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Biomedical subjects

A Ruf

Publications and source records attributed to A Ruf.

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

Effect of glycoprotein IIb-IIIa receptor antagonism on platelet membrane glycoproteins after coronary stent placement.

Platelet membrane glycoproteins play a crucial role in ischemic complications after coronary stenting. Glycoprotein IIb-IIIa blockade reduces adverse clinical events after angioplasty but is associated with rare but profound thrombocytopenia that might increase hemorrhagic complications. Changes in platelet membrane glycoproteins of patients with angina who underwent coronary stenting and were treated with the GPIIb-IIIa antagonist abciximab (n=20) or with heparin (n=23) were studied. GPIb-IIIa receptor blockade and membrane glycoproteins were evaluated with immunological markers in venous blood samples taken before. 10, 24, 48, 72, and 96 h after initial treatment with either abciximab or heparin. Patients receiving abciximab therapy showed a rapid inhibition of binding of fluorochrome-conjugated mAb CD41 and c7E3 concomitant with a reduction in platelet aggregation which was restored in part in the days after termination of abciximab infusion. Induction of ligand-induced binding sites on GPIIb-IIIa was increased in patients receiving abciximab. The expression of ligand-induced binding sites correlated inversely with platelet count. No significant change in platelet membrane markers were found in the heparin group. In vitro studies showed that abciximab induces ligand-induced binding sites on isolated platelets and on nuclear cells bearing recombinant GPIIb-IIIa. Abciximab rapidly achieves GPIIb-IIIa receptor blockade after coronary stent placement that might be beneficial in high-risk settings to bridge the delayed action of ticlopidine. Significant alterations of platelet membrane glycoproteins during GPIIb-IIIa antagonism might contribute to development of acute profound thrombocytopenia.

Abciximab↗
PubMed1997

Photoaffinity labelling of human poly(ADP-ribose) polymerase catalytic domain.

Photoaffinity labelling of the human poly(ADP-ribose) polymerase (PARP) catalytic domain (40 kDa) with the NAD+ photoaffinity analogue 2-azido-[alpha-32P]NAD+ has been used to identify NAD+-binding residues. In the presence of UV, photo-insertion of the analogue was observed with a stoichiometry of 0.73 mol of 2-azido-[alpha-32P]NAD+ per mol of catalytic domain. Competition experiments indicated that 3-aminobenzamide strongly protected the insertion site. Residues binding the adenine ring of NAD+ were identified by trypsin digestion and boronate affinity chromatography in combination with reverse-phase HPLC. Two major NAD+-binding residues, Trp1014 of peptide Thr1011-Trp1014 and Lys893 of peptide Ile979-Lys893, were identified. The site-directed mutagenesis of these two residues revealed that Lys893, but not Trp1014, is critical for activity. The close positioning of Lys893 near the adenine ring of NAD+ has been confirmed by the recently solved crystallographic structure of the chicken PARP catalytic domain [Ruf, Menissier-de Murcia, de Murcia and Schulz (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7481-7485].

Affinity Labels↗
PubMed1997

In-vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with beta-thalassaemia major.

Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with beta-thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p-selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual-colour flow cytometry. A significantly (P < 0.01) higher fraction of FITC-annexin V-labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation (P < 0.001) was found in TM patients between the number of RBC-bound annexin V molecules and the fraction of CD62p (p-selectin) or CD63-positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelerate thrombin generation in vivo which, in turn, triggers platelet activation.

Adolescent↗
PubMed1996

Structure of the catalytic fragment of poly(AD-ribose) polymerase from chicken.

The crystal structures of the catalytic fragment of chicken poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] with and without a nicotinamide-analogue inhibitor have been elucidated. Because this enzyme is involved in the regulation of DNA repair, its inhibitors are of interest for cancer therapy. The inhibitor shows the nicotinamide site and also suggests the adenosine site. The enzyme is structurally related to bacterial ADP-ribosylating toxins but contains an additional alpha-helical domain that is suggested to relay the activation signal issued on binding to damaged DNA.

Animals↗
PubMed1996

Freeze-dried human leukocytes stabilized with uranyl acetate during low temperature embedding or with OsO4 vapor after embedding.

Two new simple stabilization procedures for freeze-dried biological material are introduced which are compatible with low temperature embedding (LTE) in Lowicryl. The first method uses a Lowicryl K11M/HM20 mixture supplemented with 0.3% uranyl acetate for LTE. For the second method polymerized Lowicryl blocks containing the freeze-dried material are exposed to OsO4 vapor which penetrates into the Lowicryl block and stabilizes the embedded specimen. The quality of structural preservation is demonstrated with human leukocytes.

Acrylic Resins↗
PubMed1995

In vivo degradation of human fibrinogen A alpha: detection of cleavage sites and release of antithrombotic peptides.

Several degradation products of fibrinogen have been shown to possess regulatory functions. Using peptide extracts from human blood filtrate, a large number of fibrinogen A alpha fragments was identified. These fragments are generated at known plasmin attack sites and at several novel cleavage sites especially at hydrophobic and basic amino acid residues. One fragment containing the cell attachment site (RGD sequence) of fibrinogen A alpha efficiently inhibits fibrinogen binding and platelet aggregation (IC50:20-50 microM) in vitro. We conclude that in vivo degradation of fibrinogen A alpha results in generation of endogenous antithrombotic peptides with local importance in fibrinolysis and platelet aggregation.

Adenosine Diphosphate↗
PubMed1995

Poly(ADP-ribose) polymerase: structure-function relationship.

Dissection of the human poly(ADP-ribose) polymerase (PARP) molecule in terms of its structure-function relationship has proved to be an essential step towards understanding the biological role of poly(ADP-ribosylation) as a cellular response to DNA damage in eukaryotes. Current approaches aimed at elucidating the implication of this multifunctional enzyme in the maintenance of the genomic integrity will be presented.

Animals↗
PubMed1995

Ultrastructural aspects of platelet adhesion on subendothelial structures.

Platelet adhesion to ligands within the subendothelium induces platelet activation. Ultrastructural and immunocytochemical investigations were carried out to obtain information on the microtopography of the adhesive interactions. Rabbit platelets which react in vivo with endothelial lesions (V. jugularis), and human platelets which react in vitro with collagen were observed. It was shown that the reaction of adhering platelets depends on the micro-topography of the adhesive surface 1. On plane surfaces the platelets spread 2. Collagen fibers induce the formation of focal contacts. They are initiated by binding of ligands to certain transmembrane receptors. The contact mediates the formation of the contractile gel, which acts as a constricting sphere internalizing surface bound ligands, i.e., retraction of collagen networks. Most of the experiments hitherto dealt with platelets that spread. The described findings show that the reaction mentioned above plays a more important role than previously imagined and may be the physiological response of platelets interacting with subendothelial components in vessel lesions.

Animals↗
PubMed1995

Flow cytometric detection of activated platelets: comparison of determining shape change, fibrinogen binding, and P-selectin expression.

Platelet activation plays an important role in the pathomechanisms of arterial vascular disorders including stroke, peripheral arterial disease (PAD), and myocardial infarction. Circulating activated platelets may be useful markers of local thrombotic events occurring in these diseases. Using flow cytometry circulating activated platelets can be detected by determining: 1. the platelets' shape change on the basis of the different light scatter properties of discocytes and spherocytes, 2. the expression of platelet bound fibrinogen or conformation specific neoantigens on fibrinogen and on its platelet receptor, and 3. the exposure of granule membrane proteins such as P-selectin as a result of platelet secretion. The concentration-effect relationships were determined for the ADP and U46619 induced shape change, fibrinogen binding, and expression of P-selectin. The EC50 for the shape change was 4 times lower than the EC50 for the fibrinogen binding and 29 times lower than the EC50 for the expression of P-selectin. These data clearly demonstrate that the shape change is a more sensitive indicator of platelet activation in vitro than fibrinogen binding or P-selectin expression. Both the shape change and fibrinogen binding were reversible, whereas the expression of P-selectin was irreversible upon stimulation. Reversibility of the shape change may be responsible for the fact that in patients with stroke or PAD the fraction of discocytes did not differ from controls, whereas more than 80% of them revealed a significantly higher fraction of P-selectin positive platelets. Thus the determination of the P-selectin expression reveals a higher diagnostic sensitivity for detecting a platelet activation in vivo than the determination of the shape change.

Antigens, CD↗
PubMed1995

Platelet-induced neutrophil activation: platelet-expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18.

Mutual contacts and platelet-expressed fibrinogen seem to be required for the stimulation of neutrophils by activated platelets. The beta 2-integrins CD11b/CD18 and CD11c/CD18 are potential receptors for fibrinogen on neutrophils. In order to investigate whether binding of fibrinogen to these integrins is involved, monoclonal antibodies (MoAbs) and Gly-Pro-Arg-Pro (GPRP) peptide that inhibits fibrinogen binding to CD11c/CD18 were checked for their effects on the interaction of activated platelets and neutrophils. The luminol-amplified chemiluminescence (CL) as a measure for the oxidative burst of neutrophils was recorded simultaneously to the platelet aggregation in mixed cell suspensions. The adhesion of platelets and neutrophils was determined microscopically. The thromboxane A2 mimetic U46619 was used as a potent platelet agonist but that does not stimulate neutrophils. aggregation and a strong CL of neutrophils. The platelet-induced activation of neutrophils required added fibrinogen which fibronectin or thrombospondin could not substitute for. Cytochalasin D (Cyto D) that blocks actin polymerization totally abrogated the platelet-induced Cl of neutrophils. The MoAb OKM1 against CD11b, which blocks fibrinogen binding to CD11b/CD18 as well as the MoAbs IOT16 and IOT18 directed against CD11a and CD18, respectively, had no effect. In contrast, the MoAb LeuM5 which inhibits the binding of fibrinogen to CD11c/CD18 revealed a strong inhibition. Furthermore, GPRP peptide which CD11c/CD18 recognizes on the A alpha-chain of fibrinogen also strongly inhibited the platelet-induced CL of neutrophils, whereas control peptides such as Gly-His-Arg-Pro (GHRP) or Gly-Pro-Gly-Gly (GPGG) had no effect. In contrast to the platelet-induced CL of neutrophils, Cyto D, MoAb against CD11c and GPRP peptide did not inhibit the CL induced by FMLP and PAF in pure neutrophil suspensions. They also did not affect U46619-induced platelet aggregation. The adhesion of platelets and neutrophils was neither dependent on added fibrinogen nor inhibited by Cyto D, MoAb against CD11c and the GPRP-peptide. Therefore fibrinogen and actin polymerization seem not to be required for the adhesion of neutrophils to platelets. However, the activation of neutrophils depends on the interaction of CD11c/CD18 with the A alpha-chain of platelet-expressed fibrinogen and the contractile system of neutrophils.

Blood Platelets↗
PubMed1992

Contact-induced neutrophil activation by platelets in human cell suspensions and whole blood.

Platelet-dependent activation of polymorphonuclear neutrophils (PMNL) was investigated with a lumi-aggregometer in heparinized whole blood and platelet-PMNL suspensions. The lumi-aggregometer allowed us to simultaneously monitor increases in impedance or light transmission as consequences of platelet aggregation and luminol-enhanced chemiluminescence (CL) as a measure of the oxidative burst in PMNL. Aggregation and platelet-PMNL contacts were also checked by light and electron microscopy. In whole blood, adenosine diphosphate (ADP) and the thromboxane A2 mimetic U 46619 induced the aggregation (increase in impedance) and the CL, which were both suppressed by EDTA, arginyl-glycyl-aspartyl-serine (RGDS) peptide, and the absence of stirring. In contrast, FMLP caused only CL that was unaffected by EDTA, RGDS peptide, and nonstirring. Similar observations were obtained with mixed suspensions containing washed platelets and PMNL at their physiologic concentrations. ADP, U 46619, and thrombin induced both aggregation (increase in light transmission) and CL, whereas FMLP caused CL but only very weak aggregation. Exogenous fibrinogen strongly enhanced the effects of ADP and U 46619. Iloprost, EDTA, RGDS peptide, red blood cell (RBC) ghosts, and nonstirring inhibited the effects induced by the platelet agonists, but were ineffective on the CL induced by FMLP. Treatment of platelets with aspirin did not affect the CL of PMNL induced by platelets. Microscopic examination, the requirements of stirring, Ca2+, and fibrinogen, and the inhibitory effects of RGDS peptide and RBC ghosts show that stimulated platelets activate PMNL in a contact-dependent manner that depends on fibrinogen binding. This was confirmed by the immunochemical demonstration of fibrinogen (but not of fibronectin) in the contact spaces between activated platelets and PMNL. Because supernatants and lysates of resting or thrombin-stimulated platelets did not induce the CL of PMNL, soluble agonists did not appear to be involved. Nonstimulated washed platelets also caused CL of PMNL that required stirring and Ca2+ and was inhibited by RBC ghosts. No CL occurred in unstimulated stirred whole blood, suggesting that a preactivation of platelets during the preparation may be responsible for the effects of unstimulated washed platelets. The results show that platelets provide a strong stimulus for PMNL that requires intercellular contact. Fibrinogen exposure on the platelet surface seems to be necessary for the activation of PMNL by stimulated platelets.

Adult↗
PubMed1992

Transport of anti-glycoprotein IIb/IIIa-antibodies into the alpha-granules of unstimulated human blood platelets.

The redistribution of the antibody-glycoprotein (GP) IIb/IIIa complex was investigated with the immuno-gold labeling technique in order to trace its transport in resting platelets. Washed platelets were incubated in the presence of aspirin and a prostacyclin analogue (iloprost) with three different monoclonal antibodies (Gi3, J15 and P2) against GPIIb/IIIa. The examination of ultrathin serial sections showed that the surface labeling was internalized into the surface connected membrane system (SCS). Labels were found within the alpha-granules after 40 min and the number of labels increased during longer incubation periods (max. 120 min). The transport possibly involved coated membranes. The alpha-granules were neither found to be altered during this process nor were any morphological signs of platelet activation detectable. The anti-GPIIb/IIIa complex remained membrane-associated during the transfer. These observations indicate that the membrane-GPIIb/IIIa complex was stable and transported from the surface into the alpha-granules of resting platelets. Since this transport was not influenced by iloprost or by aspirin it may be interpreted as constitutive endocytosis.

Antibodies, Monoclonal↗
PubMed1991

Role of internalization in platelet activation induced by collagen fibers--differential effects of aspirin, cytochalasin D, and prostaglandin E1.

Washed human platelets were stimulated with fibrillar collagen and platelet aggregation was prevented by non-stirring conditions. In these samples, electron microscopy revealed three fractions of platelets: 1) a majority without contacts to the collagen fibers, 2) with focal contacts to collagen, and 3) a small fraction of platelets with internalized collagen. All platelets had undergone shape change, and exhibited an internal contraction and granule release. However, only those with internalized collagen were completely degranulated. The internalized collagen was found to be in close contact to the contractile sphere in the platelet center, as it was demonstrable with computer assisted 3-D reconstruction from serial sections. Aspirin inhibited neither the adhesion to collagen nor its internalization by internal contraction. Also it did not impair the shape change and degranulation in the platelet fractions that internalized collagen. However, aspirin blocked the shape change and internal contraction of the other platelets and inhibited the [3H]serotonin release. Cytochalasin D 0.1 microM also suppressed the internalization of collagen, the shape change, the formation of a contractile gel, the degranulation, and the [3H]serotonin release in all platelets, whereas the number of platelets that adhered to collagen remained unchanged. The same effects were produced by prostaglandin E1. If the platelets were stimulated with the TXA2 mimetic, U46619, cytochalasin D at 0.1 microM had no effect; but at 20 microM it strongly enhanced the degranulation and the [3H]serotonin release, although the platelets remained discoid. It is concluded that collagen triggers a focal activation of an adherent platelet at the site of its initial contact to collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Alprostadil↗
PubMed1990

Ultrastructure of the interaction between human platelets and polymerizing fibrin within the first minutes of clot formation.

In the presence of fibrinogen, thrombin induces the aggregation of platelets, the formation of fibrin, and the retraction of the fibrin network. Since these phenomena are initiated in the first minutes after stimulation, the platelet-fibrin interaction in the early stages of clot formation was investigated. To avoid the formation of high fibrin masses, which may obstruct the morphological evaluation, suspensions of washed platelets were diluted with homologous platelet-poor plasma and stimulated with thrombin. The incubation was stopped by fixation after different times and the clots were studied in series of ultra-thin sections. Between 30 and 60 s after stimulation, polymerizing fibrin was found to be situated predominantly within the contact spaces in aggregates of degranulating platelets. Assembling fibres were seen also in plasmalemmal invaginations located in the close vicinity of the constricting contractile cytoskeleton. Between 5 and 10 min after stimulation, fibres with increasing thickness were observed between the platelets and were internalized into deep-surface invaginations, which remained attached to the outer rim of the contractile cytoskeleton. Surface areas without connection to the cytoskeleton revealed no binding or internalization of fibres. Clots obtained after addition of cytochalasin (36 microM) showed no retraction. Fibrin was found to be bound on the whole surface of discoid and degranulated platelets but no internalization occurred. These results suggest that the association of the clustered fibrin(ogen)-receptor complexes to the contractile cytoskeleton and the formation of a constricting sphere leads to the retraction of the clot by internalization of the fibres.

Adenosine Diphosphate↗
PubMed1986

Morphology of the interaction of collagen fibrils with normal human platelets and thrombasthenic platelets.

The ultrastructure of the platelet contacts with collagen fibrils (CF) as well as the course taken by CF on the platelet surface were studied on ultrathin sections of platelets and CF. Platelets from normal donors and from a patient with thrombasthenia were incubated in citrated plasma with collagen. For electron microscopy a protein-stabilizing fixation procedure was applied. Platelet-collagen contacts (PCC) are characterized by a distance of 7 +/- 3 nm between the platelet membrane and the CF; the gap contains electron-dense bridges. The PCC of normal and thrombasthenic platelets are morphologically identical. Hence, it is unlikely that the glycoproteins IIb/IIIa-complex, which is absent in thrombasthenic platelets, plays a significant role in the platelet collagen interaction. The CF induce random movements of the platelets and their pseudopods, whereby the fibrils, which often show multisite attachment, coil up around the platelet surface; they become bent and often display drastic directional changes. CF are found inside invaginations of the platelet membrane as well as in depressions of the platelet surface. These processes require an involvement of the platelet's contractile system, which appears to interact reversibly with the platelet plasma membrane.

Blood Platelet Disorders↗