 93.1+/-3.7% of control, p<0.001). In washed Fura-2-loaded platelets procaine not only brought about the significantly reduced Ca2+ release from intraplatelet storage pools after platelet stimulation with 15 micromol/l ADP (25.3+/-12.5% of control, p<0.001), but also it significantly increased the reduction in Ca2+ concentration upon the addition of Ca2+ chelator, EDTAK2 (48.9+/-13.5% vs. 40.9+/-12.1% of initial [Ca2+]i concentration, p(1,alpha)<0.025). Overall, procaine considerably reduced calcium mobilization from intraplatelet storage pools and Ca2+ efflux across platelet membrane. Based on these data, we suggest that the preventive effects of procaine on platelet release reaction and calcium mobilization might relate to the changes in the organization of membrane components embedded into a lipid bilayer, which are crucial in triggering of platelet release reaction. Procaine-mediated dislocations of some membrane components and/or distortion of lipid-protein interactions could generate a steric hindrance, which might interfere with platelet signal transduction, thus leading to impaired mobilization of Ca2+ and other components from intraplatelet storage pools.

The Specificity and Function of the Metal-binding Sites in the Integrin β3 A-domain

The A-domains within integrin β subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the β3 A-domain, β3-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn2+- and two Ca2+-binding sites with the LIMBS being the site that did not bind Ca2+. Although the ADMIDAS could bind Ca2+, it did not bind Mg2+. These results indicate that the Ca2+-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the β3 A-domain fragment bound fibrinogen in the presence of 0.1 mm Ca2+ but not in 3 mm Ca2+. This behavior recapitulated the effects of Ca2+ on fibrinogen binding to αvβ3 but not αIIbβ3. Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the β3 A-domain.

Protein disulfide isomerase directly interacts with β-actin Cys374 and regulates cytoskeleton reorganization.

Background: PDI regulates cytoskeleton reorganization by the thiol-disulfide exchange in β-actin. Results: PDI directly binds to Cys374 of β-actin during cell adhesion and spreading. Conclusion: Interaction of PDI with β-actin is induced by integrin-mediated cell adhesion and promotes cytoskeleton reorganization. Significance: PDI is a new regulator of the intramolecular disulfide-thiol rearrangement of β-actin in response to αIIbβ3 integrin engagement. Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the β-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with β-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and β-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbβ3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the β-actin Cys374 thiol. Formation of the β-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with β-actin when MEG-01 cells adhere via the αIIbβ3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in β-actin via a redox-dependent mechanism.

Fibrinogen Contains Cryptic PAI-1 Binding Sites That Are Exposed on Binding to Solid Surfaces or Limited Proteolysis

Objective—In this work, we identified the fibrinogen sequence that on exposure serves as the primary binding site for functionally active PAI-1 and to a lesser extent for its latent form. In contrast, this site only weakly interacts with PAI-1 substrate. Methods and Results—The binding site is located in the N-terminal α (20-88) segment of fibrinogen, in the region exposed on (1) adsorption of fibrinogen to solid surfaces; (2) the release of fibrinopeptide A during thrombin conversion of fibrinogen to fibrin; and (3) plasmin degradation of fibrinogen. This region was first identified by the yeast 2-hybrid system, then its binding characteristics were evaluated using the recombinant α(16-120) fragment and its shorter version, the α(20-88) fragment, in a solid phase binding assay and plasmon surface resonance measurements. Because fibrinogen fragment E does not bind PAI-1, it suggests that sequences of Aα chain interacting with PAI-1 are located in the N-terminal part of the α(20-88) segment. Conclusions—Therefore, PAI-1 directly bound to the α(20-88) and thus concentrated in fibrinogen/fibrin, particularly at sites of injury and inflammation, may account for the recent observations that both its active and latent forms stimulate cell migration and wound healing.

Association of pp60cc‐src with αIIbβ3 in resting platelets

To detect whether 125I‐αIIbβ3 is associated with tyrosine kinases in platelets, antibodies specific to pp60c‐src, pp54/58lyn, and pp62Fyn were used to precipitate their homologous antigens. In contrast to Lyn and Fyn kinases, pp60c‐src appears to be complexed with αIIbβ3. Both proteins, pp60c‐src and αIIbβ3, coprecipitated when antibodies to pp60c‐src were used in the immunoprecipitation experiments. This conclusion was further supported by immunoprecipitation of αIIbβ3 from Triton X‐100 extracts of nonlabelled platelets with P2 antibodies. There was no pp60c‐src detectable in immunoprecipitates obtained with antibodies specific to α2β1 or GPIb. Since PGE1 was used to prevent platelet activation in buffers throughout all procedures and there was no phosphorylation of pp72syk we assume that the platelets were in the resting state. Therefore, we conclude that αIIbβ3 and pp60c‐src can form a complex in resting platelets suggesting that pp60c‐src is directly involved in initiating the outside‐in signaling cascades in blood platelets.