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Biomedical subjects

L Muszbek

Publications and source records attributed to L Muszbek.

At least 37 records · Page 2Linked to original sources
PubMed1999

The pool of fatty acids covalently bound to platelet proteins by thioester linkages can be altered by exogenously supplied fatty acids.

The goals of this investigation were, first, to develop a chemical strategy to identify and quantitate the mass of fatty acid which is covalently bound to proteins by thioester linkage in unactivated platelets, and, second, to determine whether exogeneously added fatty acids can alter the fatty acid composition of thioester bound fatty acids. Studies with radiolabeled fatty acids cannot identify and quantitate the actual fatty acids bound to proteins because they permit analysis of only the radiolabeled fatty acids added and their metabolites. Therefore, in the absence of metabolic labeling by radiolabeled fatty acids, we isolated the thioester-linked fatty acids from platelet proteins using hydroxylamine at neutral pH to form fatty acid hydroxamates. The hydroxamates were subsequently converted to fatty acid methyl esters by acid methanolysis for quantitation by gas chromatography-mass spectrometry. Using platelet specimens from 14 subjects, 74% of the fatty acid recovered from the unactivated platelet proteins as thioester linked was palmitate. Importantly, however, 22% was stearic acid, and oleate was 4% of the total thioester bound fatty acid. There was minimal variability (2.6-fold at maximum) between the subjects in the amount of the thioester-linked palmitate and thioester-linked stearate. However, there was substantial variability (>100-fold at maximum) between subjects in the amount of thioester-linked oleate. We also demonstrated that incubation of platelets with exogenous fatty acids can alter the profile of fatty acids bound to platelet proteins by thioester linkages. Incubation of platelets with 100 microM palmitate for 3 h increased the amount of thioester-linked palmitate by up to 26%, and incubation of platelets with 100 microM stearate increased the amount of thioester-linked stearate up to 30%. In support of the observation that radiolabeled fatty acids other than palmitate were shown to be capable of binding to platelet proteins by thioester linkage, our results indicate that the fatty acids actually bound to unactivated platelet proteins include a significant amount of stearate, and variable amounts of oleate, as well as palmitate. In addition, the data show that palmitate and stearate can be increased, as a percentage of total protein-bound fatty acid, by incubation with exogenous palmitate and stearate, respectively.

Blood Platelets↗
PubMed1997

[Thrombocyte dysfunction in children with albinism].

Seven children with albinism were examined for haemorrhagic diathesis. In 6 patients coagulation screening tests (prothrombin time, activated partial thromboplastin time, thrombin time) were normal, in 1 patient prolongation of activated partial thromboplastin time due to mild factor XI deficiency was found. Platelet counts were in the reference range, however, the template bleeding time of 6 out of 7 patients was prolonged. Evaluation of platelet aggregation and secretion in a lumi-aggregometer demonstrated that only the child with normal bleeding time had normal platelet function, and 6 children with prolonged bleeding time had impaired aggregation and release reaction. Five out of these 6 patients had severe decrease of ATP secretion even when high dose of thrombin was used as agonist. The number of dens granules in the platelets of these patients, as measured by the uptake of mepacrine, was significantly less than in normal controls. These findings clearly suggested storage pool deficiency. In one of these patients storage pool deficiency was associated with mild factor XI deficiency. The high frequency of haemorrhagic diathesis in albino children emphasizes, the importance of the screening of patients with albinism for bleeding diathesis.

Adolescent↗
PubMed1997

Molecular mechanism of a mild phenotype in coagulation factor XIII (FXIII) deficiency: a splicing mutation permitting partial correct splicing of FXIII A-subunit mRNA.

Congenital factor XIII (FXIII) deficiency is potentially a severe bleeding disorder, but in some cases, the symptoms may be fairly mild. In this study, we have characterized the molecular mechanism of a mild phenotype of FXIII A-subunit deficiency in a Finnish family with two affected sisters, one of whom has even had two successful pregnancies without regular substitution therapy. In the screening tests for FXIII deficiency, no A-subunit could be detected, but by using more sensitive assays, a minute amount of functional A-subunit was seen. 3H-putrescine incorporation assay showed distinct FXIII activity at the level of 0.35% of controls, and also the fibrin cross-linking pattern in the patients clotted plasma showed partial gamma-gamma dimerization. In Western blot analysis, a faint band of full-length FXIII A-subunit was detected in the patients' platelets. The patients have previously been identified as heterozygotes for the Arg661 --> Stop mutation. Here we report a T --> C transition at position +6 of intron C in their other allele. The transition affected splicing of FXIII mRNA resulting in low steady state levels of several variant mRNA transcripts. One transcript contained sequences of intron C, whereas two transcripts resulted from skipping of one or two exons. Additionally, correctly spliced mRNA lacking the Arg661 --> Stop mutation of the maternal allele could be detected. These results demonstrate that a mutation in splice donor site of intron C can result in several variant mRNA transcripts and even permit partial correct splicing of FXIII mRNA. Further, even the minute amount of correctly processed mRNA is sufficient for producing protein capable of gamma-gamma dimerization of fibrin. This is a rare example of an inherited functional human disorder in which a mutation affecting splicing still permits some correct splicing to occur and this has a beneficial effect to the phenotype of the patients.

DNA Mutational Analysis↗
PubMed1997

Posttranslational modification of proteins with fatty acids in platelets.

Direct modification of proteins by fatty acid can occur as cotranslational N-myristoylation of an N-terminal glycine residue or as posttranslational thioesterification of cysteine residue(s). Platelets provide an excellent model system for studying the posttranslational type of modification in the absence of active protein synthesis and in the absence of protein synthesis-related protein modifications with lipids. Using this model system it was shown that thioesterification of proteins with fatty acid is less specific for palmitate than it was thought earlier and that other saturated, mono- and even polyunsaturated long chain fatty acids can also participate. The chain length and the extent of unsaturation of the protein-linked fatty acid moiety can, very likely, modulate hydrophobic protein-membrane lipid and protein-protein interactions. CD9, HLA class I glycoprotein, glycoproteins Ib, IX and IV, P-selectin and alpha subunits of G proteins have been demonstrated unequivocally as S-fatty acid acylated platelet proteins.

Acylation↗
PubMed1997

Fatty acid acylation of platelet proteins.

A variety of fatty acids can become covalently attached to platelet proteins by thioester linkage. These fatty acids include palmitate, myristate, stearate, arachidonate, and eicosapentaenoate. More than 20 platelet proteins can be acylated by fatty acids. Several of the acylated platelet proteins have been identified, including glycoprotein Ib beta, glycoprotein IX, P-selectin, G-protein alpha subunits, and CD9. This report reviews the fatty acid acylation of platelet proteins.

Acylation↗
PubMed1997

Molecular mechanisms of mutations in factor XIII A-subunit deficiency: in vitro expression in COS-cells demonstrates intracellular degradation of the mutant proteins.

Factor XIII deficiency is an autosomal recessive bleeding disorder that is largely caused by various mutations in FXIII A-subunit gene. Characteristically, the patients lack both A-subunit activity and antigen in the circulation. Here we have analysed the consequences of four missense mutations (Met242-->Thr, Arg252-->Ile, Arg326-->Gln, Leu498 to Pro) and one stop mutation (Arg661-->Stop) in the FXIII A-subunit gene by expression in COS-cells. After transient transfection each mutant cDNA expressed mRNA at an equal level to the wild type FXIII. However, the mutant polypeptides accumulated in the cells in significantly reduced quantities and demonstrated only very low enzymatic activity. Analysis of immunoprecipitated metabolically labelled polypeptides demonstrated remarkable instability and intracellular degradation of all mutant FXIII proteins. These results verify the deleterious nature of the individual amino acid changes and confirm that protein instability and susceptibility to proteolysis are consequences of the mutations, as predicted from the three-dimensional model of crystallised FXIII A-subunit.

Animals↗
PubMed1996

Thioesterification of platelet proteins with saturated and polyunsaturated fatty acids.

We have demonstrated that more than 20 platelet proteins can be acylated with fatty acids via thioester linkages. These include the glycoprotein IX beta chain of glycoprotein Ib, components of the von Willebrand factor receptor on the platelet surface, P-selectin, and alpha subunits of Gz, Gq, and Gi. Our studies have shown that platelet proteins can be posttranslationally acylated in thioester linkages not only with palmitate but with myristate and also with the eicosanoid precursor fatty acids arachidonate and eicosapentaenoate. Thioesterification of platelet proteins with fatty acids other than palmitate may have significant functional consequences for reversible binding of proteins to membranes.

Arachidonic Acid↗
PubMed1996

ETRO Working Party on Factor XIII questionnaire on congenital factor XIII deficiency in Europe: status and perspectives. Study Group.

A questionnaire was sent out in 1993 to more than 350 European institutions caring for patients with hemorrhagic disorders with the request to provide data of patients with congenital factor XIII deficiency, to pursue the following aims: (1) establish a registry of congenital factor XIII deficiency patients, (2) promote exchange between clinicians and basic researchers, (3) improve diagnostic and therapeutic approaches, and (4) stimulate research on gene defects and their impact on factor XIII function. So far, 72 patient questionnaires from 60 families have been collected. Their bleeding pattern is typical, with frequent involvement of the umbilical cord and the central nervous system. Forty-nine patients receive regular factor XIII replacement, but obviously some patients with mild symptoms do not require prophylactic substitution, despite low factor XIII levels. On the other hand, 18 patients had factor XIII activities of > or = 5% of normal, but only 3 of those patients were reported to have no bleeding symptoms. Furthermore, 17 symptomatic, apparently heterozygous relatives in eight families were observed. Seven out of 30 females aged over 18 years had experienced spontaneous abortions; wound healing problems were seen in 26 patients. Currently, a second questionnaire is being distributed to obtain more detailed information on bleeding and other symptoms, diagnostic approaches, and exclusion of concurrent other bleeding diatheses. Future activities will be validation and standardization of assays, and study of gene defects and their impact on the structure of factor XIII and symptoms of patients. We intend to expand the survey to countries outside Europe.

Abortion, Habitual↗
PubMed1996

Novel aspects of blood coagulation factor XIII. I. Structure, distribution, activation, and function.

Blood coagulation factor XIII (FXIII) is a protransglutaminase that becomes activated by the concerted action of thrombin and Ca2+ in the final stage of the clotting cascade. In addition to plasma, FXIII also occurs in platelets, monocytes, and monocyte-derived macrophages. While the plasma factor is a heterotetramer consisting of paired A and B subunits (A2B2), its cellular counterpart lacks the B subunits and is a homodimer of potentially active A subunits (A2). The gene coding for the A and B subunits has been localized to chromosomes 6p24-25 and 1q31-32.1, respectively. The genomic as well as the primary protein structure of both subunits has been established, and most recently the three-dimensional structure of recombinant cellular FXIII has also been revealed. Monocytes/macrophages synthesize their own FXIII, and very likely FXIII in platelets is synthesized by the megakaryocytes. Cells of bone marrow origin seem to be the primary site for the synthesis of subunit A in plasma FXIII, but hepatocytes might also contribute. The B subunit of plasma FXIII is synthesized in the liver. Plasma FXIII circulates in association with its substrate precursor, fibrinogen. Fibrin(ogen) has an important regulatory role in the activation of plasma FXIII. The most important steps of the activation of plasma FXIII are the proteolytic removal of activation peptide by thrombin, the dissociation of subunits A and B, and the exposure of the originally buried active site on the free A subunits. The end result of this process is the formation of an active transglutaminase, which cross-links peptide chains through epsilon(gamma-glutamyl)lysyl isopeptide bonds. Cellular FXIII in platelets becomes activated through a nonproteolytic process. When intracytoplasmic Ca2+ is raised during platelet activation, the zymogen--in the absence of subunit B--assumes an active configuration. The protein substrates of activated FXIII include components of the clotting-fibrinolytic system, adhesive and contractile proteins. The main physiological function of plasma FXIII is to cross-link fibrin and protect it from the fibrinolytic plasmin. The latter effect is achieved mainly by covalently linking alpha 2 antiplasmin, the most potent physiological inhibitor of plasmin, to fibrin. Plasma FXIII seems to be involved in wound healing and tissue repair, and it is essential to maintaining pregnancy. Cellular FXIII, if exposed to the surface of the cells, might support or perhaps take over the hemostatic functions of plasma FXIII; however, its intracellular role has remained mostly unexplored.

Animals↗
PubMed1996

Promotion of the crosslinking of fibrin and alpha 2-antiplasmin by platelets.

Factor XIII (FXIII) is of high importance in the regulation of fibrinolysis. It crosslinks alpha 2-antiplasmin (alpha 2AP) and fibrin and by this way protects fibrin from the prompt elimination by plasmin. Although FXIII of platelets has been implicated in this protective mechanism, the role of platelets and platelet FXIII in the crosslinking process is far from being elucidated. As demonstrated by SDS PAGE and by immunoblotting for alpha 2AP, intact normal platelets resuspended in FXIII-free plasma or FXIII-free fibrinogen solution catalyzed the crosslinking of fibrin chains and also the crosslinking of alpha 2AP to fibrin alpha-chains. With FXIII-deficient platelets no crosslinking reaction could be observed indicating that the crosslinking with normal platelets was, indeed, due to platelet FXIII and not to another, putative platelet transglutaminase. However, the crosslinking of alpha 2AP to fibrin induced by the FXIII of intact platelets resuspended in FXIII-free plasma was considerably less extensive than the crosslinking carried out by the FXIII of normal plasma in the presence of FXIII-free platelets. Furthermore, the replacement of FXIII-free platelets by normal platelets in normal FXIII-containing plasma resulted in little, if any, difference in the crosslinking process. When crosslinking was induced by highly purified plasma FXIII the presence of intact FXIII-free platelets significantly accelerated the formation of alpha-chain polymers as well as the incorporation of alpha 2AP-fibrin alpha-chain hetero-dimer into these polymers. The results indicate that, in physiological conditions, platelet FXIII plays only a minor role in the crosslinking of alpha 2AP and fibrin; however, platelets, independently of their FXIII content, promote the crosslinking reaction by providing a catalytic surface on which the formation of highly crosslinked fibrin polymers is accelerated.

Blood Platelets↗
PubMed1995

Localization of transglutaminase in human lenses.

Transglutaminase activity has been detected in the lenses of laboratory animals and in human cataracts. However, its distribution in the lens tissue has not been investigated. Using a monoclonal antibody against tissue transglutaminase, we showed by Western blotting and immunoabsorption that transglutaminase of normal human lens is immunologically related to tissue transglutaminase but has a slightly higher M(r) than the latter enzyme. Using monoclonal or polyclonal antibody against tissue transglutaminase, lens transglutaminase was localized to the epithelial cell layer on the anterior lens surface and to a thin stripe between the capsule and the peripheral cortex on the posterior surface. Lens fibers were not stained with the antibodies. Factor XIII, another transglutaminase, could not be detected in the lens tissue. The localization of transglutaminase in the lens suggests that lens transglutaminase is synthesized in the epithelial cells and secreted into the virtual space between the capsule and the peripheral cortex spreading all around the lens substance.

Adult↗