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Biomedical subjects

C S Cierniewski

Publications and source records attributed to C S Cierniewski.

At least 55 records · Page 3Linked to original sources
PubMed1994

The epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets.

To examine whether the epidermal growth factor (EGF)-like domain Pro47-Asp87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E. coli. The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met1-Val444 at high yield in eight clones of E. coli. The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro47-Asp87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC. After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro47-Asp87 peptide fragment reduced fluidity of the membrane lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements. Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.

Base Sequence↗
PubMed1994

Expression of fibrinogen receptors on platelets of uremic patients is correlated with the content of GPIIb and plasma level of creatinine.

Platelets of uremic patients, activated with ADP, exposed less fibrinogen receptors than control platelets, i.e. 24612 +/- 5541 and 33400 +/- 4302 receptors per platelet, respectively. However, this difference was not statistically significant. When compared with the total number of GPIIb/IIIa complexes, quantified from platelet glycoprotein IIb (GPIIb) contents, active receptors on the platelet surface represented 13.6% and 35.1% of total pool of fibrinogen receptors in uremic and control platelets, respectively. The number of exposed fibrinogen receptors was positively correlated with the amount of GPIIb copies in both uremic and normal platelets. In uremic platelets, both the number of exposed receptors and the number of GPIIb copies were correlated with the plasma creatinine concentration suggesting, that binding of fibrinogen to uremic platelets depends upon the degree of renal failure. Uremic platelets contain similar amounts of fibrinogen as control ones i.e. 13.2 +/- 2.3 micrograms and 17.6 +/- 2.2 micrograms per 1 x 10(8) platelets, respectively. Whereas for beta-thromboglobulin (beta-TG) there was a significant difference of 392 +/- 102 ng and 803 +/- 202 ng per 1 x 10(8) platelets, respectively. Reduced beta-TG content in uremic platelets suggests limited platelet activation in vivo. These results support the concept that uremic platelets have impaired functions and indicate that there is a relationship between the progression in renal failure and disability of platelets in thrombosis.

Adenosine Diphosphate↗
PubMed1993

Localization of the cross-linking site of GPRVVERHK in the gamma-chain of human fibrinogen.

The peptide alpha 17-24-Lys (GPRVVERHK) corresponding to the N-terminus of the alpha chain of fibrin was synthesized and used to localize its binding site in the fibrinogen molecule. The peptide was radioiodinated, incubated with fibrinogen, cross-linked with a bifunctional reagent disuccinimidyl suberate and the resulting product was analyzed in several ways, including plasmin digestion. The binding of the radioactive peptide was mainly to the gamma-chain and was inhibited by unlabelled GPRVVERHK and GPRP. After plasmin digestion, the radioactivity was present in fragment D1 and also in its gamma-chain remnant, but not in fragments D2, D3 or E3. Fragment D1 cross-linked with iodinated GPRVVERHK was purified by affinity chromatography on immobilized anti-fragment D IgG, further digested with plasmin in the presence of EGTA and the peptides were fractionated by reverse-phase HPLC. The amino acid sequence analysis of the radioactive peak revealed the presence of two peptides, gamma 357-373 and GPRVVERHK. It was concluded that the binding site for GPRVVERHK is in the sequence gamma 357-373 which is present in fragment D1 but absent in fragments D2 and D3.

Amino Acid Sequence↗
PubMed1993

Microenvironment changes in human blood platelet membranes associated with binding of tissue-type plasminogen activator.

Whole washed platelets were labelled with the free radicals [2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinylox y] (16-DOXYL-Ste) or [2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3- oxazolidinyloxy] (5-DOXYL-Ste) and incubated with recombinant tissue-type plasminogen activator (rt-PA). Changes in the membrane fluidity caused by rt-PA were detected by alterations in h+1/h0 calculated from the ESR spectra for 16-DOXYL-Ste and 5-DOXYL-Ste incorporated into the lipid bilayer (h+1 and h0 are the heights of the low-field and middle-field lines of the spectra, respectively). Interaction of rt-PA with both resting and stimulated platelets resulted in increased rigidity of the membrane lipid bilayer as indicated by the reduced value of h+1/h0. This phenomenon can be explained either by conformational changes of membrane receptors caused by the attachment of rt-PA and the subsequent rearrangement of the lipid matrix of platelet membranes, or by the direct association of rt-PA with membrane phospholipids and thus partial embedding of protein molecules into the lipid bilayer restraining lipid mobility.

Blood Platelets↗
PubMed1993

Antibodies to recombinant fragment 212-276 of protein C specifically recognize the intact human molecule.

The peptide fragment Pro212-Ile276 of human protein C was produced as a part of a fusion protein in Escherichia coli. The identity of the peptide was confirmed by immunoblotting experiments using specific antibodies to intact protein C. The peptide Pro212-Ile276 was isolated from the fusion protein after mild hydrolysis with formic acid by gel filtration and reverse-phase HPLC. This peptide fragment was used to produce antibodies specific for the heavy chain of protein C which recognized native protein C present in blood plasma. Antibodies to intact protein C reacted also with the Pro212-Ile276 peptide fragment, indicating that this region is immunogenic in intact protein C and may represent a native epitope.

Amino Acid Sequence↗
PubMed1993

Tissue-type plasminogen activator induces alterations in structure and conformation of membrane proteins upon its interaction with human platelets.

In our very recent ESR study we reported that upon rt-PA binding to platelets the H+1/h0 ratios of 16-doxylstearate and 5-doxylstearate spin labels incorporated into the lipid bilayer of platelet membranes were significantly decreased. It corresponded to the increased rigidity of platelet lipid bilayer. In order to further explore this phenomenon we employed a fluorescence-quenching technique which enabled us to estimate the energy transfer efficiency and the apparent interchromophore distance between membrane protein tryptophan and 1-anilino-8-naphthalenesulphonate (ANS) molecules embedded in the membrane lipid bilayer. As t-PA interacts with the platelet membrane this distance decreases, resulting in the relevant increase of energy transfer efficiency. Thus, the data indicate that upon t-PA binding the membrane tryptophan residues are more exposed to the external environment and the quenchable fraction of membrane tryptophan becomes greater. Furthermore, the spectrum of ANS is slightly shifted towards longer wavelengths, which can be accounted for by an increase in the polarity of the environment. It suggests a diminished contact of membrane tryptophan with phospholipid fatty acids. Based on these observations we concluded that the interaction of rt-PA with platelet membranes might induce conformational changes in the membrane proteins, and consequently result in rearrangements of lipid matrix and the alterations in lipid-protein interactions in platelet membranes.

Blood Platelets↗
PubMed1993

Increased platelet-fibrinogen interaction in patients with hypercholesterolemia and hypertriglyceridemia.

Binding of fibrinogen to platelets washed from the blood of patients with hypercholesterolemia and hypertriglyceridemia (n = 25) and control donors (n = 12) was compared. In addition, the content of platelet glycoprotein IIb was determined by radioimmunoassay. Fibrinogen was bound in significantly higher amounts (P < 0.02) to hyperlipidaemic platelets activated by ADP than to control ones (107,112 +/- 16,371 and 45,612 +/- 6495 molecules per platelet, respectively). The mean content of GPIIb was the same in hyperlipidaemic and in control platelets (2.06 +/- 0.16 and 1.94 +/- 0.21 micrograms/10(8) platelets, respectively). The amount of fibrinogen bound to the activated hyperlipidaemic platelets showed a positive correlation with total plasma cholesterol and LDL (r = 0.45 and 0.47, respectively) whereas a negative correlation with plasma HDL was found (r = -0.50). The increased expression of fibrinogen binding sites similar to that of hyperlipidaemic platelets could be produced by preincubation of normal platelets with palmitic acid. This was evidenced by a significant increase of fibrinogen binding sites in control platelets. This suggests that either palmitoylation of the receptor or microenvironment changes in the membrane lipid bilayer may be responsible for the enhanced platelet receptor capacity to bind fibrinogen.

Adenosine Diphosphate↗
PubMed1993

Do the spectra of maleimide spin-labelled whole blood platelets reflect the structure and conformation of membrane proteins?

The maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl, MSL), the commonly used reagent specific for cysteine thiol groups in proteins, penetrates cell membranes and binds to both the membrane and cytoplasmic protein moieties. In order to differentiate the labelling of these two subpopulations of cell proteins, we developed three different methodological approaches varying in the consequent distribution of this label in platelets. (A) The labelling of platelet proteins was negligible when bovine serum albumin was used in the Tyrode's buffer for the isolation of platelets, as the majority of the spin label was bound to the albumin coated on the platelets. (B) Preblocking of the reactive thiol groups in albumin with non-spin maleimide analog, N-ethylmaleimide (NEM), caused a considerable amount of MSL to bind with whole platelets but the impartment of membrane component was below 50%. It suggests that the majority of the spin label penetrated platelets and was bound to the intrinsic platelet proteins. (C) In order to prevent labelling of intrinsic platelet proteins with MSL, platelets were preincubated with N-ethylmaleimide, which was able to penetrate platelets and block the reactive thiol groups inside the cells. Such a treatment resulted in a saturation of the intrinsic protein residues with this non-spin analog. The subsequent incubation of thus-treated albumin-free platelets with MSL was to enhance considerably the likelihood of the attachment of MSL molecules to the thiol groups available in platelet-membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The amino acids that constitute sequence gamma 268-282 of fibrinogen are not involved in fibrin monomer polymerization.

Congenitally abnormal fibrinogens with impaired fibrin monomer polymerization have been described to contain single amino-acid substitutions localized in certain positions of the gamma 275-330 peptide region. To evaluate the role of the amino-acid sequence in the vicinity of Arg275 in fibrin monomer polymerization, the peptide fragment corresponding to gamma 268-282 was synthesized and used to obtain peptide-specific antibodies. These antibodies, when purified immunochemically on the immobilized peptide, bound to the intact fibrinogen and fibrin monomers with the same binding affinity. However, they did not recognize the gamma 268-282 epitopes on the denatured and reduced fibrinogen molecules. The lack of influence of antipeptide antibodies on fibrin monomer polymerization indicates that the gamma 268-282 peptide is not directly involved in the structure of the polymerization site in the D domain of fibrinogen. It is suggested that substitution of Arg275 either by His or Cys in abnormal fibrinogens results probably in conformational changes which disturb a proper orientation of the polymerization site and reduce its expression.

Amino Acid Sequence↗
PubMed1992

Production and characteristics of human tissue plasminogen-activator antibodies with region-oriented synthetic peptides.

We selected six peptide sequences as belonging to potential epitopes of tissue plasminogen activator (tPA) using, as the main criterion for their choice, the location of the peptide sequences on the surface of the protein molecule. The six peptides (corresponding to amino acids 4-8, 11-16, 96-101, 272-277, 371-376 and 514-519) were synthesized, coupled to carrier proteins and injected into rabbits. All of these peptides elicited antibodies and 15-75% binding of the corresponding iodinated peptide was obtained with a 1:100 dilution of antiserum. Only two anti-(peptide) sera [anti-(tPA96-101) and anti-(tPA272-277)] reacted with intact tPA and its heavy chain in Western immunoblotting analysis. These two peptides sequences and fragment tPA11-16 appear to be involved in the structure of native antigenic epitopes of tPA, since they were recognized and antibodies present in antisera raised against native tPA. There was no interaction between anti-(tPA4-8) and anti-(tPA371-376) sera with intact one-chain or two-chain tPA. In the case of anti-(tPA4-8) cleavage of one-chain tPA to two-chain tPA and reduction of disulfide bonds exposed this epitope.

Antibodies↗
PubMed1992

Involvement of the alpha chain in fibrin clot formation. Effect of monoclonal antibodies.

Murine monoclonal antibodies 9C3, 7B1, and 9E9 have been obtained using native human fibrinogen as the antigen. The antibodies reacted with the epitopes in the COOH-terminal domain of the A alpha chain. Fragmentation of the A alpha chain with plasmin, and, as in the case of the 9E9 epitope, with V8 protease, followed by isolation of the smallest reacting peptides, allowed the localization of the epitopes for 9C3, 7B1, and 9E9 to the amino acid sequences of alpha 240-268, alpha 425-440, and alpha 541-574, respectively. All three monoclonal antibodies strongly inhibited the rate of fibrin polymer assembly from monomers, both in the purified system and in the human plasma. The mechanism of this strong inhibition implied a rapid formation of fibrin protofibrils, followed by capping with IgG molecules of protofibrils containing approximately ten monomers. These observations demonstrated that certain regions in the COOH terminus of the alpha chain may play an important role in the assembly of a fibrin clot, presumably being involved in lateral aggregation of protofibrils.

Amino Acid Sequence↗
PubMed1992

[Tissue-type plasminogen activator (T-PA). Structure and function].

The paper describes the results of the studies on structure and function of the tissue-type plasminogen activator collected for the past 10 years. The properties of one- and two-chain t-PA as well as the structure of the particular domains in the t-PA molecule have been characterized and discussed.

Amino Acid Sequence↗
PubMed1991

The decreased circulating platelet mass and its relation to bleeding time in chronic renal failure.

The mean platelet volume, measured optically and by the electrical impedance method, was analysed in the patients with chronic renal failure. All parameters describing the circulating platelet biomass except the platelet volume heterogeneity were decreased in these patients. There is a strong inverse relation between the Ivy bleeding time and the platelet mass in the patient group. Our results indicate that the smaller platelets circulating in the patients with chronic renal failure may contribute to the uremic bleeding diathesis, and disturbed thrombocytopoiesis occurs in chronic renal failure.

Adult↗