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C Tapparelli

Publications and source records attributed to C Tapparelli.

At least 19 recordsLinked to original sources
PubMed2000

Selective boron-containing thrombin inhibitors--X-ray analysis reveals surprising binding mode.

Based on the structural comparison of the S1 pocket in different trypsin-like serine proteases, a series of Boc-D-trimethylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being different amino boronic acids, have been synthesized as inhibitors of thrombin. Among the novel compounds, a number of derivatives were synthesized which appeared to have side-chain variants too big to fit into the S1 pocket. Nevertheless, these compounds inhibited thrombin in the nM range. The X-ray structure of one of these inhibitors bound to the active side of thrombin reveals that a new binding mode is responsible for these surprising results.

Amides↗
PubMed1999

Design, synthesis and biological evaluation of selective boron-containing thrombin inhibitors.

Based on the structural comparison of the S-1 pocket in different trypsin-like serine proteases, a series of Boc-D-trimethylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being different amino boronic acids, have been synthesised as inhibitors of thrombin. The influence of hydrogen donor/acceptor properties of different residues in the P-1 side chain of these inhibitors on the selectivity profile has been investigated. This study confirmed the structure-based working hypothesis: The hydrophobic/hydrophilic character of amino acid residues 190 and 213 in the neighbourhood of Asp 189 in the S-1 pocket of thrombin (Ala/Val), trypsin (Ser/Val) and plasmin (Ser/Thr) define the specificity for the interaction with different P-1 residues of the inhibitors. Many of the synthesised compounds demonstrate potent antithrombin activity with Boc-D-trimethylsilylalanine-proline-boro-methoxypropylglycine++ + pinanediol (9) being the most selective thrombin inhibitor of this series.

Binding Sites↗
PubMed1996

Characterization of the P2' and P3' specificities of thrombin using fluorescence-quenched substrates and mapping of the subsites by mutagenesis.

The importance of substrate residues P2' and P3' on thrombin catalysis has been investigated by comparing the hydrolysis of a series of fluorescence-quenched substrates. Each consisted of a 10-residue peptide, carrying a 2-aminobenzoyl (Abz) group at the N-terminus, and a penultimate 2,4-dinitrophenyl (Dnp) derivatized lysine. Cleavage of such a peptide relieves the intramolecularly-quenched fluorescence, allowing determination of the kinetic parameters. The nature of the P2' residue was found to have a major influence on the rate of cleavage: the Kcat/Km value for the hydrolysis of the Arg-Ser bond in Abz-Val-Gly-Pro-Arg-Ser-Phe-Leu-Leu-Lys(Dnp)-Asp-OH was nearly 3 orders of magnitude higher than that for the hydrolysis of the same substrate with aspartate instead of phenylalanine at the P2' position. Comparatively, the P3' side chain was less important: the kcat/Km value for the substrate with the least effective residue (aspartate) was only 33 times lower than that of the substrate with the most favorable amino acid (lysine). The role of thrombin residues Arg35, Lys36, Glu39 and Lys60f in the putative P2' and P3' binding sites was also examined. Replacement of Lys60f by glutamine improved the rate of cleavage for peptides with P2' lysine or leucine. Compared with thrombin, mutants E39K and E39Q hydrolyzed faster substrates with an acidic residue in P2' or P3', but slightly slower those with a lysine at either position. Mutations R35Q and K36Q only improved the hydrolysis of substrates with an acidic P2' residue. Overall, thrombin prefers bulky hydrophobic side chains in subsite S2' and positively charged residues in S3', whereas acidic residues are markedly antagonistic to both subsites.

Amino Acid Sequence↗
PubMed1995

Thrombin inhibitors as antithrombotic agents: the importance of rapid inhibition.

For use as an antithrombotic agent, a thrombin inhibitor must be potent and specific, i.e., it should not significantly inhibit the proteases of the anticoagulation (activated protein C) and fibrinolytic systems (plasminogen activator and plasmin). Previous evaluation of potency and specificity has been based on inhibition constants (Ki values). However, consideration of the kinetic parameters for natural plasma serine protease inhibitors indicates that a low Ki value with thrombin is not sufficient; the inhibited complex must also form rapidly. Moreover, potent inhibition of activated protein C and plasmin could be tolerated providing the inhibited complex only forms slowly. An ideal profile of kinetic parameters with thrombin, activated protein C and plasmin is formulated and discussed in relation to various classes of thrombin inhibitors. Examination of kinetic data for thrombin inhibitors currently in clinical trials (hirudin and hirulog) indicates that they possess this ideal profile of kinetic parameters.

Amino Acid Sequence↗
PubMed1995

Antithrombotic activity in vivo of SDZ 217-766, a low-molecular weight thrombin inhibitor in comparison to heparin.

Antithrombotic potency of SDZ 217-766, a potent inhibitor of thrombin and other trypsin-like serine proteases, was studied in comparison with heparin in rat models of thrombin induced lung platelet accumulation, of thrombosis in arterio-venous shunt, and of venous thrombosis induced by tissue factor. Thrombin-induced platelet accumulation in the lung was inhibited dose-dependently by SDZ 217-766 following intravenous (i.v.) administration of 0.03 mg/kg to 0.3 mg/kg as well as by intraduodenal (i.d.) administration of 3 mg/kg and 10 mg/kg. Comparable inhibitory effects were observed with heparin at 30 IU/kg and 100 IU/kg. In the rat arterio-venous shunt, following i.v. administration of SDZ 217-766, thrombus formation was inhibited by 40% at 0.1 mg/kg, by 60% at 0.3 mg/kg and was abolished at 1.0 mg/kg whilst APTT was prolonged 1.1 fold over the control value at 0.1 mg/kg and 2.7 fold at 1.0 mg/kg. Similar inhibitory effects were observed following i.d. administration of 10 and 30 mg/kg with only marginal (1.2 to 1.8 fold) APTT elevation. In the same model, heparin administered either i.v., 30-300 IU/kg, or subcutaneously, 100 and 300 IU/kg, inhibited thrombus formation dose dependently but in contrast to SDZ 217-766, the inhibitory effect was paralleled by 5-to > 10 fold APTT elevation over baseline. In the venous thrombosis model, SDZ 217-766 infused at 10 micrograms/kg/min and 20 micrograms/kg/min, reduced thrombus formation by 35% and 70%, respectively. In comparison, thrombus formation was decreased by 22% when heparin was infused at 1 IU/kg/min, and abolished at 3 IU/kg/min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Platelet aggregation and fibrinogen binding in human, rhesus monkey, guinea-pig, hamster and rat blood: activation by ADP and a thrombin receptor peptide and inhibition by glycoprotein IIb/IIIa antagonists.

Platelet aggregation and fibrinogen binding in whole blood, induced either by ADP or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin receptor (TRP, thrombin receptor activating peptide), have been studied with blood from different species. Aggregation was assessed by counting the number of single platelets in blood before und after addition of the agonist with an automated cell counter. Both ADP (0.02-0.5 microM) and TRP (1-10 microM) were found to be potent agonists of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal aggregatory response within 2 min of agonist addition. In contrast, hamster and rat platelets were much less responsive to both ADP and TRP in terms of the whole blood aggregation. Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857, inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50's of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50's (as determined by flow cytometric analysis of platelet fibrinogen binding) were 4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around 100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1993

In vitro and in vivo characterization of a neutral boron-containing thrombin inhibitor.

Peptide boronic acid derivatives have proven to be very potent inhibitors of serine proteases with boroarginine derivatives being particularly potent thrombin inhibitors. The importance of the charged side chain of arginine has been investigated by synthesizing a derivative in which this side chain has been replaced by a neutral one. This boronic acid derivative, D-benzyloxycarbonyl (Z)-Phe-Pro-methoxypropylglycine-pinanediol (MpgC10H16), inhibited thrombin by a competitive mechanism with an inhibition constant (Ki) of 8.9 nM. In comparison to boroarginine derivatives, Z-D-Phe-Pro-boroMpgC10H16 displayed higher selectivity for thrombin over trypsin (Ki = 1.1 microM) and plasmin (Ki = 15.7 microM). Prolongation of thrombin time and activated partial thromboplastin time were observed with micromolar concentrations of Z-D-Phe-Pro-boroMpgC10H16. In a thrombin-dependent in vitro aggregation assay with human platelets, Z-D-Phe-Pro-boroMpgC10H16 inhibited aggregation with an IC50 of 85 nM. When tested in a thrombin-dependent platelet accumulation model in the rat, a bolus injection of (Z)-D-Phe-Pro-boroMpgC10H16 (0.3-3 mg/kg) inhibited platelet accumulation. Thus, the substitution of the charged guanidino group in the P1 side chain by the neutral methoxy group resulted in a potent and highly selective thrombin inhibitor with an interesting pharmacological profile with in vitro as well as in vivo models.

Animals↗
PubMed1993

Synthetic low-molecular weight thrombin inhibitors: molecular design and pharmacological profile.

Thrombin is a multifunctional protein: in addition to its role in coagulation, thrombin has important biological effects on platelets, endothelial and smooth muscle cells, leukocytes, the heart and neurones. A detailed understanding of the structure of thrombin, of related serine proteases and of enzyme-inhibitor complexes has aided in the discovery of potent and selective new inhibitor molecules. Some of these novel thrombin inhibitors are active when administered orally and have shown remarkable efficacy as antithrombotic agents in animal models, offering a greater therapeutic potential than presently available drugs. This potential extends also to non-thrombotic indications where thrombin may be involved, namely inflammation, cancer and neurodegenerative diseases. The recent identification of specific thrombin receptors on different cells provides an alternative strategy for inhibiting thrombin's cellular actions, without necessarily compromising its role in haemostasis. In this review, Carlo Tapparelli and colleagues present a comprehensive update of these recent developments in the field of thrombin biology and pharmacology suggesting a new era of therapeutic drugs is on the horizon.

Animals↗
PubMed1992

High level expression of human apolipoprotein A-I in transgenic rats raises total serum high density lipoprotein cholesterol and lowers rat apolipoprotein A-I.

To examine the consequences of increased apolipoprotein A-I production on cholesterol and lipoprotein metabolism, we have produced two lines of transgenic rats; one expressing moderate and one very high levels of human apolipoprotein A-I. The rats were produced by microinjection of a 13 kbp DNA fragment containing the human apolipoprotein A-I gene plus 10 kbp of its 5' flanking sequence and 1 kbp of its 3' flanking sequence. Both lines of transgenic rats express human apolipoprotein A-I mRNA in liver and human apolipoprotein A-I in plasma. Sera from these rats contain significantly higher levels of total apolipoprotein A-I, high density lipoprotein cholesterol and phospholipid than sera from non-transgenic littermates. Transgenic rats expressing high levels of human apolipoprotein A-I have reduced levels of serum rat apolipoprotein A-I suggesting a mechanism exists to down-regulate apolipoprotein A-I production. These transgenic rats provide a unique animal model to examine the effects of increased apolipoprotein A-I production on lipid and lipoprotein metabolism.

Animals↗
PubMed1990

Hemodynamic, antivasoconstrictor, and antiatherosclerotic effects of calcium antagonists in animal models of atherosclerosis.

Calcium antagonists retard the development of atherosclerosis in cholesterol-fed rabbits and modestly enhance regression after their return to a normal diet. Proliferative lesions following endothelial damage (from, for example, balloon catheter, electrical stimulation) are also diminished. Many mechanisms for these effects have been proposed and their relative importance is not yet clear. However, changes in blood lipid levels do not play an important role. Only a few investigations into how atherosclerosis affects the hemodynamic actions of calcium antagonists have been carried out. Thus, the effects of isradipine were compared in atherosclerotic and normal rabbits. Isradipine increased heart rate and cardiac output less in atherosclerotic rabbits than in normal ones while having no effect on the surface electrocardiogram (ECG). In contrast, the arteriolar vasodilator, dihydralazine, induced ST-segment depression with similar falls in blood pressure, partly explainable by reflex tachycardia and intramyocardial maldistribution of coronary blood flow. Flow to the brain increased with isradipine and decreased with dihydralazine. In atherosclerotic animals, the pressor effects of norepinephrine, phenylephrine, and angiotensin II (Ang II) were amplified. Isradipine partly corrected this enhanced responsiveness. Calcium antagonists thus elicit beneficial hemodynamic and antivasoconstrictor effects in atherosclerotic experimental animals, in addition to having a long-term prophylactic antiatherosclerotic action.

Animals↗
PubMed1989

Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein.

Exocytotic secretion requires the interaction and fusion of secretory vesicles with the plasma membrane. This process could be mediated by specific recognition molecules acting as intracellular, membrane-bound receptors and ligands. One possible component of such a recognition site on the plasma membrane is a protein of relative molecular mass (Mr) 51,000 (51K) that has been isolated from bovine adrenal chromaffin cells. This protein binds strongly to chromaffin granules, the secretory vesicles of these cells. To determine the function of this membrane-anchored chromaffin granule-binding protein in exocytosis, we tested the effect of intracellularly injected antibodies on secretion. Here we show, by two independent techniques in two different cell types, that antibodies against this protein inhibit exocytosis. In rat pheochromocytoma cell cultures, monospecific antibodies, applied by erythrocyte ghost fusion, impair the release of 3H-noradrenaline. The same antibodies, introduced into individual chromaffin cells through a patch pipette, block exocytosis, as revealed by the measurement of membrane capacitance. These results demonstrate the functional involvement in exocytosis of a plasma membrane protein with high affinity for secretory vesicles.

Adrenal Medulla↗
PubMed1989

Release of platelet Met-enkephalin, but not serotonin, in migraine. A platelet response unique to migraine patients?

Plasma-induced release of platelet-serotonin and Met-enkephalin was studied by incubating platelets from migraineurs, tension headache patients and healthy volunteers with water, plasma collected during migraine attacks and with plasma collected during attack-free periods. Migraine-attack plasma, but not migraine attack-free plasma, released Met-enkephalin from platelets of migraineurs. Platelets of healthy controls or tension headache sufferers did not release their Met-enkephalin in response to either incubation medium. In contrast to Met-enkephalin, serotonin was not released from platelets in relevant quantities by migraine-attack plasma.

Adult↗
PubMed1989

Serotonin metabolism in migraine.

To investigate systemic serotonin (5-HT) metabolism in migraine, we determined platelet and platelet-free plasma concentrations of 5-HT, its precursors tryptophan and 5-hydroxytryptophan, and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA), as well as the activities of the platelet enzymes monoamine oxidase and phenolsulfotransferase in classic and common migraineurs. Between attacks, migraineurs had lower plasma 5-HT and higher 5-HIAA levels than did healthy controls and patients with tension headache. During migraine attacks, plasma 5-HT levels were substantially higher than during attack-free periods, while 5-HIAA concentrations and platelet enzyme activities were lower. Platelet 5-HT was reduced only during common, but not classic, migraine attacks. We hypothesize that systemic 5-HT metabolism is enhanced in migraineurs during headache-free periods and transiently decreases during attacks, presumably due to a fall in enzymatic degradation. Furthermore, platelet behavior differs during migraine attacks with and without aura, and release of platelet 5-HT cannot (exclusively) be held accountable for the rise of plasma 5-HT during migraine attacks.

5-Hydroxytryptophan↗
PubMed1989

Nonimmunochemical quantitation of mammalian apolipoprotein A-I in whole serum or plasma by nonreducing gel electrophoresis.

A rapid and convenient method for the quantitation of mammalian apoA-I has been developed. The method involves nonreducing sodium dodecyl sulfate gel electrophoresis and Coomassie blue staining, and takes advantage of the relative abundance of apoA-I in whole serum or plasma. ApoA-I was sufficiently resolved to allow quantitation by laser densitometry or spectrophotometry. The assay was linear from 0.25 to 4.0 micrograms of apoA-I. Analytic recovery was 98%. Within-assay variability was 3.1% and between-assay variability was 7.5%. A high degree of positive correlative (r = 0.98) was observed with a human apoA-I radioimmunoassay. For several species investigated, the apoA-I values obtained correlated strongly and positively with high density lipoprotein cholesterol values. When applied to a study of nutritional perturbation in the Mongolian gerbil, the method detected sensible and significant changes in serum apoA-I that paralleled changes in HDL cholesterol.

Animals↗
PubMed1987

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes.

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.

Adenosine Triphosphate↗
PubMed1986

Comparison of the actions of serotoninergic agents on human saphenous veins and platelets.

Changes in tension of helical strips from human saphenous veins and reversible aggregation of human platelets were recorded in vitro. Comparison of the activities of 12 serotonin receptor agonists revealed that only 4 of the investigated tryptamine derivatives acted as full agonists on both tissues. 5-Carboxamidotryptamine, a drug with selective affinity for both 5-HT1A and 5-HT1B binding sites, though the most potent agonist on veins, failed to produce platelet aggregation but acted as a weak antagonist of the 5-HT-induced reversible aggregation. The tetralin derivative 8-OH-DPAT, a drug with selective affinity for 5-HT1A binding sites, was a weak partial agonist on veins and completely devoid of any activity on the platelets. The antagonism of 5-HT by spiperone was monophasic on platelets but biphasic on veins. These data are in line with the contention that the 5-HT-induced reversible aggregation of human platelets is initiated by 5-HT2-like recognition sites while the evidence suggests that the contractile response of human saphenous vein to 5-HT reflects activation of both 5-HT2- and 5-HT1-like recognition sites.

Blood Platelets↗
PubMed1986

Pharmacological studies on frozen stored canine saphenous veins and basilar arteries.

Canine saphenous veins were either placed in Krebs-Henseleit solution and stored for 24 h at +4 degrees C, or immersed in FCS (fetal calf serum) containing 1.8 mol/l DMSO (dimethyl sulfoxide), slowly frozen to -70 degrees C and stored for 4 weeks at -70 degrees C or -190 degrees C. Canine basilar arteries were either stored in Krebs-Henseleit solution for 24 h at +4 degrees C or slowly frozen and stored for 3 months in FCS plus 1.8 mol/l DMSO at -70 degrees C. Subsequent pharmacological investigations revealed a considerable attenuation of the contractile force of frozen-stored vessels but the evidence suggests that there may be a very good preservation of the main biochemical properties, such as monoamine oxidase activity, endogenous prostaglandin synthesis and uptake1 mechanisms in veins stored at -190 degrees C and there is an excellent correlation of the pD2 values for various tryptamine derivatives on canine basilar arteries stored for 3 months at -70 degrees C with those calculated on fresh preparations. It is concluded that freezing isolated blood vessels may be considered an effective means of preserving and storing vascular tissues for pharmacological investigations.

Animals↗