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Biomedical subjects

C S Cierniewski

Publications and source records attributed to C S Cierniewski.

At least 37 records · Page 2Linked to original sources
PubMed1998

Association of pp60c-src with alpha IIb beta 3 in resting platelets.

To detect whether 125I-alpha IIb beta 3 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 alpha IIb beta 3. Both proteins, pp60c-src and alpha IIb beta 3, coprecipitated when antibodies to pp60c-src were used in the immunoprecipitation experiments. This conclusion was further supported by immunoprecipitation of alpha IIb beta 3 from Triton X-100 extracts of nonlabelled platelets with P2 antibodies. There was no pp60c-src detectable in immunoprecipitates obtained with antibodies specific to alpha 2 beta 1 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 alpha IIb beta 3 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.

Adult↗
PubMed1997

Differential effects of the tyrosine kinase inhibitors on collagen type 1-induced platelet aggregation and adhesion to this protein.

Herbimycin A, lavendustin A, and methyl 2,5-dihydroxycinnamate were used to study the role of protein tyrosine kinases in collagen-platelet interaction. All three compounds produced a concentration dependent inhibition of platelet aggregation induced by collagen type I, characterized by values of IC50 equaled to 0.9, 10.0, and 5.0 microM, respectively. This effect was accompanied by strong inhibition of phosphorylation of p125FAK, p90, p72syk, p60c-arc, and p56lyn. In the absence of the inhibitors, phosphorylation of these proteins is evoked by aggregation of platelets. In addition to the antiaggregatory effect, the tyrosine kinase inhibitors reduced adhesion of platelets to collagen although to much lower extent than aggregation. Platelets which adhered to collagen showed also the presence of phosphorylated p125FAK, p90, p72syk, p60c-arc, and p56lyn. Of these proteins, the extent of phosphorylation of p90 was particularly high. Adhesion of platelets was associated with inhibition of phosphorylation of p125FAK, p60c-arc, and p56lyn only when high concentration of lavendustin A and methyl 2,5-dihydroxycinnamate were used. Herbimycin A did not affect adhesion-evoked protein tyrosine phosphorylation. Phosphorylation of p90 and p72syk was not affected by inhibitors. This study indicates that collagen type I can induce different transmembrane signalling dependent upon whether platelet aggregates formation or adhesion of platelets to this protein occurs.

Benzoquinones↗
PubMed1997

Stereodependent inhibition of plasminogen activator inhibitor type 1 by phosphorothioate oligonucleotides: proof of sequence specificity in cell culture and in vivo rat experiments.

Hexadecadeoxyribonucleotides complementary to a fragment of human PAI-1 mRNA located upstream of the start codon and their phosphorothioate analogs were studied in cultured HUVECs as sequence-dependent inhibitors of PAI-1 expression. The activity of the random mixture of diastereomers of phosphorothioate hexadecanucleotide PS-16H has been compared with that of isosequential, stereoregular [All-Sp] and [All-Rp] isomers. The highest inhibitory effect on PAI-1 synthesis was observed with the [All-Sp] diastereomer. Stereorandom phosphorothioate oligonucleotide PS-16R complementary to the same region of rat PAI-1 mRNA, when injected into tail vein of rats, substantially decreased the level of PAI-1 in blood plasma.

Animals↗
PubMed1997

Platelet membrane fluidity and intraplatelet Ca2+ mobilization are affected in uraemia.

In present investigations, platelet membrane fluidity and intraplatelet Ca2+ mobilization were analysed in uraemic platelets by fluorescence techniques. Thirteen non-dialyzed uraemic patients and 16 control subjects were examined. Anisotropy of DPH-probe, measured at 37 degrees C, was significantly higher in control (0.2236 +/- 0.0050) than in uraemic platelets (0.1969 +/- 0.0082; p < 0.01). There was no difference between control (109.8 +/- 6.0 nM) and uraemic platelets (100.0 +/- 7.3 nM) when the basal [Ca2+]i in resting platelets was determined. Activation of platelets by ADP (12.5 microM) or by thrombin (0.1 U/ml) resulted in an increase in [Ca2+]i. It was significantly higher (p* < 0.003 for ADP and p* < 0.009 for thrombin, respectively) in control platelets (383.6 +/- 56.3 nM and 2031.0 +/- 298.8 nM, respectively) than in uraemic ones (191.0 +/- 21.3 nM and 838.7 +/- 144.1 nM, respectively). The amount of released Ca2+ was higher in control platelets activated by both ADP and thrombin (157.6 +/- 21.4 nM and 409.3 +/- 71.0 nM, respectively) than in uraemic platelets (76.7 +/- 15.7 nM and 203.0 +/- 29.3 nM, respectively) and the differences were significant (p < 0.01 and p* < 0.01, respectively). These results indicate an abnormal intracellular Ca2+ mobilization in uraemic platelets. Both increased membrane fluidity and decreased Ca2+ mobilization should be considered as a possible reason of reduced fibrinogen receptor exposure on uraemic platelets.

Adenosine Diphosphate↗
PubMed1997

Peptide-specific antibodies as probes of the topography of the Ca2+-binging motifs in alphaIIbbeta3.

Antibodies against synthetic peptides corresponding to four Ca2+-binding motifs of the alphaIIb subunit have been obtained and used as molecular probes to analyze the topography of the alphaIIbeta3 complex. The specificity of the antibodies has been characterized by ELISA and Western immunoblotting in terms of binding capacity and affinity to the isolated alphaIIbbeta3 and its alphaIIb subunit. Our data suggest that: (a) all four Ca2+-binding motifs of the alphaIIb are partially exposed on the surface of the intact molecule and accessible to antipeptide antibodies. However, they are not in close vicinity to the ligand recognition domain since the antibodies do not produce complete inhibition of platelet aggregation. (b) The conformation of amino acid stretches which form the second Ca2+-binding motif of alphaIIb is particularly dependent upon the presence of cation, and this region undergoes significant conformational alterations upon Ca2+ expulsion.

Amino Acid Sequence↗
PubMed1996

Antigenic properties of fibrinogen component of Hemaseel HMN subjected to the antiviral severe dry heat treatment.

The experiments in this work were focused on determination of the extent to which fibrinogen component of fibrin glue (Hemaseel HMN, Haemacure Biotech.) retained the conformation of the parent molecule after dry heat antiviral treatment (one hour at 100 degrees C). For this purpose antigenic structure of the fibrinogen component, heated and non-heated, was compared to that of control fibrinogens, i.e. the isolated one and the fibrinogen present in fresh human blood plasma. The analytic parameters CI50 and CIs were calculated from the competitive inhibition data obtained in ELISA systems using antisera to fibrinogen, plasmic fragments D and E, and to polypeptide chains A alpha, B beta, and gamma. These immunochemical analyses indicated that there was a modified expression of some fibrinogen epitopes probably resulting from unfolding of the A alpha chain and the better exposure of the E domain to the hydrated environment induced upon a heating procedure. Our data show that dry heat treatment of fibrinogen component is not associated with a significant overall conformational change of the molecule.

Epitopes↗
PubMed1996

Microenvironmental changes in platelet membranes induced by the interaction of fibrinogen-derived peptide ligands with platelet integrins.

A few studies have confirmed the influence of peptides containing either the RGD or dodecapeptide H-12-V (HHLGGAKQAGDV) sequence on cell membrane structure and function. In order to consider previous findings and to explore microenvironmental changes associated with the interaction of these two fibrinogen-derived peptides with platelet membranes, we employed fluorescence quenching and electron paramagnetic resonance techniques to monitor the possible alterations in platelet membrane dynamics induced by RGDS and H-12-V. The interaction of RGDS with platelet membranes resulted in reduced values of the h+1/ho parameter in both 5-doxylstearic acid and 16-doxylstearic acid spectra indicating a significant rigidification of the membrane lipid bilayer. Otherwise, the fibrinogen-derived peptide that contained the gamma chain C-terminal sequence H-12-V had a fluidizing effect on the platelet membrane lipid bilayer. The labelling of platelet membranes with 1-anilino-8-naphthalenesulphonate (ANS) enabled us to estimate the energy transfer efficiency and the apparent interchromophore distance between membrane protein tryptophan and ANS embedded into the membrane lipid bilayer. As RGDS interacts with platelet membrane this distance decreases, resulting in the relevant increase of energy transfer efficiency. The opposite alterations were recorded upon interaction of platelet membranes with H-12-V. Furthermore, a small shift towards longer wavelengths, which accompanies the spectra of ANS in control platelet membranes, vanishes during the interaction with the peptide H-12-V. This observation can be accounted for by a decrease in the polarity of the ANS environment, and may suggest an enhanced contact of the membrane tryptophan with phospholipid fatty acids. Thus, the data indicate that after the action of H-12-V on platelet membrane receptors, the membrane tryptophan residues become exposed to the external environment and the quenchable fraction of membrane tryptophan becomes smaller. The increase (a) in the relative rotational correlation time (tau c) of 4-(ethoxyfluorophosphinyloxy)-2,2,6,6-tetramethylpiperidine- 1-oxyl (ethoxyfluorophosphinyloxy-TEMPO) and (b) in the hw/hs ratio in the spectra of 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (maleimido-TEMPO) indicate that under these conditions there is an effective immobilization of some domains located on the hydrated surface of membrane proteins and mobilization of those domains buried inside the membrane protein molecules. The interaction of RGDS with platelet membrane integrins resulted in contrary effects, as compared to H-12-V. In conclusion, our spectroscopic data indicate that these two fibrinogen-derived peptides induce opposite effects in the dynamics of platelet membrane components.

Amino Acid Sequence↗
PubMed1996

The immunosuppressory and adhesive miniregion of the human major histocompatibility protein, human leukocyte antigen DQ.

Our previous studies showed that the nonapeptide fragment of human leukocyte antigen DQ with the TPQRGDVYT sequence strongly suppresses the immune response [Z. Szewczuk, I. Z. Siemion, and Z. Wieczorek (1996) Molecular Immunology, 33, 903-9081]. The fragment contains the RGDVY sequence, which is very similar to thymopentin (pentapeptide RKDVY, an active fragment (32-36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the RGD sequence, known as an inhibitor of adhesion processes. In the present study we tested an influence of the nonapeptide and its shorter fragments on binding the activated platelets and K562 cells to fibrinogen and fibronectin, respectively. We also designed and synthesized a cyclic thymopentin-like peptide. C*RGDVYC* (where C* indicates Cys participating in disulfide bridge) to restrict its conformation. The cyclization product strongly suppresses the humoral and cellular immune response and selectively inhibits the adhesion of K562 cells to fibronectin. The results are discussed in the light of CD conformational studies.

Animals↗
PubMed1996

Diabetes mellitus alters the effect of peptide and protein ligands on membrane fluidity of blood platelets.

The increased nonenzymatic glycosylation of platelet membrane proteins has been suggested to underlie platelet hypersensitivity in diabetes and the relationship of this to the reduced membrane lipid fluidity has been reported. As the modulation in membrane fluidity may determine the degree of accessibility of membrane receptors, the consequent alterations in membrane lipid-protein interactions in diabetes mellitus may also underlie the differentiated effects of various thrombotic and fibrinolytic agents on platelet membrane lipid bilayer. In the present study we employed electron paramagnetic resonance and fluorescence spectroscopy to explore the ligand-induced platelet membrane fluidity changes in diabetic state, i.e. under conditions when the membrane architecture is considerably altered. The yield of the excimer formation of pyrenemaleimide (PM), which depends directly upon the collisional rate and distances between molecules, was elevated in diabetic platelet membranes, thus pointing to the occurrence of some constraints in the structure/conformation of platelet membrane proteins in diabetes mellitus. Such an immobilization of PM was accompanied by the significant elevation in membrane protein glycation in diabetic platelets. The effects of various interacting ligands on platelet membrane fluidity were significantly lower in diabetic platelets, and the differences were much more distinct at the lower depths of a lipid bilayer. Nevertheless, the alterations in membrane lipid fluidity observed upon the interaction of a given ligand occurred with an approximately equal frequency in control and diabetic platelets. Moreover, the probability that these alterations were less profound in diabetic platelets was the same for all types of ligands studied. In diabetic patients the interaction of RGDS and tissue-type plasminogen activator with platelet membranes resulted in much smaller reductions of the h+/h0 parameters in 5-DOXYL-Ste acid-labelled platelets, thus indicating a lesser rigidization of membrane lipid bilayer in diabetes. Likewise, the fluidizing effect of both fibrinogen itself and fibrinogen-derived peptides containing gamma-chain carboxy-terminal sequence H-12-V was less pronounced in diabetic platelet membranes.

Adult↗
PubMed1996

Internalization of oligodeoxynucleotide antisense to type-1 plasminogen activator inhibitor mRNA in endothelial cells: a three-dimensional reconstruction by confocal microscopy.

A three-dimensional reconstruction analysis of localization of phosphodiester and phosphorothioate oligonucleotide antisense to type-1 plasminogen activator inhibitor (PAI-1) mRNA within endothelial cells is described. When EA.hy 926 cells were incubated with fluorescently labelled phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness composition of the images of the particular samples was much higher for PS-16 than PO-16 and dependent upon the extracellular concentration and the incubation time. The 3-D reconstructions based on the series of optical sections of the samples, spaced every 1.5 microns, showed the punctuate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. Even after 24 h incubation of endothelial cells with 2.5 microM of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide localization within the cytoplasm and only traces of oligonucleotides could be seen in the cell nucleus and/or perinuclear organelles.

Antisense Elements (Genetics)↗
PubMed1995

Inhibition by modified oligodeoxynucleotides of the expression of type-1 plasminogen activator inhibitor in human endothelial cells.

Antisense phosphodiester and phosphorothioate oligodeoxynucleotides (23-residue or 24-residue oligodeoxynucleotides) were constructed for sequences of type-1 plasminogen-activator-inhibitor mRNA to assess their capability to modulate type-1 plasminogen-activator-inhibitor-mediated fibrinolysis. Antisense oligodeoxynucleotides were targeted at the mRNA sequence coding a signal peptide, at a part of the reactive center Ile342-Pro349, and at an internally translated segment Asn265-Leu272. The effect of antisense oligonucleotides on the concentration of type-1 plasminogen activator inhibitor in conditioned media and human endothelial cells was determined by the activity test with fibrin as a substrate, and by immunoprecipitation after metabolic labelling of cells with [35S]methionine. Three phosphorothioate oligodeoxynucleotides were specifically inhibitory while phosphodiester oligodeoxynucleotides with the same sequence did not show any activity. Phosphorothioate oligodeoxynucleotides 2, 4 and 6 inhibited the synthesis of type-1 plasminogen activator inhibitor in endothelial cells in a time-dependent and concentration-dependent manner. These data suggest that antisense oligodeoxynucleotides may be useful in the design of antithrombolytic therapeutics.

Base Sequence↗
PubMed1995

Expression of fibrinogen receptors and GPIIb molecules on uraemic platelets: effect of recombinant human erythropoietin therapy.

In 14 chronic uraemic patients on regular haemodialysis treatment mean numbers of fibrinogen (Fg) receptors and glycoprotein IIb (GPIIb) molecules on one blood platelet were 8370 +/- 1282 and 15,694 +/- 2102 respectively, and they were significantly lowered in comparison with those found in 14 healthy persons (37,826 +/- 966 and 57,994 +/- 6824 respectively). A high positive correlation was found between numbers of GPIIb molecules and those of Fg receptors both in chronic uraemic patients and healthy subjects. Four-month treatment of haemodialysis patients with recombinant human erythropoietin (rHuEpo) induced significant rises in haematocrit from 22.3 +/- 1.2 to 31.2 +/- 1.7%, blood platelet count from 164,000 +/- 27,000/microliters to 206,000 +/- 16,000/microliters and mean platelet volume from 7.08 +/- 0.29 fl to 8.26 +/- 0.22 fl. However, in haemodialysis patients numbers of the investigated surface platelet receptors, measured at 8 and 16 week of rHuEpo treatment, remained unchanged.

Adenosine Diphosphate↗
PubMed1994

Concentration of RGDS-containing degradation products in uremic plasma is correlated with progression in renal failure.

A concentration of protein degradation products containing the RGDS sequence, which could contribute to a lower reactivity of uremic platelets, has been estimated in both uremic (n = 16) and control (n = 7) plasmas. Degradation products and other small molecules were separated from plasma by filtration through AMICON YM-10 filter. RGDS antigen was determined in filtered material using the radioimmunoassay method based on monospecific anti-RGDS rabbit polyclonal antibodies. The concentration of RGDS-containing degradation products in uremic plasma ranged from 0.8 to 353 nM with mean value 58.6 +/- 24.9 nM and was higher than in control (0.7 to 5.9 nM, mean value 2.1 +/- 0.9 nM). Moreover, the level of RGDS-antigen positively correlated with plasma creatinine concentration (R = 0.87, p < 0.001). The filtered material showed an inhibitory effect on fibrinogen binding to control platelets in respect to RGDS-antigen concentration. We conclude that the elevated concentration of RGDS-containing degradation products in uremic plasma is partially responsible for bleeding tendency in renal failure.

Adult↗
PubMed1994

Characterization of cation-binding sequences in the platelet integrin GPIIb-IIIa (alpha IIb beta 3) by terbium luminescence.

The binding of cations to purified GPIIb-IIIa (alpha IIb beta 3) and synthetic peptides corresponding to the potential cation-binding sites within this integrin has been assessed by terbium luminescence spectroscopy. Tb3+ supported fibrinogen binding to purified GPIIb-IIIa, at lower concentrations than Ca2+, consistent with its higher affinity for cation-binding motifs. Titration analyses indicated the presence of five Tb(3+)-binding sites of relatively high affinity in the receptor. These sites also could be filled by divalent cations. Six sequences within GPIIb-IIIa have the appropriate spacing of five of the usual six coordination sites for cations in functional Ca(2+)-binding EF-hand motifs. Peptides containing Tyr and/or Trp at selected positions as fluorescence energy donors were synthesized, and their Tb(3+)-binding capacity was assessed. The four potential Ca(2+)-binding sequences in the GPIIb subunit, GPIIb 242-255, 296-309, 364-377, and 425-438, were functional, despite lacking the usual Glu residue at the terminal coordination position. These peptides bound Tb3+ with the same affinity as typical Ca(2+)-binding loop peptides and also bound Ca2+ and other divalent cations without preference. Of the two candidate GPIIIa sequences, 118-131 and 208-221, the former bound Tb3+ and divalent cations with an affinity similar to that of the GPIIb peptides, whereas the latter peptide was not functional. This functional difference, as well as data obtained with substituted peptides, emphasizes the importance of the first coordination position for interaction of synthetic peptide loops with cations. Together, these data identify the five cation-binding sites within intact GPIIb-IIIa.

Amino Acid Sequence↗
PubMed1994

GPIIIa(90-102) and GPIIIa(631-653) epitopes as markers of conformational changes occurring during the activation of the glycoprotein IIb/IIIa complex.

To evaluate the range of conformational changes in the GPIIIa subunit upon the interaction of GPIIb/IIIa complex with fibrinogen, we employed two polyclonal antipeptide antibodies against synthetic peptides spanning chain segments (90-102) and (631-653) of GPIIIa. The first peptide fragment derives from the region preceding the putative binding site for the RGD sequence, while the second one is located in the vicinity of membrane lipid bilayer. The resting platelets bound about 13,700 and 11,500 molecules of anti-[GPIIIa(90-102)] and anti-[GPIIIa(631-653)] antibodies, respectively. The anti-[GPIIIa(90-102)] antibodies preferentially bound to the active form of GPIIb/IIIa complex. This binding was significantly reduced after dissociation of the complex, indicating that the recognized epitopes became partially hidden in the GPIIIa subunit. In contrast to anti-[GPIIIa(90-102)], the anti-[GPIIIa(631-653)] antibodies reacted optimally with the EDTA-treated GPIIb/IIIa. Upon stimulation of platelets with ADP, the number of binding sites anti-[GPIIIa(631-653)] was reduced 2.8-fold. The binding affinity of anti-[GPIIIa(90-102)] antibodies to the resting and ADP-stimulated platelets differed 4.7-fold, indicating that the stimulation of the platelets with ADP resulted in better exposure of this epitope. This binding was inhibited almost totally by fibrinogen in a dose-dependent fashion. Similarly, fibrinogen inhibited binding of the anti-[GPIIIa(631-653)] antibodies to the platelets. In the reversed system, binding of fibrinogen to platelets was inhibited only to 60% by the anti-[GPIIIa(90-102)] antibodies. Such inhibition of fibrinogen binding was sufficient to block aggregation of ADP-stimulated platelets by the anti-[GPIIIa(90-102)] antibodies in platelet-rich plasma. In contrast, the anti-[GPIIIa(631-653)] potentiated fibrinogen binding by 20%, confirming that there were additional interacting sites for fibrinogen in other regions of the GPIIb/IIIa complex. The presented data indicate that the activation of the fibrinogen receptor is associated with extensive conformational changes in the GPIIIa subunit, detectable by use of antipeptide antibodies recognizing not only the epitopes located next to the putative ligand-binding site but also the epitopes in the vicinity of the platelet membrane lipid bilayer.

Adult↗
PubMed1994

[Progress in knowledge about fibrinolysis regulation].

Plasminogen activator inhibitor-1 (PAI-1) plays particularly important role in regulation of homeostasis of fibrinolytic system. It neutralizes active molecules of tissue-type and urokinase-type plasminogen activators. PAI-1 is synthesized mainly in endothelial cells but it is present also in other cells. It was found in vitro that elevated expression of PAI-1 gene and increase in PAI-1 concentration released from endothelium is caused by many different biologically active substances. Among them there are thrombin, lipopolysaccharides, cell growth factors, cytokines and also glucocorticoids and phorbol esters. In this work mechanisms of regulation of PAI-1 synthesis and role of this protein in homeostasis of fibrinolytic system are described.

Animals↗