Biomedical subjects

L Muszbek

Publications and source records attributed to L Muszbek.

At least 55 records · Page 3Linked to original sources

PubMed1995

Transformation of cellular factor XIII into an active zymogen transglutaminase in thrombin-stimulated platelets.

The cellular form of blood coagulation factor XIII (FXIII) is present in platelets, monocytes and macrophages. During long-term stimulation of platelets by thrombin cellular FXIII becomes activated and cross-links proteins, however, the mechanism of its activation has not been elucidated. It was shown that, contrary to plasma FXIII, the intracellular activation of platelet FXIII does not involve proteolysis. FXIII remained intact in thrombin-activated platelets, i.e., the activation peptide was not removed from the molecule. Part of the zymogen FXIII molecules, however, assumed an active configuration as was demonstrated both by the measurement of transglutaminase activity and by active-site-SH titration. These findings clearly indicate that during platelet activation, when intracellular Ca2+ concentration is raised, a slow non-proteolytic transformation of FXIII zymogen into an active transglutaminase occurs.

Blood Platelets↗

PubMed1994

[Simultaneous occurrence of lupus anticoagulant and acquired "storage pool" disease of thrombocytes].

The authors describe the case of a female patient with a history of simultaneous abortion and deep vein thrombosis as well as moderate bleeding disturbances. Investigations revealed the presence of lupus anticoagulant while a thrombocyte "storage pool" disease was confirmed. This case demonstrates the association of these two haemostatic disturbances, and points to a possible relationship between them. Since detailed analyses failed to demonstrate the presence of antiplatelet antibodies, the authors suggest a possible damaging effect of lupus anticoagulant to the endothelium leading to thrombocyte activation.

Abortion, Spontaneous↗

PubMed1994

Covalent binding of arachidonate to G protein alpha subunits of human platelets.

The alpha subunits of GTP-binding regulatory proteins (G proteins) are subject to lipid modifications required for anchorage to membrane and/or interactions with other proteins. With the knowledge that alpha subunits are palmitoylated, which we demonstrate here for human platelets, we sought to determine whether these subunits also bind arachidonate and myristate in a covalent, post-translational manner. All alpha subunits examined were found to incorporate radioactivity upon incubation of human platelets with [3H]palmitate, [3H]arachidonate, and [3H]myristate. The identity of [3H]palmitate and [3H]arachidonate as covalently bound fatty acids was confirmed by high pressure liquid chromatography following alkaline methanolysis. With [3H]myristate, however, the bound fatty acid proved to be [3H]palmitate, presumably generated by a 2-carbon chain elongation. Protein-bound [3H]palmitate and [3H]arachidonate were released by hydroxylamine at neutral pH, implying a thioester linkage between protein and fatty acid. Thus, post-translational modifications of G protein alpha subunits include palmitoylation and arachidonoylation, but not myristoylation. Given the different physical properties of saturated and unsaturated fatty acids and the large-scale release of arachidonate during platelet activation, changes in arachidonate incorporation may serve as an important regulator of alpha subunit function.

Alprostadil↗

PubMed1993

Covalent modification of proteins by arachidonate and eicosapentaenoate in platelets.

The posttranslational modification of proteins by fatty acids has been shown to involve long chain-saturated fatty acids, predominantly palmitate. In the present study, we demonstrated by metabolic labeling of human platelets with [3H]arachidonate and [3H]eicosapentaenoate that these polyunsaturated fatty acids can also become covalently linked to proteins. The extent of binding of arachidonate to proteins was somewhat less than that of palmitate. Arachidonate binding to platelet proteins was not significantly influenced by the inhibition of cyclooxygenase and lipoxygenase. This finding and the high performance liquid chromatography analysis of radiolabeled products removed from proteins by selective cleavage techniques established that arachidonate, and not its metabolic products, was the protein-linked radiolabeled moiety in [3H]arachidonate-labeled platelets. A 7.5-fold higher concentration of unlabeled palmitate competed to a small extent with [3H] arachidonate for protein labeling. Both arachidonate and eicosapentaenoate were bound to proteins almost exclusively through ester linkages. It was further demonstrated that 61 and 66% of total protein-linked arachidonate and eicosapentaenoate, respectively, were bound via thioester bonds. In contrast, 91% of the binding of palmitate to proteins occurred via thioester linkages. As demonstrated by SDS-polyacrylamide gel electrophoresis and fluorography, the patterns of palmitoylated and arachidonoylated proteins were similar but not identical, with selected proteins only palmitoylated or only arachidonoylated. [3H]Eicosapentaenoate labeled the same set of proteins as [3H]arachidonate. The fluorographic pattern of 3H-arachidonoylated proteins was not changed by cyclooxygenase and lipoxygenase inhibitors. The binding of a polyunsaturated fatty acid to a protein in place of a saturated fatty acid could significantly influence the hydrophobic interactions of the protein and, thereby, have important functional implications.

Arachidonic Acid↗

PubMed1993

P-selectin is acylated with palmitic acid and stearic acid at cysteine 766 through a thioester linkage.

We report that the adhesion receptor P-selectin can be metabolically labeled with [3H]palmitic acid in human platelets. Analysis of alkaline methanolysis products from labeled protein demonstrated that the radioactivity associated with P-selectin was covalently bound palmitic acid. [3H]Palmitic acid was cleaved by hydroxylamine treatment at neutral pH and by reducing agents, indicating that acylation occurred through a thioester linkage. Both stearic acid and palmitic acid were detected by gas chromatography-mass spectrometry analysis of alkaline hydrolysates of purified P-selectin. Deletion or mutation of Cys766 eliminated [3H] palmitic acid labeling of P-selectin in transfected COS-7 cells. We conclude that the cytoplasmic domain of P-selectin is acylated at Cys766 through a thioester bond. Fatty acid acylation may regulate intracellular trafficking or other functions of P-selectin.

Acylation↗

PubMed1993

Myristoylation of proteins in platelets occurs predominantly through thioester linkages.

We have demonstrated by several lines of evidence that in platelets myristate is linked to proteins predominantly via thioester bonds as is palmitate, and the covalent binding of the two long chain saturated fatty acids to proteins involves the same mechanisms. The first piece of evidence to support the thioester linkage between myristate and proteins is that [3H]myristate could be removed from proteins via alkaline methanolysis, which disrupts ester bonds but not amide bonds. The second piece of evidence is that unlabeled palmitate, which can form only thioester bonds in physiologic concentrations, competitively inhibits the formation of alkaline methanolysis-sensitive covalent bonds between [3H]myristate and proteins. Third, by SDS-polyacrylamide gel electrophoresis and fluorography, the patterns of labeled proteins from [3H]myristate- and [3H]palmitate-labeled platelets are identical. Fourth, [3H]myristate-labeled proteins, like [3H]palmitate-labeled proteins, both release their fatty acid moieties when exposed to hydroxylamine at neutral pH, which disrupts thioester but not hydroxyester bonds. These findings indicate that although the covalent binding of palmitate to proteins was found to occur at a faster rate than that of myristate, protein S fatty acid acylation that occurs posttranslationally is not specific for palmitate.

Blood Platelets↗

PubMed1993

Platelet factor XIII becomes active without the release of activation peptide during platelet activation.

The potentially active A subunit of factor XIII of blood coagulation has also been detected in platelets and monocytes/macrophages through the exact function of this cellular protransglutaminase has not yet been elucidated. In physiological conditions the first step in the activation of plasma factor XIII is the removal of an activation peptide from the N-terminal end of subunit A by thrombin. The A subunit then, in the presence of Ca2+, dissociates from the inhibitory B subunit and assumes an active conformation. Cellular factor XIII, which lacks B subunit, can be proteolytically activated in vitro by thrombin and the intracellular Ca2+ sensitive protease, calpain, in the same way as plasma factor XIII subunit A, and calpain has been suggested as the intracellular protease involved in the activation of cellular factor XIII in platelets. In the present experiments it was shown by SDS PAGE that during long-term stimulation of platelets with thrombin nondisulfide-crosslinked high M(r) protein polymers not penetrating the concentrating gel were formed. The lack of these polymers in thrombin-stimulated factor XIII deficient platelets clearly indicated that their formation in normal platelets was due to factor XIII that became active during platelet activation. However, no release of the activation peptide could be detected by Western blotting during this process. Similarly, no proteolytic cleavage of factor XIII was detectable when platelets were stimulated by Ca2+ ionophore through this stimulus activated calpain as it was clearly demonstrated by the breakdown of major intracellular calpain substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗

PubMed1992

Cytoskeletal assembly and vinculin-cytoskeleton interaction in different phases of the activation of bovine platelets.

Vinculin is an Mr 130 kDa protein that has been implicated in membrane-cytoskeleton interaction in various cell types. It has been demonstrated that vinculin is not a cytoskeletal component in resting platelets, but part of it becomes associated with the cytoskeleton during thrombin-induced activation. In this study, using a quantitative immunoblotting technique, the relation of vinculin to the cytoskeleton in different phases of activation of bovine platelets was explored, and the process of incorporation of vinculin into the cytoskeleton was related to that of cytoskeletal assembly. The assembly of cytoskeleton proceeded at a significantly faster rate than the association of vinculin with it, which shows that the latter process is not due to passive trapping of vinculin into the Triton-insoluble residue, but certain biochemical changes had to occur before such an interaction became possible. When the formation of pseudopodia was prevented by cytochalasin B, but neither aggregation nor the release reaction induced by thrombin were inhibited, the recovery of vinculin in the Triton-insoluble residue even increased. In both time- and thrombin-concentration-dependent studies, poor correlation was found between vinculin-cytoskeleton association and the extent of aggregation. Activation with phorbol-myristate-acetate, which is a strong stimulus for aggregation but produces only a slight release in the granular content, resulted in the association of only a negligible amount of vinculin with the cytoskeletal fraction. The incorporation of vinculin into the cytoskeletal fraction of thrombin activated platelets started with the release reaction but still proceeded, and the greatest part of the reaction occurred after secretion had gone to completion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c-src in platelets has led to speculation that this kinase is responsible for tyrosine-specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c-src is detergent-soluble in fibroblasts overexpressing the c-src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c-src we have investigated the subcellular distribution of pp60c-src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c-src is detergent-soluble in resting platelets, while 40% of total platelet pp60c-src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c-src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c-src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor-fibrinogen binding. We suggest that the translocation of pp60c-src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.

Biological Transport↗

PubMed1992

Marker profile, enzyme activity, and function of a human myelomonocytic leukemia cell line.

Morphological and functional characteristics of a permanent human leukemia cell line (DD) that possesses myelomonocytic features were investigated. The cells bear a second type Fc gamma receptor and form rosettes with sheep erythrocytes sensitized with rabbit IgG (EA). However, the surface-bound EA is not internalized. The cell line lacks the surface markers CD2, CD19, CD14, HLA-DR, Fc gamma receptor I, Fc gamma receptor III, and CR3. alpha 1-Antitrypsin, lysozyme, Factor XIII a subunit of blood coagulation, and acid phosphatase reactions were negative. A terminal differentiation of the DD cell line was observed when the expression of CD14, CR3, Fc gamma receptor I, and Fc gamma receptor III was induced. The DD cells induced with 12-O-tetradecanoylphorbol-13-acetate or Escherichia coli lipopolysaccharide can internalize EA via Fc gamma receptor II and complement-coated yeast in the function of the inducers. The phagocytic ability appears to be parallel with the appearance of enzymes which participate in phagocytosis.

Antigens, Surface↗

PubMed1991

Isolation and partial characterization of a cystine-rich basic heparin-binding protein from bovine platelets.

We report the isolation and partial characterization of a so far unidentified basic platelet protein. Delipidated bovine platelets were extracted at pH 2.1. The extract was subjected to differential precipitation at pH 5.4-5.5 and by ammonium sulfate, then it was further purified by ion exchange chromatography on DEAE and CM cellulose columns in an urea containing medium. The major protein peak eluted from the CM cellulose column by NaCl gradient contained a protein in electrophoretically homogeneous form. It consists of a single polypeptide chain with an Mr of 28,000 as estimated by SDS PAGE. It was shown to be extremely rich in lysine and cystine and possessed a highly basic character (pI 9.8-10.1). On this basis the term cystine-rich basic protein (CRBP) was proposed for the new protein. Unlike some other low Mr basic proteins it did not bind calmodulin and troponin C, however, it showed significant heparin neutralizing activity.

Amino Acids↗

PubMed1990

Non-proteolytic activation of cellular protransglutaminase (placenta macrophage factor XIII).

Plasma Factor XIII is a zymogen (plasma protransglutaminase) with the tetrametric structure A2B2, whereas the cellular protransglutaminase, i.e. Factor XIII in the platelet and monocyte/macrophage, consists exclusively of A subunits (A2). It is generally accepted that at Ca2+ concentrations comparable with that in plasma the proteolytic removal of an N-terminal activation peptide is the prerequisite for the Ca2(+)-induced formation of a catalytically active configuration of subunit A. In this study it was demonstrated that at high concentrations NaCl or KCl induced a non-proteolytic activation of cellular (placental macrophage) but not plasma protransglutaminase. The activation depended on time and salt concentration, and Ca2+, in the range 0-20 mM, greatly enhanced the activation process. At 1.25 M-NaCl maximal activation occurred within 60 min in the presence of 2 mM-CaCl2, and even at physiological NaCl concentration a slow progressive activation could be observed in the presence of Ca2+. The specific activity of salt-activated Factor XIII was 1.5-2.0-fold higher than that obtained after thrombin activation. The non-proteolytic activation of cellular protransglutaminase was abolished by the addition of subunit B of plasma Factor XIII in stoichiometric amount, which suggests that (one of) the physiological function(s) of the B subunit in plasma Factor XIII is to prevent the slow spontaneous activation of A subunit that would occur in a plasmatic environment.

Calcium↗

PubMed1990

Increased density of histiocytes in uterine leiomyomas.

The histiocyte content of leiomyomata and adjacent, histologically normal myometrium has been quantified morphometrically with the help of recently developed immunostaining for tissue macrophages. Histiocytes identified by immunoreaction for Factor XII subunit A and by double immunofluorescent labelings were found to be identical with tissue macrophages present in normal uteri. Histiocyte counts were significantly higher in leiomyomatous areas than in adjacent normal myometrium, and the distribution of these cells was also changed. In the normal uterine wall, histiocytes could be detected only in the connective tissue septa separating smooth muscle bundles, while in the myomatous nodules they were diffusely scattered throughout the whole area. In addition to providing new information on the cellular composition of leiomyomas, the present observations also raise several questions concerning the possible pathogenetic role of FXIII-positive histiocytes in neoplastic proliferation of uterine smooth muscle.

Factor XIII↗

PubMed1989

Factors of the extrinsic pathway of blood coagulation in tumour associated macrophages.

Despite the growing evidence implicating intratumoural fibrin formation in the progression of malignant tumours, the origin of coagulation factors that participate in extravascular clotting has not been elucidated. Using immunohistochemical methods we attempted to detect and localize clotting factors of extrinsic pathway in different human malignant tumours. Coagulation factors II, V, VII and X (FII, FV, FVII and FX) were detected in a huge number of cells showing spindle-shaped or stellate morphology. By double immunohistochemical labellings it was demonstrated that cells containing these clotting factors express monocyte/macrophage differentiation marker antigens recognized by Leu M3, Ki M7 and DAKO-macrophage monoclonal antibodies, i.e. they represent monocyte-derived, phagocytic tumour associated macrophages (TAMs). These findings suggest that TAMs can be viewed as clot cells, which in addition to the initiation of extravascular clotting by expressing procoagulant activity can also provide all the components necessary for the extrinsic thrombin formation.

Animals↗

PubMed1989

Glycoprotein Ib and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages.

The glycoprotein (GP) Ib-IX complex is a major component of the platelet membrane which mediates adhesion of platelets to exposed subendothelium. GP Ib is a heterodimer with a large alpha chain (Mr = 135,000-145,000) and small beta chain (Mr = 22,000-27,000) linked by a disulfide bond(s). GP Ib is bound in a noncovalent 1:1 complex with GP IX (Mr = 17,000-22,000). We labeled isolated human platelets with [3H] palmitate or surface-labeled platelet membrane glycoproteins with sodium periodate-[3H]sodium borohydride and immunoprecipitated the GP Ib-IX complex from radiolabeled platelet lysates using a mouse monoclonal antibody (SZ.1) which recognizes the intact complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates from [3H]palmitate-labeled platelets revealed two radiolabeled bands under reducing conditions at 24 and 19 kDa and two bands under nonreducing conditions at 170 and 19 kDa. As demonstrated by the parallel analysis of immunoprecipitates from periodate-[3H]sodium borohydride-labeled platelets, the [3H]palmitate-labeled bands obtained under reducing conditions corresponded to GP Ib beta and GP IX and the ones obtained under nonreducing conditions to intact GP Ib and GP IX, respectively. Using alkaline methanolysis followed by high pressure liquid chromatography analysis of the methanolysis products, we demonstrated that the radioactivity associated with the GP Ib-IX complex from [3H]palmitate-labeled platelets was, in fact, covalently bound [3H]palmitate in ester linkage to protein. The protein-fatty acid linkage was also disrupted by hydroxylamine at neutral pH. Thus, this study demonstrates that GP Ib beta and GP IX in human platelets are both fatty acid-acylated with palmitate through thioester linkages.

Acylation↗

PubMed1989

Immunohistochemical detection of factor XIII subunit a in histiocytes of human uterus.

As spontaneous abortion is a frequent finding in females with Factor XIII (FXIII) deficiency it has been presumed that this clotting factor is essential to normal pregnancy. FXIII subunit a (FXIII A) has been demonstrated in the homogenate of human uterus, but no information on its cellular distribution has been published, so far. In the present study first FXIII A was detected in paraformaldehyde-fixed, paraffin-embedded sections of human uterus by immunoperoxidase technique. Cells containing FXIII A were localized between collagen fibrils stained by Picrosirius Red F3B in the connective tissue. To characterize them the immunofluorescent detection of FXIII A was combined by the visualization of different marker antigens of monocytes and macrophages recognized by Leu-M3, RFD7, anti-HLA-DR and DAKO-anti-macrophage monoclonal antibodies on frozen sections. The coexpression of FXIII A with monocyte and macrophage differentiation marker antigens clearly proves that cells containing FXIII A in the uterus are monocyte-derived tissue macrophages. The results well agree with our previous findings demonstrating FXIII A in human monocytes and different types of macrophages. On the basis of these results, the presence of FXIII A does not seem to be a specificity of the uterus but a characteristic of monocyte/macrophage cell line including tissue macrophages, in general.

Adult↗

PubMed1989

Congenital factor XIII deficiency with multiple benign breast tumours and successful pregnancy with substitutive therapy. A case report.

A 34-year-old woman with congenital factor XIII (FXIII) deficiency and multiple connective tissue tumours is reported. The subunit a of FXIII was totally absent in her plasma, platelets and histiocytes of breast fibroadenomas and considerably reduced in the monocytes (below 5%). The plasmatic level of subunit b was also reduced (25%). She had a bleeding tendency and habitual abortions. Fresh frozen plasma therapy permitted a successful pregnancy.

Adult↗

PubMed1989

Characterization of rapidly adhering amniotic fluid cells by combined immunofluorescence and phagocytosis assays.

Culture of human amniotic-fluid cells from cases of fetal neural tube defects produces a population of rapidly adhering cells that were initially thought to be macrophages and later interpreted to be of neural origin. In this study double and triple labeling systems for the simultaneous detection of glial and macrophage differentiation marker antigens have been used to demonstrate that rapidly adhering cells cannot be considered a homogeneous population but instead represent two distinct cell types. One of these cell populations is of glial origin and shows specific staining for glial fibrillary acidic protein, while the other population is monocyte-derived macrophages which express marker antigens recognized by Leu M3, KiM7, and Dako antimacrophage monoclonal antibodies.

Amniotic Fluid↗