Intra-individual, randomised comparison of the MRI contrast agents gadobutrol versus gadoteridol in patients with primary and secondary brain tumours, evaluated in a blinded read.

OBJECTIVE: To prove that 1.0 M gadobutrol provides superior contrast enhancement and MRI image characteristics of primary and secondary brain tumours compared with 0.5 M gadoteridol, thereby providing superior diagnostic information. METHODS: Brain MRI was performed in two separate examinations in patients scheduled for neurosurgery. Independent injections of 1.0 M gadobutrol and 0.5 M gadoteridol at doses of 0.1 mmol Gd/kg body weight were administered per patient in randomised order. Evaluation was performed in an off-site blinded read. RESULTS: Fifty-one patients in the full analysis set (FAS) were eligible for efficacy analysis and 44 for the per-protocol analysis. For the primary efficacy variable "preference in contrast enhancement for one contrast agent or the other", the rate of "gadobutrol preferred" was estimated at 0.73 (95 % confidence interval 0.61; 0.83), showing significant superiority of gadobutrol over gadoteridol. Calculated lesion-to-brain contrast and the results of all qualitative secondary efficacy variables were also in favour of gadobutrol. Keeping a sufficient time delay after contrast application proved to be essential to get optimal image quality. CONCLUSION: Compared with 0.5 M gadoteridol, 1.0 M gadobutrol was proven to have significantly superior contrast enhancement characteristics in a routine MRI protocol of primary and secondary brain tumours.

Human platelet electrorotation change induced by activation: inducer specificity and correlation to serotonin release.

Electrorotation of single platelets was compared with [14C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0.10(-7) S.m-1 to 1.0.10(-4) S.m-1) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.

Changes in G-actin after platelet activation in platelet rich plasma.

We have used the DNase I inhibition assay to study changes in G-actin after platelet activation in platelet-rich plasma (PRP) induced by ADP. Because of problems associated with depolymerization of F-actin after lysis of ADP-activated platelets in the presence of plasma, G-actin was measured using a lysis buffer that contained formaldehyde to prevent any depolymerization of F-actin. Different patterns of response were seen depending on the concentration of ADP used, and these were modified by avoiding aggregation by either not stirring the sample or by adding EDTA. The results show rapid conversion of G-actin to F-actin in association with shape change, and there is a further decrease in G-actin associated with irreversible platelet aggregation. Thus evidence is presented that actin polymerization occurs in two phases after ADP stimulation.

Acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIb-IIIa complex--1. Glycoprotein analysis.

A patient with idiopathic thrombocytopenic purpura developed after splenectomy a thrombasthenia-like severe haemorrhagic diathesis characterized by a normal or subnormal platelet count, prolonged bleeding time, strongly reduced platelet adhesion to glass and defective platelet aggregation in response to ADP and collagen. In contrast to hereditary thrombasthenia membrane glycoproteins (GP) IIb and IIIa were normally present in the patient's platelets. Immunoelectrophoretic analysis revealed an abnormal behaviour of the patient's GP IIb-IIIa complex. Autoantibodies against GP IIb-IIIa were detected in Triton-extracted washed platelets. Incubation of normal platelets with plasma from the patient resulted in a similar immunoelectrophoretic abnormality of the GP IIb-IIIa complex indicating that bound autoantibodies (IgG) are responsible for the abnormal immunoelectrophoretic behaviour of the patient's GP IIb-IIIa complex. Platelet fibrinogen was severely reduced similar to classical thrombasthenia suggesting that the GP IIb-IIIa complex is involved in platelet fibrinogen storage.

The platelet glycoprotein IIb/IIIa complex is involved in the adhesion of activated platelets to leukocytes.

The adhesion of activated platelets to leukocytes (rosette formation) seems to be mediated by CD62 on platelets and its counter-receptor (CD15 or a sialic acid-containing glycoprotein) on polymorphonuclear leukocytes (PMNL). However, neither treatment of platelets with an anti-CD62 antibody or fucoidan nor treatment of PMNL with anti-CD15 antibody or neuraminidase are able to inhibit completely the adhesion. Therefore, we have studied the platelet GPIIb/IIIa complex (CD41a) for its involvement in the adhesion of activated platelets to PMNL. The following evidences point to a participation of CD41a in the adhesion of activated platelets to leukocytes: a) inhibition of adhesion by monoclonal antibodies (mab) raised toward CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of the CD41a complex with EGTA, and d) inhibition of rosette formation using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62. It is likely that fibrinogen is involved in the adhesion of platelets to PMNL via CD41a, since fibrinogen increases the rosette formation of ADP-stimulated platelets. Furthermore, the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein IIIa which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

Adhesion of activated platelets to polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNL) are components of the blood which as such interact extensively with other blood cells, with endothelial cells or with plasma. Here, we consider the interaction between PMNL and platelets which is efficient during adhesion of platelets to PMNL and which can be studied in vitro using the rosette formation assay. The adhesion of activated platelets to PMNL seems to be mediated mainly by a protein of platelets (CD62) and its counterreceptor on PMNL, but also other platelet receptors are involved. Here we demonstrate the participation of the glycoprotein IIb-IIIa complex (CD41a) in the adhesion of activated platelets to PMNL due to the following findings: a) inhibition of adhesion by monoclonal antibodies raised against CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of CD41a with EGTA and d) inhibition of adhesion using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62 upon activation. The adhesion of activated platelets to PMNL via CD41a seems to be mediated by fibrinogen due to the following findings: a) addition of fibrinogen to ADP-stimulated and fixed platelets increases significantly the rosette formation and b) the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein IIIa which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

The role of the GSH-disulfide status in the reversible and irreversible aggregation of human platelets.

Disturbance of cellular SH/SS status of blood platelets by diminution of the level of reduced glutathione is very sensitively reflected in changes of the in vitro aggregation. Additionally, disulfide-linked protein polymers are formed. One of these polymers participates in mediating platelet disaggregation.

Chemical modification of cytoskeletal proteins of human blood platelets by diamide.

Incubation of human blood platelets with diamide (azo-dicarboxylic acid-bis-dimethylamide, DIA) influences the aggregation behaviour considerably. Depending on concentration and incubation time DIA induces a reversible aggregation or brings about complete inhibition. The effect is reversible and may be due to the regeneration of reduced glutathione (GSH) which will be oxidized to GSSG by DIA. DIA causes disulfide-linked polymer formation of certain cytoskeletal proteins. At least three polymer families (Pa, Pb, Pc) with different molecular weights are formed depending on dosage and incubation time of DIA. The appearance of a double band in Pa correlates with reversible aggregation, the formation of Pc is always accompanied by a complete inhibition of aggregation. A disturbance of cytoskeleton-membrane interaction by polymer formation can be assumed. GSH serves as a reductant of disulfide-linked polymers whereby a direct link between the maintenance of SH/SS status of platelets and GSH can be established.

Functional studies on platelets of a patient with an acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIB/IIIA complex.

Platelet functions have been studied of a 63 year old woman with a severe acquired thrombopathy. The platelets did not adhere to siliconized glass. Aggregation could not be induced by either ADP (1 microM) nor collagen (2 micrograms/ml), no release of serotonin was found under these conditions. Thrombin caused only a weak aggregation response. Quantitative analysis of platelet actin revealed a very low total actin content (473 micrograms/10(9) platelets) and an extremely low F-actin value (3% of total actin). Stimulation of platelets with 0.1 U/ml thrombin for 3 min resulted in an increase of only 5% F-actin, whereas ADP and collagen did not induce any actin polymerization. Ca2+ movement in the patient's platelets is severely impaired after ADP and collagen stimulation, whereas a normal Ca2+ movement was obtained by 0.1 U/ml thrombin. The inhibition of the functions of normal platelets (aggregation and actin polymerization) by addition of patient's serum (5-10% final concentration) points to receptor blockade by platelet autoantibodies in the patient's serum. The antibody was purified by adsorption on Protein-A-Sepharose. Addition of IgG-suspension (5% final concentration) to washed control platelets resulted in similar effects on aggregation and actin polymerization compared to the effects of patient's serum.

Effect of GSH depletion by 1-chloro-2,4-dinitrobenzene on human platelet aggregation, arachidonic acid oxidative metabolism and cytoskeletal proteins.

Platelet reduced glutathione (GSH) is completely depleted by 1-chloro-2,4-dinitrobenzene (CDNB), which is a substrate for GSH-S-transferase. GSH-depleted platelets: a) aggregate normally at high inducer concentration; b) respond with increased (after arachidonic acid) or depressed (after collagen) aggregability at low inducer concentration; c) show almost no arachidonic acid-induced stimulation of the hexose monophosphate shunt; d) are sensitized to oxidant agents such as diamide, which elicits a faster cytoskeletal protein oxidative polymerization and reversible aggregation. Our results suggest that GSH acts as a reducing cofactor and/or free radical scavenger in the PG-hydroperoxidase step of the cyclooxygenase pathway; moreover, GSH protects membrane and cytoskeletal protein -SH groups from oxidation.

Platelet membrane defects in fawn hooded bleeder rats.

An inbred strain of fawn hooded rats with a congenital platelet defect shows a marked bleeding tendency with prolonged bleeding time. This haemorrhagic disorder has been exclusively related to a deficiency of nucleotides in platelet dense granules. When tested in cell electrophoresis platelets from fawn hooded bleeder rats showed a significantly lower electrophoretic mobility than normal rat platelets. Subsequent studies on the platelet membrane protein pattern by high resolution two-dimensional gel electrophoresis revealed the deficiency of a membrane glycoprotein (apparent molecular mass 90.000, isoelectric point 5.6), which is detectable in normal rat platelets after surface labeling by periodate-tritiated sodium borohydride. It seems likely, that this glycoprotein defect contributes at least partially to the disorder of platelet function in fawn hooded bleeder rats.

Substances that polymerize or depolymerize cytoskeletal proteins affect platelet spreading and thrombus-formation on surfaces coated with human collagen isotypes I, IV, and V.

The effect of substances that affect platelet cytoskeleton on the interaction of gel-filtered platelets with surfaces coated with human monomeric type I, IV, and V collagen was studied. The sulfhydryl group oxidizing agent azodicarboxylic acid-bis-dimethylamide (diamide) which causes disulfide-linked polymer formation of certain cytoskeletal proteins, the actin-polymerization inhibitor, cytochalasin B, and 2-mercaptopropionylglycine (2-MPG), a cell-permeable SH-reagent, completely abolish adhesion-induced platelet spreading and mural platelet aggregate formation on collagen-coated surfaces. Extrusion of pseudopods was inhibited by cytochalasin B and 2-MPG as well as by diamide, but only the latter caused spherulation of platelets, whereas cytochalasin B and 2-MPG left the discoid shape of resting platelets intact. These effects are dose-dependent and are not accounted for by a chemical modification of the collagenous substrates by the cytoskeletal perturbing substances. The present data indicate that (i) cytoskeletal rearrangements are essential in adhesion-induced platelet spreading and aggregate formation on surfaces coated with collagen, but not in supporting the initial attachment of native platelets to the substrate; (ii) both, polymerization and depolymerization of actin filaments affect platelet activation; (iii) the sulfhydryl-disulfide status of the platelet seems to be a possible target for anti-platelet drugs, since chemical modification of platelets by the GSH-GSSG-active substances, diamide and 2-MPG, leads to a reversible inhibition of adhesion-induced platelet activation.

Influence of diamide on aggregation, cytoskeletal proteins, and arachidonic acid metabolism in human platelets.

Simultaneous addition of diamide (azodicarboxylic acid-bis-dimethylamide, DIA), a SH-oxidizing agent, and collagen causes a deaggregation of otherwise irreversibly aggregating platelets. Thromboxane B2 (TXB2) and 12-HE-TE formation is inhibited depending on the concentration ratio between collagen and DIA. Thus, at 0.25 mM DIA and 20 micrograms/ml collagen neither TXB2 nor 12-HETE were measurable, but a full scale reversible aggregation is induced. Deaggregation is further attained by adding DIA to collagen-induced aggregates at a time, when maximum amplitude has been achieved. Investigation of arachidonic acid (AA) metabolites under these conditions revealed no influence of DIA on AA metabolism. Therefore, AA metabolization seems to play a minor role in collagen-induced aggregation and DIA-induced deaggregation. Polymerization of certain cytoskeletal proteins of the platelets, after addition of DIA, parallels DIA-induced deaggregation. DIA inhibits endogenous AA release, probably by interaction with platelet plasma membrane. DIA seems to inhibit the release of the alpha-granula protein thrombospondin.

Changes in the composition of the platelet cytoskeleton in response to ADP: effects of MK-852 and ARL 66096.

Platelet activation by adenosine diphosphate (ADP) results in an alteration in the composition of the cytoskeleton. Here we have determined the effects of MK-852 and ARL 66096 on the cytoskeletal changes that occur. MK-852 is a GPIIb/IIIa antagonist that inhibits aggregation by interfering with fibrinogen binding ARL 66096 is a P2T antagonist that selectively inhibits ADP-induced aggregation. Neither agent inhibits the shape change response. Experiments were performed in hirudinized platelet-rich plasma. Platelet activation led to a significant and sustained increase in the cytoskeletal content of actin binding protein (ABP), myosin, alpha-actinin, a 66K protein and actin, and a significant decrease in a 31K protein. In the presence of MK-852 there was no increase in ABP or the 66K protein and no decrease in the 31K protein. The increase in myosin and alpha-actinin became reversible but there was still incorporation of actin into the cytoskeleton. In the presence of ARL 66096 there was no increase in ABP or the 66K protein and no decrease in the 31K protein. ARL 66096 also prevented incorporation of alpha-actinin and actin. As with MK-852, myosin incorporation became reversible. The results suggest that (1) myosin is incorporated into the cytoskeleton transiently during shape change, (2) ADP interaction with the ADP aggregation receptor (but not that for shape change) is associated with alpha-actinin and actin incorporation into the cytoskeleton, and (3) further changes that occur are consequent to fibrinogen binding and platelet aggregation.

Contact-induced modulation of neutrophil elastase secretion and phagocytic activity by platelets.

It has been reported that platelets stimulate generation of reactive oxygen species in neutrophils and monocytes by a mechanism that requires mutual cell-cell contact and the presence of P-selectin on the platelet surface. In the present study we investigated the effect of platelet-neutrophil contacts on neutrophil elastase secretion and phagocytic activity. Non-activated or thrombin-activated platelets were fixed with formaldehyde, washed and incubated with neutrophils in the absence or presence of various neutrophil agonists. Elastase secretion was determined by measuring the enzyme activity in cell-free supernatants using a chromogenic substrate. Platelet-neutrophil adhesion and ingestion of zymosan particles by neutrophils were quantitated by light microscopy. Platelets significantly reduced elastase secretion from neutrophils but had no effect on the elastase activity in the supernatant of neutrophil lysates. When neutrophils were stimulated with the ionophore A23187 or the chemotactic peptide FMLP, thrombin-activated platelets were more potent to inhibit elastase secretion when compared with non-activated platelets. Neutrophils that were not able to bind platelets to their surface had a significantly lower phagocytic activity when compared with neutrophil with adherent platelets or neutrophils that were incubated in the absence of platelets. The results indicate that platelet-neutrophil contacts may also lead to an inhibition of neutrophil functions and that such inhibition could be due to a transient contact rather than due to a firm platelet-neutrophil adhesion.

A sensitive procedure for quantifying the phagocytic competence of blood granulocytes.

The phagocytic activity of blood granulocytes can be quantitatively assayed by ingestion of opsonised paraffin oil droplets containing the dye "Oil Red-O" (Stossel, T.P., Mason, R.J., Hartwig, J., and Vaughan, M. (1972) J. Clin. Invest. 51: 615-624). We have modified this assay by incorporating [3H]glycerol into the oil droplets which allows a more sensitive and reproducible measurement of the phagocytic competence of blood granulocytes even at very low cell counts. Comparative studies after one day storage of the blood at 4 degrees C is feasible since they retain 84% of the phagocytic capacity measured when isolated from fresh blood.

Actin filament content in platelets--a sensitive index of cellular reactivity.

Blood platelets have the capacity to participate in a number of physiological as well as pathological processes within the circulation. In order to evaluate their cellular reactivity a number of platelet function tests have been developed. The main in vitro function tests are assessment of aggregation and adhesion, secretion, arachidonate metabolism, coagulant activities and the characterization of surface membrane glycoproteins (Day and Rao, 1986). Here we measure alterations of the G-/F-actin equilibrium of platelets. High F-actin values of unstimulated platelets indicate a hyperreactivity of the cell as examined in platelets from diabetics. Determination of the actin filament content in platelets can be considered as a new sensitive function test.

Comparative study of new alpha-galactosidases in transglycosylation reactions.

We have studied the potential of several newly cloned alpha-galactosidases to catalyze the regioselective synthesis of disaccharides using 4-nitrophenylgalactoside as a donor. The kinetics of the reactions were followed by in situ NMR spectroscopy. The following thermophilic enzymes have been tested: Aga A and an isoenzyme Aga B obtained from the strain KVE39 and Aga 285 from the strain IT285 of Bacillus stearothermophilus; Aga T is an alpha-galactosidase from Thermus brockianus (strain IT360). Two other non-thermophilic alpha-galactosidases have also been evaluated: Aga 1 (Streptococcus mutans, strain Ingbritt) and Raf A (Escherichia coli, strain D1021). For all of the enzymes studied, high regioselectivity was observed leading to two (1 --> 6)-disaccharides: 4-nitrophenyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside and methyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside, which were obtained in 54% (Aga B) and 20% (Aga T) yields, respectively.

Extracts of feverfew may inhibit platelet behaviour via neutralization of sulphydryl groups.

It has been suggested that extracts of feverfew may inhibit platelet behaviour via effects on platelet sulphydryl groups. In the present study we have obtained evidence for such a mode of action. Compounds that contain sulphydryl groups such as cysteine and N-(2-mercaptopropionyl)glycine prevented the inhibition of platelet behaviour by feverfew. Feverfew and parthenolide (one of the active components of feverfew) dramatically reduced the number of acid-soluble sulphydryl groups in platelets. This effect occurred at concentrations similar to those that inhibited platelet secretory activity. Feverfew itself did not induce the formation of disulphide-linked protein polymers in platelets but polymer formation occurred when aggregating agents were added to feverfew-treated platelets. Feverfew evoked changes in the metabolism of arachidonic acid that were similar to those observed in glutathione-depleted platelets.

High spatial resolution magnetic resonance imaging of experimental cerebral venous thrombosis with a blood pool contrast agent.

PURPOSE: The purpose of this study was to investigate the feasibility of clot visualization in small sinus and cortical veins with contrast enhanced MRA in a cerebral venous thrombosis animal model using a blood pool contrast agent, Gadofosveset, and high spatial resolution imaging. MATERIAL AND METHODS: For induction of cerebral venous thrombosis a recently developed combined interventional and microsurgical model was used. Cerebral sinus and cortical vein thrombosis was induced in six pigs. Two further pigs died during the procedure. Standard structural, time-of-flight- and phase contrast-angiograms were followed by fast time resolved high resolution 3D MRA (4D MRA) and subsequent high spatial resolution 3D MRA in the equilibrium phase with and without addition of parallel imaging. Visualization of the clots using the different sequences was subjectively compared and contrast-to-noise ratio (CNR) was assessed. RESULTS: In the remaining six animals the procedure and MR-imaging protocol including administration of Gadofosveset was successfully completed. The 3D high resolution MRA in the equilibrium phase without the addition of parallel imaging was superior to all the other applied MR measurement techniques in terms of visualization of the clots. Only applying this sequence bridging vein thromboses were also seen as a small filling defect with a high CNR of >18. CONCLUSION: Only the non-accelerated high spatial resolution 3D MRA in the equilibrium in conjunction with the blood pool agent Gadofosveset allows for high-contrast visualization of very small clots in the cerebral sinus and cortical veins. STATEMENT CLINICAL IMPACT: Detection of cortical vein thrombosis is of high clinical impact. Conventional MRI sequences often fail to visualize the clot. We could demonstrate that, in contrast to conventional sequences, with high spatial resolution 3D MRA in the equilibrium in conjunction with the blood pool agent Gadofosveset very small clots in the cerebral sinus and cortical veins could be successfully visualized. We think that with the presented approach cortical vein thrombosis might also be sufficiently visualized in patients.