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C S Cierniewski

Publications and source records attributed to C S Cierniewski.

At least 73 records · Page 4Linked to original sources
PubMed1990

The conformation changes of the finger domain of tissue type plasminogen activator during the activator-inhibitor reaction.

A peptide fragment of tissue plasminogen activator (tPA) corresponding to amino acid residues 4-8 (tPA4-8) was synthesized, coupled to thyroglobulin and injected into rabbits. Antibodies specific to the peptide tPA4-8 were purified immunochemically on the pentapeptide coupled to CNBr-Sepha rose 4B. Anti-tPA4-8 antibodies, reacted with iodinated peptide tPA4-8, showing a relatively high binding affinity (KD = 2.3 x 10(-8) M). There was no interaction between anti-tPA4-8 antibodies and native one- or two-chain tPA. However, reduction of disulfide bonds unmasked the epitope on the heavy chain of tPA which became accessible to anti-tPA4-8 antibodies. Similarly, complexing of tPA with alpha 1-antitrypsin inhibitor resulted in unmasking of the epitope formed by amino acid residues in the positions 4-8. Presented data suggest that complexing of tPA with inhibitors results in conformational changes occurring in the "finger" domain of tPA molecule and such conformational transition can be detected by antipeptide antibodies. Therefore, anti-tPA4-8 antibodies may be employed as sequence-specific reporter molecules to monitor local conformational changes in tPA molecule.

Amino Acid Sequence↗
PubMed1990

The effect of anti-beta 43-47 Fab fragments on expression of the polymerization sites in the E domain of fibrinogen.

In order to assess the significance of the aminoterminal residues of the B beta chain in expression of polymerization sites of the E domain, we have prepared polyclonal antiserum against a synthetic peptide corresponding to beta 43-47 of human fibrinogen. Affinity purified immunoglobulin IgG and Fab prepared from these antibodies reacted strongly and specifically with the synthetic pentapeptide and intact fibrinogen molecule. This specificity was determined both by radioimmunoassay and Western blot analysis of fibrinogen and its plasmic fragments and described in our previous paper (Cierniewski et al., 1986, Biochim. Biophys. Acta, 884, 594-597). Immunochemically purified anti beta 43-47 antibodies and their Fab fragments were strong inhibitors of the fibrin monomer polymerization. Our results imply that amino acid sequence beta 43-47 recognized by these antibodies may be in a close vicinity to the contact sites of the E domain.

Amino Acid Sequence↗
PubMed1989

Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets.

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.

Adult↗
PubMed1989

Expression of fibrinogen receptors in platelets of migraine patients--correlation with platelet GPIIb content and plasma cholesterol.

Binding of fibrinogen to platelets washed from blood of migraine patients (n = 30) and control donors (n = 24) was compared. In addition, contents of platelet glycoprotein IIb and platelet fibrinogen were determined in both groups by radioimmunoassay. The receptor capacity for fibrinogen in platelets activated by ADP was significantly higher (p less than 0.01) in migraine patients (52,505 +/- 4,925) than in controls (33,881 +/- 3,965). The mean contents of GPIIb (3.51 +/- 0.34 micrograms/10(8) platelets) and fibrinogen (37.26 +/- 4.05 micrograms/10(8) platelets) in migraine platelets were also markedly increased (p less than 0.01 and p less than 0.001, respectively) when compared to controls (2.21 +/- 0.18 micrograms of GPIIb and 18.75 +/- 2.29 micrograms of fibrinogen per 10(8) platelets, respectively). There was a high correlation between the number of fibrinogen receptors exposed by ADP and the total amount of platelet GPIIb both in migraine patients (R = 0.69, p less than 0.01) and controls (R = 0.62 p less than 0.01), as well as plasma cholesterol in the case of migraine patients (R = 0.82, p less than 0.001).

Adult↗
PubMed1988

Anti-(Arg-Gly-Asp-Ser) antibody and its interaction with fibronectin, fibrinogen and platelets.

An antibody population recognizing the sequence Arg-Gly-Asp-Ser (RGDS) in fibronectin, anti-(RGDS)N, was isolated by immunoadsorption. Between 2.5% and 4.9% of antibodies were obtained from two different anti-fibronectin sera indicating that this region represents an antigenic epitope in native fibronectin. Complete inhibition of binding of 125I-fibronectin to anti-(RGDS)N was produced only by nonreduced and reduced fibronectin. Fibrinogen and synthetic RGDS tetrapeptide, each at concentration of 10 microM, showed only a slight inhibition of 22% and 17%, respectively. Measurements of the conformational constant, the equilibrium constant for the interconversion of the non-native and native conformations of this epitope, showed that less than 0.0001% of the RGDS molecules adopt the native conformation in aqueous solutions. It indicates that long-range interactions in fibronectin and fibrinogen result in different conformations of the RGDS sequence in both proteins. Anti-(RGDS)N antibodies purified from anti-fibronectin serum had a strong inhibitory effect on thrombin-stimulated platelet aggregation. They also inhibited binding of fibronectin and fibrinogen to thrombin-stimulated platelets, supporting the primary role of the RGDS sequence in the direct interaction of these proteins with platelet membrane receptors.

Antibodies↗
PubMed1988

Mathematical modelling of the transport of low molecular weight solutes across biological membranes. The transport of Leu, His and Glu into human blood platelets.

A model describing the transport of low molecular weight solutes across cell membranes is presented. The model accounts for many different systems which may mediate the fluxes of various solutes, for the effect of Na+ ions, and for time dependence of the processes. It generalizes the classical three-parameter equation for transport. Solutions to the model were employed to interprete experimental data obtained for the uptake of DL-leu, L-his and L-glu by human blood platelets.

Biological Transport↗
PubMed1987

Activation of blood platelets and increased plasma fibrinogen and fibronectin in men exposed to infrasounds, acoustic noise and airborne dust in electric steelworks.

Concentration of platelet factor 4 (PF4), beta-thromboglobulin (beta TG), fibrinogen (Fg), and fibronectin (Fn) was determined in samples of blood plasma taken from healthy men employed in two electric steelworks in Poland. They had been working at electric arc furnaces and thus particularly exposed to intense infrasounds, acoustic noise and airborne dust during 2 to 15 years for 6-8 hours per day. We found a significant increase in levels of beta TG, Fn, and Fg, but not of PF4, in blood plasma of men exposed to these agents. The beta TG/PF4 ratio, significantly higher in studied groups (p less than 0.001) when compared to control ones, was considerably correlated (r = 0.47, p less than 0.02) with their duration of work. We also found weak correlations between plasma beta TG levels (r = 0.33, p less than 0.05) or concentration of plasma Fn (r = 0.38, p less than 0.02) and time of work under such hazardous conditions.

Acoustic Stimulation↗
PubMed1987

Conformational equilibria in the gamma chain COOH terminus of human fibrinogen.

The conformations of the gamma chain COOH terminus of intact fibrinogen and various fragments containing this region have been compared by an immunochemical analysis. Location of a major epitope in the sequence gamma 391-405 was successfully predicted from a hydrophobicity profile. An antibody population specific for the native epitope within the gamma 391-405 segment was isolated by immunoadsorption. Between 19.2 and 22.8% of antibodies were obtained from three different antisera, indicating that this region represents one of the major epitopes of native fibrinogen. Anti-gamma 391-405(N) antibodies were used to determine the value of Kconf, the equilibrium constant for the interconversion of the non-native and native conformations of this epitope. The measurements were done using native fibrinogen, fragments D1 and DD, gamma chain, and gamma 391-405. In addition, the effect of 5 M guanidine HCl on the conformation of fragments D1 and DD, which is known to abolish their antipolymerizing activity, was studied. Radioiodinated fibrinogen was used in the determination of Kconf, CI50%, and CIs (quantitative analytical parameters calculated from competitive inhibition radioimmunoassays) by measuring the competition between 125I-fibrinogen and the fibrinogen derivatives under study for binding to the immunochemically purified antibody. The measurements indicated that the epitope is unperturbed by iodination of fibrinogen and that 38.5% of fragment D1, 8.9% of fragment DD, 3.6% of the gamma chain, and less than 0.008% of the gamma 391-405 molecules adopt in aqueous solution the native conformation within the epitope. Denaturation of fragment D1 with 5 M guanidine HCl affected only slightly the conformation of this gamma chain determinant. More significant changes in the conformation were observed when fragment DD was denatured. The results suggest that long-range interactions are necessary for the stabilization of the native structure in the region of fibrinogen that interacts with the antibody and which is in close vicinity to the polymerization site, cross-linking site, and platelet recognition site.

Amino Acid Sequence↗
PubMed1987

Synthesis and biological activity of substance P C-terminal hexapeptide analogues: structure-activity studies.

The synthesis of six hexapeptide analogues of C-terminal Substance P fragment containing alpha, beta-dehydrophenylalanine (delta Phe) in the position 7 or 8 is described. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of delta Phe in various analogues of C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive activity. The analogues [Glp6, delta Phe7]SP6-11 and [Glp6, delta Phe8]SP6-11 retained 70% and 45% of hypotensive activity of the C-terminal hexapeptide of Substance P, respectively but they showed a completely destroyed antigenic determinant. The analogues containing additionally Sar or His in the position 9 showed a complete lack of both: hypotensive activity and expression of the antigenic determinant of Substance P.

Amino Acid Sequence↗
PubMed1987

Kinetics of the beta-thromboglobulin release from alpha-granules of blood platelets activated by ADP.

Mathematical model of the beta-thromboglobulin (BTG) release from alpha-granules stimulated with ADP is compared with experimental data. Secretion of BTG was analysed using platelets washed by different procedures in the presence or absence of inhibitors of platelet activation. The release of BTG was monitored in response to 5-100 microM ADP. The rate constant of the release of BTG (K = 1.83 X 10(3) M-1 min-1) and efficient amount of ADP molecules secreted by a single platelet (W = 5.2 X 10(-18) mole) were estimated. The model precisely describes the release of BTG associated with the isolation of platelets (blood collection, washing procedure) and allows to estimate the extent of activation of blood platelets in the suspension.

Adenosine Diphosphate↗
PubMed1987

Quantitation and characterization of human platelet glycoprotein IIIa by radioimmunoassay.

Glycoprotein IIIa was quantitated in human platelets by radioimmunoassay using antisera specific to platelet membranes and purified glycoprotein IIIa. Glycoprotein IIIa and glycoprotein IIb were isolated from washed platelets by Triton X-114 extraction followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioiodinated glycoprotein IIIa was further purified by affinity chromatography on Lentil lectin-Sepharose 4B. Purified glycoprotein IIb showed little crossreactivity with 125I-labeled glycoprotein IIIa using the anti-platelet membrane or anti-glycoprotein IIIa antisera on a competition inhibition radioimmunoassay. The expression of glycoprotein IIIa epitopes were the same for the purified glycoprotein IIIa and glycoprotein IIIa in Triton X-100 solubilized platelets. A 66 kDa protein derived from glycoprotein IIIa by limited proteolysis of platelet membranes also expressed the same epitopes as intact glycoprotein IIIa. Solubilized platelets contained approximately 16 micrograms of total glycoprotein IIIa antigen per 10(9) cells. The level of glycoprotein IIIa determined by radioimmunoassay in one patient with Glanzmann's thrombasthenia amounted to 6.7% of normal and it was close to the values obtained by other methods.

Antigen-Antibody Complex↗