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Notes: Abstract Background: Current strategies in the treatment of patients with acute coronary syndromes include antiplatelet agents and thrombin antagonists, most commonly aspirin and heparin, respectively. Cardiac events, however, occur despite what is considered to be maximal medical treatment.Methods: We determined the percentage of activated platelets in whole blood samples taken from 22 patients with unstable angina and non-Q-wave myocardial infarction participating in the TIMI III B trial. Platelet activation was assessed using a monoclonal antibody to the surface-expressed α-granule protein, P-selectin, and flow cytometry. All patients received a full complement of antiischemic medications as well as intravenous heparin and oral aspirin, and were then randomized to tissue plasminogen activator or placebo.Results: Platelet activation prior to randomization was increased threefold to fourfold compared with healthy volunteers (11.4 ± 11.4% vs. 2.5 ± 2.0%; p 〈 0.01). Serial measurements performed 12, 24, 48, and 96 hours after treatment initiation revealed that platelet activation persisted. No differences in patients experiencing recurrent ischemic events (n=9) or those randomized to a 90-minute, accelerated infusion of tissue plasminogen activator (n=12) were observed.Conclusions: A modest degree of platelet activation is seen for at least 96 hours and possibly longer in patients with unstable angina and non-Q-wave myocardial infarction, despite being treated with intravenous heparin and oral aspirin. These findings support current efforts to identify more potent and selective antithrombotic treatment strategies.

Notes: Abstract Background: Myocardial ischemia at rest is typically associated with atherosclerotic coronary artery disease, atheromatous plaque rupture, and intracoronary thrombosis. In areas of advanced disease and vascular injury, the extent of thrombus is influenced largely by a delicate balance of procoagulant factors, favoring thrombus initiation, growth, and development, and anticoagulant factors, attempting to limit potentially flow-limiting coronary thrombosis. Thrombin, a 308 amino acid serine protease, is considered the most potent procoagulant factor in the setting of acute vessel wall injury, playing an essential role in the conversion of fibrinogen to fibrin, accelerating the prothrombinase complex, activating platelets, and stabilizing fibrin polymers. The purpose of this study was to determine the relationship between electrocardiographic abnormalities and markers of thrombin activity and generation among patients with unstable angina and non-Q-wave myocardial infarction.Methods and Results: In a study of 36 patients (59.1 ± 11.0 years) with myocardial ischemia at rest participating in the Thrombolysis in Myocardial Ischemia (TIMI) IIIB trial, thrombin activity in plasma, as determined by fibrinopeptide A (FPA), prothrombin fragment 1.2 (F 1.2), and thrombin-antithrombin III complexes (TAT) concentrations, were found to be increased significantly when compared with healthy volunteers (p 〈 0.004). Thrombin generation was also increased modestly compared with age-matched patients with stable coronary artery disease undergoing elective cardiac catheterization. Given that the surface 12-lead electrocardiogram (ECG) is frequently abnormal in patients with ischemic chest pain at rest and represents a readily available, first-line diagnostic test for assessing disease activity and treatment response, we investigated whether ECG abnormalities and thrombin activity/ generation in plasma were correlated. Twenty-six patients (72%) had ECG changes compatible with myocardial ischemia at the time of study entry, including 18 (50%) with newly inverted T waves (or pseudonormalization), 14 (39%) with reversible ST-segment depression, and 4 (11%) with transient (〈30 minutes) ST-segment elevation. Within the predefined ECG groups there were no differences in plasma thrombin activity between patients with and those without confirmed abnormalities. Similarly, there were no differences in either plasma thrombin activity or generation between the predefined ECG groups.Conclusion: Although ECG abnormalities supporting the presence of myocardial ischemia occur commonly in patients with chest pain at rest, they do not correlate closely with markers of thrombin activity and generation in plasma. The diagnostic and prognostic capabilities of these diagnostic tools, considered either alone or together, require further investigation.

Notes: The syntheses of 2′-O-succinyl, 2′-O-succinyltyrosinyl methyl ester and 2′-O-succinyliodotyrosinyl methyl ester derivatives of a cyclic nucleotide, derivatives necessary for the successful development of a specific radioimmuno-assay, are described. Fast atom bombardment with collision-induced dissociation and mass-analysed ion kinetic energy spectroscopy were used to verify the positions of substitution and the retention of the 3′,5′-cyclic phosphate moiety. Comparison of spectra produced after different iodination times permitted the optimization of the reaction conditions.

Notes: Analysis of urine from cancer patients by capillary gas chromatography/mass spectrometry positively identified 14 urinary nucleosides including several modified nucleosides. Levels of the modified nucleosides 1-methyl-adenosine, 2-methylguanosine, N2,N2-dimethylguanosine and 1-methylinosine as well as the total nucleoside level were elevated in the urine when a malignant tumour was present; the levels of N2,N2-dimethylguanosine were found to correlate with the stage of the cancer.

Notes: Continuous-flow fast-atom bombardment mass Spectrometry has been developed to directly monitor cyclic nucleotide (substrate) and its product levels from an on-going phosphodiesterase reaction. Analysis of cAMP and cCMP phosphodiesterase incubates have been performed where the temporal evolution of the enzymic reaction is monitored and the effect of enzyme concentration upon the rate of reaction determined. Quantitative data on the enzyme kinetics have been obtained, in the form of Lineweaver-Burke plots, that are shown to correlate well with well-established radiometric methods.

Notes: Cyclic nucleotides are biochemical second messengers which mediate the actions of many hormones and neurotransmitters. Fast-atom bombardment (FAB) mass spectrometry and mass-analysed ion kinetic energy (MIKE) spectra have provided a means of unambiguous identification of these compounds, and in addition to identifying endogenous putative cyclic nueleotides and their synthetic analogues FAB/MIKE analysis has now been applied to good effect to both qualitative and quantitative studies of cyclic nuclcotide-related enzymes. Here the application of this mass spectrometric technique to cyclic nucleotides is described and its impact upon this field reviewed.

Notes: Tandem mass spectrometry is applied to the tocopherols and representative tocotrienols of the vitamin E family. The collision-induced dissociation/mass analysed ion kinetic energy spectra generated from three ions in the electron impact ionization spectra of 5,7,8-trimethyltocol, 5,7,8-trimethyltocotrienol, 5,7-, 5,8- and 7,8-dimethyltocol and 7,8-dimethyltocotrienol are described. The technique allowed direct physical characterization of each class of tocochromanol, and in the case of monomethyltocols differentiation of 5-methyltocol from the 7- and 8-methyltocol isomers, and its value in analysis of biological tissue extracts is established.

Notes: A protein kinase, stimulated by cytidine 3′, 5′ -cyclic monophosphate, is conventionally assayed by monitoring the incorporation of radiolabelled phosphate from adenosine triphosphate into a histone substrate. Here the assay of the protein kinase is carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data so obtained show good agreement with data obtained by the conventional radiometric assay: the intrinsic advantage of the mass spectrometric assay is the capacity for multiple component monitoring; the ability of the kinase to bind competing cyclic nucleotides togeter with integral adenosine triphosphatase (ATPase) and phosphodiesterase activity can also be assessed.

Notes: Two isomers of adenosine 3′,5′-cyclic monophosphate, which show agonist and antagonist activity with cyclic AMP-dependent protein kinase, were found to yield essentially identical positive-ion fast-atom bombardment (FAB) mass spectra, but S1 and S2 fragments of differing relative intensities in their collision-induced dissociations, studied using mass-analysed ion kinetic energy (CID/MIKE) spectra. Halogen-substituted cyclic nucleotides, used in differentiating between protein kinase cyclic nucleotide binding sites, produced FAB mass spectra and CID/MIKE spectra with fragmentations generally analogous to those of the parent cyclic nucleotides; the bromo-derivatives showed a greater propensity for dehalogenation than the chloro-derivatives. The adenosine triphosphate analogues, adenylyl-(β,γ-methylene)-diphosphate and adenylyl-imidodiphosphate, alternative substrates for adenylyl cyclase, showed similar fragmentations with the methylene and imido groups blocking cleavage between the β and γ phosphate groups. The fragmentations observed are discussed in the context of the use of these compounds in the assay of protein kinase and adenylyl cyclase activity by quantitative mass spectrometry.

Notes: Extracts of the red seaweed Porphyra umbilicalis are a valuable constituent of herbal medicines. The nucleotide complement of such an extract was purified and compounds identified by chromatographic behaviour, UV absorbance/pH profile, base-to-phosphate ratio, acid hydrolysis, and by fast-atom bombardment mass spectrometry with mass-analysed ion kinetic energy spectrum scanning. In addition to eighteen common nucleotides, and two cyclic nucleotides, fifteen novel nucleotides were identified, comprising eight deoxynucleotides and two cyclic deoxynucleotides, ten aminoacylnucleotides and two nucleoside trisphosphates together with 2′-phosphoadenosine-3′,5′-cyclic pyrophosphate, 5′-phosphoadenosine-2′,3′-cyclic monophosphate and N6,N6-dimethyladenosine-5′-monophosphate. The possible origin and potential actions of these novel compouds are discussed.